U.S. patent application number 13/258454 was filed with the patent office on 2012-05-17 for selective and potent peptide inhibitors of kv1.3.
This patent application is currently assigned to Amgen Inc.. Invention is credited to Taruna Arora, Edward J. Belouski, Thomas C. Boone, Colin V. Gegg, Shaw-Fen Sylvia Hu, Frederick W. Jacobsen, Yue-Sheng Li, Leslie P. Miranda, Justin K. Murray, Hung Nguyen, John K. Sullivan, Kenneth W. Walker.
Application Number | 20120121591 13/258454 |
Document ID | / |
Family ID | 42740266 |
Filed Date | 2012-05-17 |
United States Patent
Application |
20120121591 |
Kind Code |
A1 |
Sullivan; John K. ; et
al. |
May 17, 2012 |
SELECTIVE AND POTENT PEPTIDE INHIBITORS OF Kv1.3
Abstract
Disclosed are compositions of matter having an amino acid
sequence of SEQ ID NO:4, or a pharmaceutically acceptable salt
thereof, including embodiments comprising a toxin peptide analog
related to ShK, HmK, and AETX-K and pharmaceutical compositions or
medicaments containing them along with a pharmaceutically
acceptable carrier. Some embodiments include a half-life extending
moiety. Also disclosed are a method of preventing or mitigating a
relapse of a symptom of multiple sclerosis and a method of treating
an autoimmune disorder using the compositions.
Inventors: |
Sullivan; John K.; (Newbury
Park, CA) ; Miranda; Leslie P.; (Thousand Oaks,
CA) ; Gegg; Colin V.; (Newbury Park, CA) ; Hu;
Shaw-Fen Sylvia; (Thousand Oaks, CA) ; Belouski;
Edward J.; (Camarillo, CA) ; Murray; Justin K.;
(Moorpark, CA) ; Nguyen; Hung; (Camarillo, CA)
; Walker; Kenneth W.; (Newbury Park, CA) ; Arora;
Taruna; (Thousand Oaks, CA) ; Jacobsen; Frederick
W.; (Newbury Park, CA) ; Li; Yue-Sheng;
(Thousand Oaks, CA) ; Boone; Thomas C.; (Newbury
Park, CA) |
Assignee: |
Amgen Inc.
Thousand Oaks
CA
|
Family ID: |
42740266 |
Appl. No.: |
13/258454 |
Filed: |
March 19, 2010 |
PCT Filed: |
March 19, 2010 |
PCT NO: |
PCT/US10/28061 |
371 Date: |
February 1, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61210594 |
Mar 20, 2009 |
|
|
|
Current U.S.
Class: |
424/134.1 ;
424/183.1; 514/1.7; 514/15.2; 514/15.4; 514/16.6; 514/16.7;
514/17.9; 514/18.6; 514/21.2; 514/21.3; 514/7.3; 530/325; 530/363;
530/387.3; 530/391.7; 530/409 |
Current CPC
Class: |
A61P 11/06 20180101;
A61P 13/12 20180101; A61P 1/04 20180101; C07K 16/18 20130101; C07K
2319/00 20130101; A61P 37/02 20180101; A61P 19/02 20180101; C07K
16/44 20130101; A61P 29/02 20180101; C07K 14/43504 20130101; A61P
19/08 20180101; C07K 2317/56 20130101; C07K 2317/92 20130101; C07K
2319/30 20130101; A61P 29/00 20180101; A61P 25/04 20180101; A61P
35/00 20180101; A61P 9/00 20180101; A61K 47/6843 20170801; A61P
43/00 20180101; C07K 14/66 20130101; C07K 16/00 20130101; A61P
25/00 20180101; C07K 2317/565 20130101; A61P 11/00 20180101; A61P
25/06 20180101; C07K 2319/55 20130101; A61P 17/00 20180101; A61P
17/06 20180101; C07K 14/575 20130101; A61P 3/10 20180101; A61P
37/08 20180101; C07K 2317/21 20130101; C07K 2317/94 20130101; C07K
16/248 20130101; A61K 47/6817 20170801; C07K 2317/24 20130101; C07K
14/605 20130101; A61K 39/39591 20130101; A61P 37/06 20180101 |
Class at
Publication: |
424/134.1 ;
530/325; 530/363; 530/391.7; 530/409; 530/387.3; 514/21.3;
514/17.9; 514/7.3; 514/18.6; 514/16.6; 514/1.7; 514/15.4; 514/16.7;
514/15.2; 514/21.2; 424/183.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 14/765 20060101 C07K014/765; C07K 16/00 20060101
C07K016/00; C07K 14/47 20060101 C07K014/47; C07K 19/00 20060101
C07K019/00; A61K 38/16 20060101 A61K038/16; A61P 25/00 20060101
A61P025/00; A61P 3/10 20060101 A61P003/10; A61P 17/06 20060101
A61P017/06; A61P 29/00 20060101 A61P029/00; A61P 11/06 20060101
A61P011/06; A61P 13/12 20060101 A61P013/12; A61P 19/08 20060101
A61P019/08; A61K 38/38 20060101 A61K038/38; A61K 38/17 20060101
A61K038/17; A61P 37/06 20060101 A61P037/06; A61P 17/00 20060101
A61P017/00; A61P 19/02 20060101 A61P019/02; A61P 37/08 20060101
A61P037/08; A61P 9/00 20060101 A61P009/00; A61P 43/00 20060101
A61P043/00; C07K 14/00 20060101 C07K014/00 |
Claims
1. A composition of matter comprising an amino acid sequence of the
formula: TABLE-US-00092 SEQ ID NO: 4
X.sub.aa.sup.1X.sub.aa.sup.2Cys.sup.3X.sub.aa.sup.4Asp.sup.5X.sub.aa.sup.6-
X.sub.aa.sup.7X.sub.aa.sup.8X.sub.aa.sup.9X.sub.aa.sup.10X.sub.aa.sup.11Cy-
s.sup.12X.sub.aa.sup.l3X.sub.aa.sup.l4X.sub.aa.sup.l5
X.sub.aa.sup.l6Cys.sup.17X.sub.aa.sup.18X.sub.aa.sup.19X.sub.aa.sup.20X.su-
b.aa.sup.21X.sub.aa.sup.22X.sub.aa.sup.23X.sub.aa.sup.24X.sub.aa.sup.25X.s-
ub.aa.sup.26X.sub.aa.sup.27Cys.sup.28X.sub.aa.sup.29
X.sub.aa.sup.30X.sub.aa.sup.31Cys.sup.32X.sub.aa.sup.33X.sub.aa.sup.34Cys.-
sup.35X.sub.aa.sup.36X.sub.aa.sup.37X.sub.aa.sup.38//
or a pharmaceutically acceptable salt thereof, wherein:
X.sub.aa.sup.1X.sub.aa.sup.2 is absent; or X.sub.aa.sup.1 is absent
and X.sub.aa.sup.2 is Glu, Ser, Ala, or Thr; or X.sub.aa.sup.1 is
Arg or Ala and X.sub.aa.sup.2 is Glu, Ser, Ala, or Thr;
X.sub.aa.sup.4 is an alkyl, basic, or acidic amino acid residue;
X.sub.aa.sup.6 is Thr, Tyr, Ala, or Leu; X.sub.aa.sup.7 is Leu,
Ile, Ala, or Lys; X.sub.aa.sup.8 is Pro, Ala, Arg, Lys, 1-Nal, or
Glu; X.sub.aa.sup.9 is Lys, Ala, Val or an acidic amino acid
residue; X.sub.aa.sup.10 is Ser, Glu, Arg, or Ala; X.sub.aa.sup.11
is Arg, Glu; or Ala; X.sub.aa.sup.13 is Thr, Ala, Arg, Lys, 1-Nal,
or Glu; X.sub.aa.sup.14 is Gln, Ala or an acidic amino acid
residue; X.sub.aa.sup.15 is an alkyl or aromatic amino acid
residue; X.sub.aa.sup.16 is a basic, alkyl, or aromatic amino acid
residue, other than Ala, Gln, Glu or Arg; X.sub.aa.sup.18 is Ala or
an acidic or basic amino acid residue; X.sub.aa.sup.19 is Thr, Ala
or a basic amino acid residue; X.sub.aa.sup.20 is Ser, Ala, or a
basic amino acid residue; X.sub.aa.sup.21 is an alkyl or aromatic
amino acid residue, other than Ala or Met; X.sub.aa.sup.22 is Lys
or Ala; X.sub.aa.sup.23 is Tyr or Ala; X.sub.aa.sup.24 is Arg, Lys,
or Ala; X.sub.aa.sup.25 is Tyr, Leu, or Ala; X.sub.aa.sup.26 is
Ser, Thr, Asn, Ala, or an aromatic amino acid residue;
X.sub.aa.sup.27 is Leu, Ala, Asn, or an aromatic amino acid
residue; X.sub.aa.sup.29 is 1-Nal, 2-Nal, Ala, or a basic amino
acid residue; X.sub.aa.sup.30 is Ala or an acidic or basic amino
acid residue; X.sub.aa.sup.31 is Thr, Ala, or an aromatic amino
acid residue; X.sub.aa.sup.33 is Gly, Ala, Arg, Lys, 1-Nal, or Glu;
X.sub.aa.sup.34 is Thr, Ser, Ala, Lys, or an aromatic amino acid
residue; each of X.sub.aa.sup.36, X.sub.aa.sup.37, and
X.sub.aa.sup.38 is independently absent or is independently a
neutral, basic, acidic, or N-alkylated amino acid residue; and
wherein: there is a disulfide bond between residue Cys.sup.3 and
residue Cys.sup.35, there is a disulfide bond between residue
Cys.sup.12 and residue Cys.sup.28; there is a disulfide bond
between residue Cys.sup.17 and residue Cys.sup.32; and the
carboxy-terminal residue is optionally amidated.
2. The composition of matter of claim 1 wherein X.sub.aa.sup.4 is
selected from Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit,
Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and
4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp, Arg,
N.sup..alpha. Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn,
Asn, Gln, Glu, Asp, .alpha.-aminoadipic acid, and
para-carboxyl-phenylalanine.
3. The composition of matter of claim 1, wherein X.sub.aa.sup.4 is
selected from Ala, Ile, Lys, Orn, Glu and Asp.
4. The composition of matter of claim 1, wherein the acidic amino
acid residue of X.sub.aa.sup.9 and X.sub.aa.sup.14 is each
independently selected from Glu, Asp, and .alpha.-aminoadipic
acid.
5. The composition of matter of claim 1, wherein the alkyl or
aromatic amino acid residue of X.sub.aa.sup.15 is selected from
Ala, 1-Nal, 2-Nal, Phe, Tyr, Val, Ile, and Leu.
6. The composition of matter of claim 1, wherein the alkyl or
aromatic amino acid residue of X.sub.aa.sup.15 is selected from
Phe, Ala, and Ile.
7. The composition of matter of claim 1, wherein X.sub.aa.sup.16 is
selected from Lys, Orn, Dab, Dap 1-Nal, 2-Nal, Tyr, Phe, Pip, 2Pal,
3Pal, N-Me-Lys, N-Me-Orn, alpha-methyl-lysine,
Lys(N.sup..epsilon.-Me), Lys(N.sup..epsilon.-Me).sub.2,
Lys(N.sup..epsilon.-Me).sub.3, para-Methyl-Phe, AMeF, and
homoPhe.
8. The composition of matter of claim 1, wherein the basic or
acidic amino acid residue of X.sub.aa.sup.18 and X.sub.aa.sup.30 is
each independently selected from Lys, Arg, Orn, Glu, Asp, His, Trp,
and Pac.
9. The composition of matter of claim 1, wherein the basic amino
acid residue of X.sub.aa.sup.19, X.sub.aa.sup.20 and
X.sub.aa.sup.29 is each independently selected from Lys, Arg, His,
Orn, D-Orn, Dab, Dap, 1Pip, 2Pal, 3Pal, N-Me-Lys, Na Methyl-Arg;
homoarginine, Cit, N.alpha.-Methyl-Cit, Homocitrulline, Guf, and
4-Amino-Phe, and N-Me-Orn.
10. The composition of matter of claim 1, wherein X.sub.aa.sup.21
is selected from Nle, Nva, Abu, Phe, Tyr, Asn, Gln, Met[O], Val,
Ile, Leu, Met[O.sub.2], Cha, Chg, Asn, Trp, para-Methyl-Phe,
alpha-methyl-Phe, and homoPhe.
11. The composition of matter of claim 1, wherein the aromatic
amino acid residue of X.sub.aa.sup.26, X.sub.aa.sup.27,
X.sub.aa.sup.31, and X.sub.aa.sup.34 is each independently selected
from 1-Nal, 2-Nal, Phe, Trp, and Tyr.
12. The composition of matter of claim 1, wherein X.sub.aa.sup.36,
X.sub.aa.sup.37, and X.sub.aa.sup.38, if present, is each
independently selected from Ala, Leu, Lys, Glu, Asp, Phe, Arg, Phe,
Asp-amide, Aib-amide, Ser-amide, Tyr, Thr-amide, Glu, Glu-amide,
beta-Ala, and N-Me-Ala.
13. The composition of matter of claim 1, wherein the
carboxy-terminal residue is amidated.
14. The composition of matter of claim 1, wherein X.sub.aa.sup.36
is present.
15. The composition of matter of claim 1, comprising an amino acid
sequence selected from SEQ ID NOS: 10, 15, 155, 157, 164, 165, 167
through 172, 179, 194, 196, 203 through 206, 211, 214 through 225,
231, 232, 233, 236, 238, 239, 242 through 254, 260, 263, and 265
through 273.
16. The composition of matter of claim 1, wherein: X.sub.aa.sup.4
is Ala, Ile, Lys, or Glu; and X.sub.aa.sup.16 is Lys, Orn, Dab, Dap
1-Nal, 2-Nal, Tyr, Phe, Pip, 2Pal, 3Pal, N-Me-Lys, or N-Me-Orn,
alpha-methyl-lysine, Lys(N.sup..epsilon.-Me),
Lys(N.sup..epsilon.-Me).sub.2, Lys(N.sup..epsilon.-Me).sub.3,
para-Methyl-Phe, alpha-methyl-Phe, or homoPhe.
17. The composition of matter of claim 16, wherein: X.sub.aa.sup.18
is Lys, Arg, Orn, Glu, Asp, His, Trp, Pac, or Ala.
18. The composition of matter of claim 17, wherein: X.sub.aa.sup.20
is Ser, Ala, Lys, Arg, Orn, Dab, Dap, 1Pip, 2Pal, 3Pal, N-Me-Lys,
or N-Me-Orn.
19. The composition of matter of claim 18, wherein: X.sub.aa.sup.29
is 1-Nal, 2-Nal, Ala, Lys, Arg, Orn, Dab, Dap, 1Pip, 2Pal, 3Pal,
N-Me-Lys, N-Me-Orn.
20. The composition of matter of claim 19, wherein: X.sub.aa.sup.30
is selected from Lys, Arg, Orn, Glu, Asp, His, Trp, or Pac.
21. The composition of matter of any of claims 1 or 13-20, further
comprising an optional linker moiety and a pharmaceutically
acceptable, covalently linked half-life extending moiety.
22. The composition of matter of claim 21, wherein the half-life
extending moiety is polyethylene glycol of molecular weight of
about 1000 Da to about 100000 Da, an IgG Fc domain, a
transthyretin, or a human serum albumin.
23. The composition of matter of claim 21, wherein the half-life
extending moiety comprises a human immunoglobulin or a human
immunoglobulin Fc domain, or both.
24. The composition of matter of claim 23 having a configuration as
set forth in any of FIGS. 12A-N.
25. The composition of matter of claim 23, wherein the composition
comprises a monovalent heterodimeric Fc-toxin peptide analog
fusion.
26. A pharmaceutical composition, comprising the composition of
matter of any of claims 1 or 13-20, and a pharmaceutically
acceptable carrier.
27. A pharmaceutical composition, comprising the composition of
matter of claim 21, and a pharmaceutically acceptable carrier.
28. The composition of matter of any of claims 1 or 13-20, wherein
the composition comprises a toxin peptide analog 33 to about 100
amino acid residues long.
29. The composition of matter of claim 21, wherein the composition
comprises a toxin peptide analog 33 to about 100 amino acid
residues long.
30. A method of preventing or mitigating a relapse of at least one
symptom of multiple sclerosis, comprising administering a
prophylactically effective amount of the composition of matter of
any of claims 1 or 13-20.
31. A method of treating an autoimmune disorder, comprising
administering a therapeutically effective amount of the composition
of matter of any of claims 1 or 13-20.
32. The method of treating an autoimmune disorder of claim 31,
wherein the autoimmune disorder is selected from the group
consisting of multiple sclerosis, type 1 diabetes, psoriasis,
inflammatory bowel disease, contact-mediated dermatitis, rheumatoid
arthritis, psoriatic arthritis, asthma, allergy, restinosis,
systemic sclerosis, fibrosis, scleroderma, glomerulonephritis,
Sjogren syndrome, inflammatory bone resorption, transplant
rejection, graft-versus-host disease, and lupus.
33. A method of preventing or mitigating a relapse of a symptom of
multiple sclerosis, comprising administering a prophylactically
effective amount of the composition of matter of claim 21.
34. A method of treating an autoimmune disorder, comprising
administering a therapeutically effective amount of the composition
of matter of claim 21.
35. The method of claim 34, wherein the autoimmune disorder is
selected from the group consisting of multiple sclerosis, type 1
diabetes, psoriasis, inflammatory bowel disease, contact-mediated
dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma,
allergy, restinosis, systemic sclerosis, fibrosis, scleroderma,
glomerulonephritis, Sjogren syndrome, inflammatory bone resorption,
transplant rejection, graft-versus-host disease, and lupus.
36. A composition of matter comprising a toxin peptide analog of up
to about 100 amino acid residues in length comprising an amino acid
sequence of SEQ ID NO:13, wherein: there is a disulfide bond
between residue Cys.sup.3 and residue Cys.sup.35 of SEQ ID NO:13;
there is a disulfide bond between residue Cys.sup.12 and residue
Cys.sup.28 of SEQ ID NO:13; there is a disulfide bond between
residue Cys.sup.17 and residue Cys.sup.32 of SEQ ID NO:13; and the
carboxy-terminal residue is optionally amidated.
37. The composition of matter of claim 36, further comprising an
optional linker moiety and a pharmaceutically acceptable,
covalently linked half-life extending moiety.
38. The composition of matter of claim 37, wherein the half-life
extending moiety is polyethylene glycol of molecular weight of
about 1000 Da to about 100000 Da, an IgG Fc domain, a
transthyretin, or a human serum albumin.
39. The composition of matter of claim 37, wherein the half-life
extending moiety comprises a human immunoglobulin or a human
immunoglobulin Fc domain, or both.
40. The composition of matter of claim 39 having a configuration as
set forth in any of FIGS. 12A-N.
41. The composition of matter of claim 39, wherein the composition
comprises a monovalent heterodimeric Fc-toxin peptide analog
fusion.
42. The composition of matter of claim 36, further comprising in
its primary sequence at the carboxy terminal end of SEQ ID NO:13 an
additional amino acid residue selected from Ala, Leu, Lys, Glu,
Asp, Phe, Arg, Phe, Asp-amide, Aib-amide, Tyr, Thr-amide, Glu,
Glu-amide, beta-Ala, and N-Me-Ala.
43. The composition of matter of claim 36, wherein the
carboxy-terminal residue is amidated.
44. A pharmaceutical composition, comprising the composition of
matter of any of claims 36-43, and a pharmaceutically acceptable
carrier.
45. A method of treating an autoimmune disorder, comprising
administering a therapeutically effective amount of the composition
of matter of any of claims 36-43.
46. The method of claim 45, wherein the autoimmune disorder is
selected from the group consisting of multiple sclerosis, type 1
diabetes, psoriasis, inflammatory bowel disease, contact-mediated
dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma,
allergy, restinosis, systemic sclerosis, fibrosis, scleroderma,
glomerulonephritis, Sjogren syndrome, inflammatory bone resorption,
transplant rejection, graft-versus-host disease, and lupus.
47. A method of preventing or mitigating a relapse of a symptom of
multiple sclerosis, comprising administering a prophylactically
effective amount of the composition of matter of any of claims
36-43.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/210,594, filed Mar. 20, 2009, which is hereby
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The instant application contains an ASCII "txt" compliant
sequence listing submitted via EFS-WEB on Mar. 19, 2010, which
serves as both the computer readable form (CRF) and the paper copy
required by 37 C.F.R. Section 1.821(c) and 1.821(e), and is hereby
incorporated by reference in its entirety. The name of the "txt"
file created on Mar. 18, 2010, is: A-1455-WO-PCT-SeqList031810-482
ST25.txt, and is 348 kb in size.
[0003] Throughout this application various publications are
referenced within parentheses or brackets. The disclosures of these
publications in their entireties are hereby incorporated by
reference in this application in order to more fully describe the
state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] The present invention is related to the biochemical arts, in
particular to therapeutic peptides and conjugates.
[0006] 2. Discussion of the Related Art
[0007] Ion channels are a diverse group of molecules that permit
the exchange of small inorganic ions across membranes. All cells
require ion channels for function, but this is especially so for
excitable cells such as those present in the nervous system and the
heart. The electrical signals orchestrated by ion channels control
the thinking brain, the beating heart and the contracting muscle.
Ion channels play a role in regulating cell volume, and they
control a wide variety of signaling processes.
[0008] The ion channel family includes Na+, K+, and Ca2+ cation and
C1-anion channels. Collectively, ion channels are distinguished as
either ligand-gated or voltage-gated. Ligand-gated channels include
both extracellular and intracellular ligand-gated channels. The
extracellular ligand-gated channels include the nicotinic
acetylcholine receptor (nAChR), the serotonin (5-hydroxytryptamine,
5-HT) receptors, the glycine and .gamma.-butyric acid receptors
(GABA) and the glutamate-activated channels including kanate,
.alpha.-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)
and N-methyl-D-aspartate receptors (NMDA) receptors. (Harte and
Ouzounis (2002), FEBS Lett. 514: 129-34). Intracellular ligand
gated channels include those activated by cyclic nucleotides (e.g.
cAMP, cGMP), Ca2+ and G-proteins. (Harte and Ouzounis (2002), FEBS
Lett. 514: 129-34). The voltage-gated ion channels are categorized
by their selectivity for inorganic ion species, including sodium,
potassium, calcium and chloride ion channels. (Harte and Ouzounis
(2002), FEBS Lett. 514: 129-34).
[0009] A unified nomenclature for classification of voltage-gated
channels was recently presented. (Catterall et al. (2000),
Pharmacol. Rev. 55: 573-4; Gutman et al. (2000), Pharmacol. Rev.
55, 583-6; Catterall et al. (2000) Pharmacol. Rev. 55: 579-81;
Catterall et al. (2000), Pharmacol. Rev. 55: 575-8; Hofmann et al.
(2000), Pharmacol. Rev. 55: 587-9; Clapham et al. (2000), Pharmacol
Rev. 55: 591-6; Chandy (1991), Nature 352: 26; Goldin et al.
(2000), Neuron 28: 365-8; Ertel et al. (2000), Neuron 25: 533-5).
The K+ channels constitute the largest and best characterized
family of ion channels described to date. Potassium channels are
subdivided into three general groups: the 6 transmembrane (6.TM.)
K+ channels, the 2.TM.-2.TM./leak K+ channels and the 2.TM./Kir
inward rectifying channels. (Tang et al. (2004), Ann. Rev. Physiol.
66, 131-159). These three groups are further subdivided into
families based on sequence similarity. The voltage-gated K+
channels, including (Kv1-6, Kv8-9), EAG (POTASSIUM CHANNEL,
VOLTAGE-GATED, SUBFAMILY H, MEMBER 1), KQT (Potassium voltage-gated
channel subfamily KQT member 1), and Slo (BKCa; POTASSIUM CHANNEL,
CALCIUM-ACTIVATED, LARGE CONDUCTANCE, SUBFAMILY M, ALPHA MEMBER 1),
are family members of the 6.TM. group. The 2.TM.-2.TM. group
comprises TWIK (POTASSIUM CHANNEL, SUBFAMILY K, MEMBER 1), TREK
(POTASSIUM CHANNEL, SUBFAMILY K, MEMBER 2), TASK (POTASSIUM
CHANNEL, SUBFAMILY K, MEMBER 3), TRAAK (POTASSIUM CHANNEL,
SUBFAMILY K, MEMBER 4), and THIK (POTASSIUM CHANNEL, SUBFAMILY K,
MEMBER 13, also known as TANDEM PORE DOMAIN HALOTHANE-INHIBITED
POTASSIUM CHANNEL), whereas the 2.TM./Kir group consists of Kir1-7.
Two additional classes of ion channels include the inward rectifier
potassium (IRK) and ATP-gated purinergic (P2X) channels. (Harte and
Ouzounis (2002), FEBS Lett. 514: 129-34).
[0010] Toxin peptides produced by a variety of organisms have
evolved to target ion channels. Snakes, scorpions, spiders, bees,
snails and sea anemone are a few examples of organisms that produce
venom that can serve as a rich source of small bioactive toxin
peptides or "toxins" that potently and selectively target ion
channels and receptors. In most cases, these toxin peptides have
evolved as potent antagonists or inhibitors of ion channels, by
binding to the channel pore and physically blocking the ion
conduction pathway. In some other cases, as with some of the
tarantula toxin peptides, the peptide is found to antagonize
channel function by binding to a region outside the pore (e.g., the
voltage sensor domain).
[0011] Native toxin peptides are usually between about 20 and about
80 amino acids in length, contain 2-5 disulfide linkages and form a
very compact structure. Toxin peptides (e.g., from the venom of
scorpions, sea anemones and cone snails) have been isolated and
characterized for their impact on ion channels. Such peptides
appear to have evolved from a relatively small number of structural
frameworks that are particularly well suited to addressing the
critical issues of potency, stability, and selectivity. (See, e.g.,
Dauplais et al., On the convergent evolution of animal toxins:
conservation of a diad of functional residues in potassium
channel-blocking toxins with unrelated structures, J. Biol. Chem.
272(7):4302-09 (1997); Alessandri-Haber et al., Mapping the
functional anatomy of BgK on Kv1.1, Kv1.2, and Kv1.3, J. Biol.
Chem. 274(50):35653-61 (1999)). The majority of scorpion and Conus
toxin peptides, for example, contain 10-40 amino acids and up to
five disulfide bonds, forming extremely compact and constrained
structure (microproteins) often resistant to proteolysis. The
conotoxin and scorpion toxin peptides can be divided into a number
of superfamilies based on their disulfide connections and peptide
folds. The solution structure of many of these has been determined
by Nuclear Magnetic Resonance (NMR) spectroscopy, illustrating
their compact structure and verifying conservation of their family
folding patterns. (E.g., Tudor et al., Ionisation behaviour and
solution properties of the potassium-channel blocker ShK toxin,
Eur. J. Biochem. 251(1-2):133-41 (1998); Pennington et al., Role of
disulfide bonds in the structure and potassium channel blocking
activity of ShK toxin, Biochem. 38(44): 14549-58 (1999); Jaravine
et al., Three-dimensional structure of toxin OSK1 from Orthochirus
scrobiculosus scorpion venom, Biochem. 36(6):1223-32 (1997); del
Rio-Portillo et al.; NMR solution structure of Cn12, a novel
peptide from the Mexican scorpion Centruroides noxius with a
typical beta-toxin sequence but with alpha-like physiological
activity, Eur. J. Biochem. 271(12): 2504-16 (2004);
Prochnicka-Chalufour et al., Solution structure of discrepin, a new
K+-channel blocking peptide from the alpha-KTx15 subfamily,
Biochem. 45(6):1795-1804 (2006)). Conserved disulfide structures
can also reflect the individual pharmacological activity of the
toxin family. (Nicke et al. (2004), Eur. J. Biochem. 271: 2305-19,
Table 1; Adams (1999), Drug Develop. Res.46: 219-34). For example,
.alpha.-conotoxins have well-defined four cysteine/two disulfide
loop structures (Loughnan, 2004) and inhibit nicotinic
acetylcholine receptors. In contrast, .omega.-conotoxins have six
cysteine/three disulfide loop consensus structures (Nielsen, 2000)
and block calcium channels. Structural subsets of toxins have
evolved to inhibit either voltage-gated or calcium-activated
potassium channels.
[0012] Due to their potent and relatively selective blockade of
specific ion channels, toxin peptides have been used for many years
as tools to investigate the pharmacology of ion channels. Other
than excitable cells and tissues such as those present in heart,
muscle and brain, ion channels are also important to non-excitable
cells such as immune cells. Accordingly, the potential therapeutic
utility of toxin peptides has been considered for treating various
immune disorders, in particular by inhibition of potassium channels
such as Kv1.3 and IKCa1 since these channels indirectly control
calcium signaling pathway in lymphocytes. (E.g., Kem et al., ShK
toxin compositions and methods of use, U.S. Pat. No. 6,077,680;
Lebrun et al., Neuropeptides originating in scorpion, U.S. Pat. No.
6,689,749; Beeton et al., Targeting effector memory T cells with a
selective peptide inhibitor of Kv1.3 channels for therapy of
autoimmune diseases, Molec. Pharmacol. 67(4):1369-81 (2005);
Possani Postay et al., VM23 and VM24, two scorpion peptides that
block human T-lymphocyte potassium channels (subtype kv1.3) with
high selectivity and decrease the in vivo DTH-responses in rats, WO
2008/139243; Mouhat et al., K+ channel types targeted by synthetic
OSK1, a toxin from Orthochirus scrobiculosus scorpion venom,
Biochem. J. 385:95-104 (2005); Mouhat et al., Pharmacological
profiling of Orthochirus scrobiculosus toxin 1 analogs with a
trimmed N-terminal domain, Molec. Pharmacol. 69:354-62 (2006);
Mouhat et al., OsK1 derivatives, WO 2006/002850 A2; B. S. Jensen et
al. The Ca2+-activated K+ Channel of Intermediate Conductance: A
Molecular Target for Novel Treatments?, Current Drug Targets
2:401-422 (2001); Rauer et al., Structure-guided Transformation of
Charybdotoxin Yields an Analog That Selectively Targets
Ca2+-activated over Voltage-gated K+ Channels, J. Biol. Chem. 275:
1201-1208 (2000); Castle et al., Maurotoxin: A Potent Inhibitor of
Intermediate Conductance Ca2+-Activated Potassium Channels,
Molecular Pharmacol. 63: 409-418 (2003); Chandy et al., K+ channels
as targets for specific Immunomodulation, Trends in Pharmacol.
Sciences 25: 280-289 (2004); Lewis & Garcia, Therapeutic
Potential of Venom Peptides, Nat. Rev. Drug Discov. 2: 790-802
(2003); Han et al., Structural basis of a potent peptide inhibitor
designed for Kv1.3 channel, a therapeutic target of autoimmune
disease, J. Biol. Chem. 283(27):19058-65 (2008)].
[0013] Calcium mobilization in lymphocytes is known to be a
critical pathway in activation of inflammatory responses [M. W.
Winslow et al. (2003) Current Opinion Immunol. 15, 299]. Compared
to other cells, T cells show a unique sensitivity to increased
levels of intracellular calcium and ion channels both directly and
indirectly control this process. Inositol triphosphate (IP3) is the
natural second messenger which activates the calcium signaling
pathway. IP3 is produced following ligand-induced activation of the
T cell receptor (TCR) and upon binding to its intracellular
receptor (a channel) causes unloading of intracellular calcium
stores. The endoplasmic reticulum provides one key calcium store.
Thapsigargin, an inhibitor of the sarcoplasmic-endoplasmic
reticulum calcium ATPase (SERCA), also causes unloading of
intracellular stores and activation of the calcium signaling
pathway in lymphocytes. Therefore, thapsigargin can be used as a
specific stimulus of the calcium signaling pathway in T cells. The
unloading of intracellular calcium stores in T cells is known to
cause activation of a calcium channel on the cell surface which
allows for influx of calcium from outside the cell. This store
operated calcium channel (SOCC) on T cells is referred to as "CRAC"
(calcium release activated channel) and sustained influx of calcium
through this channel is known to be critical for full T cell
activation [S. Feske et al. (2005) J. Exp. Med. 202, 651 and N.
Venkatesh et al. (2004) PNAS 101, 8969]. For many years it has been
appreciated that in order to maintain continued calcium influx into
T cells, the cell membrane must remain in a hyperpolarized
condition through efflux of potassium ions. In T cells, potassium
efflux is accomplished by the voltage-gated potassium channel Kv1.3
and the calcium-activated potassium channel IKCa1 [K. G. Chandy et
al. (2004) TIPS 25, 280]. These potassium channels therefore
indirectly control the calcium signaling pathway, by allowing for
the necessary potassium efflux that allows for a sustained influx
of calcium through CRAC.
[0014] Sustained increases in intracellular calcium activate a
variety of pathways in T cells, including those leading to
activation of NFAT (Nuclear Factor of Activated T cells), NF-kB
(NUCLEAR FACTOR OF KAPPA LIGHT CHAIN GENE ENHANCER IN B CELLS) and
AP-1 [ACTIVATOR PROTEIN 1; Quintana-A (2005) Pflugers Arch.--Eur.
J. Physiol. 450, 1]. These events lead to various T cell responses
including alteration of cell size and membrane organization,
activation of cell surface effector molecules, cytokine production
and proliferation. Several calcium sensing molecules transmit the
calcium signal and orchestrate the cellular response. Calmodulin is
one molecule that binds calcium, but many others have been
identified (M. J. Berridge et al. (2003) Nat. Rev. Mol. Cell. Biol.
4,517). The calcium-calmodulin dependent phosphatase calcineurin is
activated upon sustained increases in intracellular calcium and
dephosphorylates cytosolic NFAT. Dephosphorylated NFAT quickly
translocates to the nucleus and is widely accepted as a critical
transcription factor for T cell activation (F. Macian (2005) Nat.
Rev. Immunol. 5, 472 and N. Venkatesh et al. (2004) PNAS 101,
8969). Inhibitors of calcineurin, such as cyclosporin A (Neoral, S
and Immune) and FK506 (Tacrolimus) are a main stay for treatment of
severe immune disorders such as those resulting in rejection
following solid organ transplant (I. M. Gonzalez-Pinto et al.
(2005) Transplant. Proc. 37, 1713 and D. R. J. Kuypers (2005)
Transplant International 18, 140). Neoral has been approved for the
treatment of transplant rejection, severe rheumatoid arthritis (D.
E. Yocum et al. (2000) Rheumatol. 39, 156) and severe psoriasis (J.
Koo (1998) British J. Dermatol. 139, 88). Preclinical and clinical
data has also been provided suggesting calcineurin inhibitors may
have utility in treatment of inflammatory bowel disease (IBD;
Baumgart D C (2006) Am. J. Gastroenterol. Mar. 30; Epub ahead of
print), multiple sclerosis (Ann. Neurol. (1990) 27, 591) and asthma
(S. Rohatagi et al. (2000) J. Clin. Pharmacol. 40, 1211). Lupus
represents another disorder that may benefit from agents blocking
activation of helper T cells. Despite the importance of calcineurin
in regulating NFAT in T cells, calcineurin is also expressed in
other tissues (e.g. kidney) and cyclosporine A & FK506 have a
narrow safety margin due to mechanism based toxicity. Renal
toxicity and hypertension are common side effects that have limited
the promise of cyclosporine & FK506. Due to concerns regarding
toxicity, calcineurin inhibitors are used mostly to treat only
severe immune disease (Bissonnette-R et al. (2006) J. Am. Acad.
Dermatol. 54, 472). Kv1.3 inhibitors offer a safer alternative to
calcineurin inhibitors for the treatment of immune disorders. This
is because Kv1.3 also operates to control the calcium signaling
pathway in T cells, but does so through a distinct mechanism to
that of calcineurin inhibitors, and evidence on Kv1.3 expression
and function show that Kv1.3 has a more restricted role in T cell
biology relative to calcineurin, which functions also in a variety
of non-lymphoid cells and tissues.
[0015] Calcium mobilization in immune cells also activates
production of the cytokines interleukin 2 (IL-2) and interferon
gamma (designated interchangeably herein as IFNg, IFN-g or
IFN-.gamma.) which are critical mediators of inflammation. IL-2
induces a variety of biological responses ranging from expansion
and differentiation of CD4+ and CD8+ T cells, to enhancement of
proliferation and antibody secretion by B cells, to activation of
NK cells [S. L. Gaffen & K. D. Liu (2004) Cytokine 28, 109].
Secretion of IL-2 occurs quickly following T cell activation and T
cells represent the predominant source of this cytokine Shortly
following activation, the high affinity IL-2 receptor (IL2-R) is
upregulated on T cells endowing them with an ability to proliferate
in response to IL-2. T cells, NK cells, B cells and professional
antigen presenting cells (APCs) can all secrete IFNg upon
activation. T cells represent the principle source of IFNg
production in mediating adaptive immune responses, whereas natural
killer (NK) cells & APCs are likely an important source during
host defense against infection [K. Schroder et al. (2004) J.
Leukoc. Biol. 75, 163]. IFNg, originally called
macrophage-activating factor, upregulates antigen processing and
presentation by monocytes, macrophages and dendritic cells. IFNg
mediates a diverse array of biological activities in many cell
types [U. Boehm et al. (1997) Annu Rev. Immunol. 15, 749] including
growth & differentiation, enhancement of NK cell activity and
regulation of B cell immunoglobulin production and class
switching.
[0016] CD40L (TUMOR NECROSIS FACTOR LIGAND SUPERFAMILY, MEMBER 5)
is another cytokine expressed on activated T cells following
calcium mobilization and upon binding to its receptor on B cells
provides critical help allowing for B cell germinal center
formation, B cell differentiation and antibody isotype switching.
CD40L-mediated activation of CD40 (B CELL-ASSOCIATED MOLECULE CD40;
Also known as TUMOR NECROSIS FACTOR RECEPTOR SUPERFAMILY, MEMBER 5)
on B cells can induce profound differentiation and clonal expansion
of immunoglobulin (Ig) producing B cells [S. Quezada et al. (2004)
Annu Rev. Immunol. 22, 307]. The CD40 receptor can also be found on
dendritic cells and CD40L signaling can mediate dendritic cell
activation and differentiation as well. The antigen presenting
capacity of B cells and dendritic cells is promoted by CD40L
binding, further illustrating the broad role of this cytokine in
adaptive immunity. Given the essential role of CD40 signaling to B
cell biology, neutralizing antibodies to CD40L have been examined
in preclinical and clinical studies for utility in treatment of
systemic lupus erythematosis (SLE),--a disorder characterized by
deposition of antibody complexes in tissues, inflammation and organ
damage [J. Yazdany and J Davis (2004) Lupus 13, 377].
[0017] Small molecule inhibitors of Kv1.3 and IKCa1 potassium
channels and the major calcium entry channel in T cells, CRAC, have
also been developed to treat immune disorders (A. Schmitz et al.
(2005) Molecul. Pharmacol. 68, 1254; K. G. Chandy et al. (2004)
TIPS 25, 280; H. Wulff et al. (2001) J. Biol. Chem. 276, 32040; C.
Zitt et al. (2004) J. Biol. Chem. 279, 12427), but obtaining small
molecules with selectivity toward some of these targets has been
difficult.
[0018] The identification of selective and potent peptide Kv1.3
inhibitors with prolonged in vivo activity has been a long standing
challenge. Production of toxin peptides is a complex process in
venomous organisms, and is an even more complex process
synthetically. Due to their conserved disulfide structures and need
for efficient oxidative refolding, toxin peptides present
challenges to synthesis. Although toxin peptides have been used for
years as highly selective pharmacological inhibitors of ion
channels, the high cost of synthesis and refolding of the toxin
peptides and their short half-life in vivo have impeded the pursuit
of these peptides as a therapeutic modality.
[0019] Much of the focus has been given to analogs of the
35-residue ShK peptide, which has a short in vivo half-life of
about 30 minutes, and which is a potent inhibitor of both the Kv1.3
ion channel and the Kv1.1 ion channel, a potassium channel
expressed in the human nervous system. (E.g., Harvey et al., A
three-residue, continuous binding epitope peptidomimetic of ShK
toxin as a Kv1.3 inhibitor, Bioorganic & Medicinal Chem. Lett.
15:3193-96 (2005); Lanigan et al., Designed peptide analogues of
the potassium channel blocker ShK toxin, Biochem. 40:15528-37
(2001)). Position 22 of ShK has been identified as a key residue
which confers Kv1.3 selectivity, and ShK binding to Kv1.3 is
sensitive to substitution at Lys9 and Arg11. For example,
[Dap22]ShK (SEQ ID NO:317; also known as ShK-Dap22) is a picomolar
range inhibitor of Kv1.3 with a reported 35-fold selectivity for
murine Kv1.3 over murine Kv1.1. (See, e.g., Kem et al., ShK toxin
compositions and methods of use, U.S. Pat. No. 6,077,680).
[Dap22]ShK is reported to display about 20-fold selectivity for
human K(v)1.3 over K(v)1.1, when measured by the whole-cell voltage
clamp method but not in equilibrium binding assays (Middleton R E,
et al., Substitution of a single residue in Stichodactyla
helianthus peptide, ShK-Dap22, reveals a novel pharmacological
profile. Biochemistry. 2003 Nov. 25 42(46):13698-707). The
ShK-Dap22 molecule was reported to have similar potency to native
ShK and to provide potent blockade of Kv1.3 with an IC50 of about
23 .mu.M as measured by whole cell patch clamp electrophysiology.
(Kalman et al., ShK-Dap22, a potent Kv1.3-specific
immunosuppressive polypeptide, J. Biol. Chem. 273(49):32697-707
(1998)).
[0020] Other ShK analogs with phosphotyrosine ("pY"), or other
anionically charged chemical entities, or fluorescein modifications
at the N-terminus have reportedly resulted in some improved
selectivity for mKv1.3 over mKv1.1. An example includes Shk-L5,
which involves a phosphotyrosine-AEEA modification at the
N-terminus of the ShK peptide. (Beeton et al., Targeting effector
memory T cells with a selective peptide inhibitor of Kv1.3 channels
for therapy of autoimmune diseases, Molec. Pharmacol. 67(4):1369-81
(2005); Chandy et al., Analogs of ShK toxin and their uses in
selective inhibition of Kv1.3 potassium channels, WO 2006/042151
A2; Pennington et al., Engineering a stable and selective peptide
blocker of the Kv1.3 channel in T lymphocytes, Molecular
Pharmacology Fast Forward, published Jan. 2, 2009 as
doi:10.1124/mol.108.052704 (2009)). AEEA is
2-(2-(2-aminoethoxy)ethoxy)acetic acid (also known as
8-Amino-3,6-Dioxaoctanoic Acid) and is used as a "linker" group in
peptide chemistry in its N-Fmoc-protected form. In ShK-L5, this
AEEA hydrophilic bifunctional linker is use as a very short bridge
between a phosphotyrosine residue and the ShK peptide. However,
from a biochemical perspective, the phosphotyrosine group is not
metabolically stable, and the phospho group can be cleaved under
physiological conditions. Beeton et al. (2005) indicated that
ShK-L5 has an estimated circulating half-life of about 50 min in
rats following subcutaneous or intravascular injection, which is
comparable to that for the native ShK peptide. (See, e.g., Beeton
et al., Selective blockade of T lymphocyte K+ channels ameliorates
experimental autoimmune encephalomyelitis, a model for multiple
sclerosis, Proc. Natl. Acad. Sci. USA 98(24):13942-47 (2001)). Thus
phosphotyrosine-linked derivative of ShK with improved metabolic
stability but which also retain high potency have been sought.
(Chaurdran, Tet. Letters, 28, 4051-4054 (2007). More recently, M.
W. Pennington et al. described ShK-192, which incorporates
phosphonophenylalanine-AEEA- at the N-terminus instead of
phosphotyrosine-AEEA. (Pennington et al., Engineering a stable and
selective peptide blocker of the Kv1.3 channel in T lymphocytes,
Molec. Pharmacol. 75(4):762-73 (2009)).
[0021] A desideratum provided by the present invention is
compositions of matter including ShK peptide analogs with improved
Kv1.3 inhibition activity, in vivo stability and/or selectivity,
which may also be fused, or otherwise covalently conjugated to a
vehicle.
SUMMARY OF THE INVENTION
[0022] The present invention is directed to a composition of
matter, which comprises an amino acid sequence of the following
formula:
TABLE-US-00001 SEQ ID NO: 4
X.sub.aa.sup.1X.sub.aa.sup.2Cys.sup.3X.sub.aa.sup.4Asp.sup.5X.sub.aa.sup.-
6X.sub.aa.sup.7X.sub.aa.sup.8X.sub.aa.sup.9X.sub.aa.sup.10X.sub.aa.sup.11
Cys.sup.12X.sub.aa.sup.13X.sub.aa.sup.14X.sub.aa.sup.15X.sub.aa.sup.16Cys-
.sup.17X.sub.aa.sup.18X.sub.aa.sup.19X.sub.aa.sup.20X.sub.aa.sup.21
X.sub.aa.sup.22X.sub.aa.sup.23X.sub.aa.sup.24X.sub.aa.sup.25X.sub.aa.sup.-
26X.sub.aa.sup.27Cys.sup.28X.sub.aa.sup.29X.sub.aa.sup.30X.sub.aa.sup.31
Cys.sup.32X.sub.aa.sup.33X.sub.aa.sup.34Cys.sup.35X.sub.aa.sup.36X.sub.aa-
.sup.37X.sub.aa.sup.38//
[0023] or a pharmaceutically acceptable salt thereof,
[0024] wherein:
[0025] X.sub.aa.sup.1X.sub.aa.sup.2 is absent; or X.sub.aa.sup.1 is
absent and X.sub.aa.sup.2 is Glu, Ser, Ala, or Thr; or
X.sub.aa.sup.1 is Arg or Ala and X.sub.aa.sup.2 is Glu, Ser, Ala,
or Thr;
[0026] X.sub.aa.sup.4 is an alkyl, basic, or acidic amino acid
residue;
[0027] X.sub.aa.sup.6 is Thr, Tyr, Ala, or Leu;
[0028] X.sub.aa.sup.7 is Leu, Ile, Ala, or Lys;
[0029] X.sub.aa.sup.8 is Pro, Ala, Arg, Lys, 1-Nal, or Glu;
[0030] X.sub.aa.sup.9 is Lys, Ala, Val or an acidic amino acid
residue;
[0031] X.sub.aa.sup.10 is Ser, Glu, Arg, or Ala;
[0032] X.sub.aa.sup.11 is Arg, Glu; or Ala;
[0033] X.sub.aa.sup.13 is Thr, Ala, Arg, Lys, 1-Nal, or Glu;
[0034] X.sub.aa.sup.14 is Gln, Ala or an acidic amino acid
residue;
[0035] X.sub.aa.sup.15 is an alkyl or aromatic amino acid
residue;
[0036] X.sub.aa.sup.16 is a basic, alkyl, or aromatic amino acid
residue, other than Ala, Gln, Glu or Arg;
[0037] X.sub.aa.sup.18 is Ala or an acidic or basic amino acid
residue;
[0038] X.sub.aa.sup.19 is Thr, Ala or a basic amino acid
residue;
[0039] X.sub.aa.sup.20 is Ser, Ala, or a basic amino acid
residue;
[0040] X.sub.aa.sup.21 is an alkyl or aromatic amino acid residue,
other than Ala or Met;
[0041] X.sub.aa.sup.22 is Lys or Ala;
[0042] X.sub.aa.sup.23 is Tyr or Ala;
[0043] X.sub.aa.sup.24 is Arg, Lys, or Ala;
[0044] X.sub.aa.sup.25 is Tyr, Leu, or Ala;
[0045] X.sub.aa.sup.26 is Ser, Thr, Asn, Ala, or an aromatic amino
acid residue;
[0046] X.sub.aa.sup.27 is Leu, Ala, Asn, or an aromatic amino acid
residue;
[0047] X.sub.aa.sup.29 is 1-Nal, 2-Nal, Ala, or a basic amino acid
residue;
[0048] X.sub.aa.sup.30 is Ala or an acidic or basic amino acid
residue;
[0049] X.sub.aa.sup.31 is Thr, Ala, or an aromatic amino acid
residue;
[0050] X.sub.aa.sup.33 is Gly, Ala, Arg, Lys, 1-Nal, or Glu;
[0051] X.sub.aa.sup.34 is Thr, Ser, Ala, Lys, or an aromatic amino
acid residue;
[0052] each of X.sub.aa.sup.36, X.sub.aa.sup.37, and
X.sub.aa.sup.38 is independently absent or is independently a
neutral, basic, acidic, or N-alkylated amino acid residue;
[0053] and wherein:
[0054] there is a disulfide bond between residue Cys.sup.3 and
residue Cys.sup.35,
[0055] there is a disulfide bond between residue Cys.sup.12 and
residue Cys.sup.28;
[0056] there is a disulfide bond between residue Cys.sup.17 and
residue Cys.sup.32; and
[0057] the carboxy-terminal residue is optionally amidated.
[0058] Encompassed within the present invention are embodiments of
the composition in which one or more additional amino acid residues
are present at the N-terminal end to the left of amino acid
position 1, or at the C-terminal end to the right beyond amino acid
position 38, or both. For example, a composition of the invention
containing a toxin peptide analog of the invention up to about 100
amino acid residues long, can have one, two, three, four, five,
six, seven, eight, nine, ten, twenty, thirty, or more additional
amino acid residues present at the N-terminal end to the left of
amino acid position 1, or at the C-terminal end to the right beyond
amino acid position 38, or at both the N-terminal and C-terminal
ends.
[0059] In some embodiments, the inventive composition of matter
comprises an amino acid sequence selected from comprising an amino
acid sequence selected from SEQ ID NOS: 10, 15, 155, 157, 164, 165,
167 through 172, 179, 194, 196, 203 through 206, 211, 214 through
225, 231, 232, 233, 236, 238, 239, 242 through 254, 260, 263, and
265 through 273 as set forth in Tables 5 and 11-17,
respectively.
[0060] In some embodiments, the inventive composition of matter
comprises an amino acid sequence selected from SEQ ID NOS: 10, 11,
12, 14, 15, 16, 19 through 29, 31 through 34, 36 through 50, 52,
54, 55, 56, 59, 60, 61, 63, 65 through 100, 130 through 140, 142
through 174, 176 through 254, and 257 through 274 as set forth in
Tables 5-17, respectively.
[0061] Another aspect of the invention is a composition of matter
comprising a toxin peptide analog of up to about 100 amino acid
residues long comprising an amino acid sequence of SEQ ID NO:13,
wherein:
[0062] there is a disulfide bond between residue Cys.sup.3 and
residue Cys.sup.35 of SEQ ID NO:13;
[0063] there is a disulfide bond between residue Cys.sup.12 and
residue Cys.sup.28 of SEQ ID NO:13;
[0064] there is a disulfide bond between residue Cys.sup.17 and
residue Cys.sup.32 of SEQ ID NO:13; and
[0065] the carboxy-terminal residue is optionally amidated.
[0066] Embodiments of the inventive compositions of matter include
toxin peptide analogs as unconjugated "naked" peptides, or
covalently linked or conjugated directly or indirectly (i.e.,
through a linker moiety) to a half life-extending moiety.
[0067] Examples of useful half-life extending moieties include an
immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4), immunoglobulin Fc
domain (e.g., a human immunoglobulin Fc domain, including Fc of
IgG1, IgG2, IgG3 or IgG4) or a portion thereof, transthyretin,
human serum albumin (HSA), or poly(ethylene glycol) (PEG) of
molecular weight of about 1000 Da to about 100000 Da. These and
other half-life extending moieties described herein are useful,
either individually or in combination. The toxin peptide analogs of
the present invention are potent peptide inhibitors of Kv1.3 with
improved potency, stability, and/or selectivity over Kv1.1,
relative to native ShK or HmK toxin peptides.
[0068] The invention also relates to a pharmaceutical composition
comprising the inventive composition of matter and a
pharmaceutically acceptable carrier, and to use of the composition
of matter in the manufacture of a medicament.
[0069] The inventive composition of matter can be used for
practicing a method of treating an autoimmune disorder. For
example, the inventive composition of matter can be used in
treatment of an autoimmune disorder selected from multiple
sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease,
contact-mediated dermatitis, rheumatoid arthritis, psoriatic
arthritis, asthma, allergy, restinosis, systemic sclerosis,
fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome,
inflammatory bone resorption, transplant rejection,
graft-versus-host disease, and lupus.
[0070] The inventive composition of matter can also be used for
practicing a method of preventing or mitigating a relapse of a
symptom of multiple sclerosis.
[0071] Although mostly contemplated as therapeutic agents,
compositions of this invention can also be useful in screening for
therapeutic or diagnostic agents. For example, one can use an
Fc-peptide in an assay employing anti-Fc coated plates. The
half-life extending moiety, such as Fc, can make insoluble peptides
soluble and thus useful in a number of assays.
[0072] Numerous additional aspects and advantages of the present
invention will become apparent upon consideration of the figures
and detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0073] FIG. 1A shows the amino acid sequence of the mature ShK
toxin peptide (SEQ ID NO:1), which can be encoded for by a nucleic
acid sequence containing codons optimized for expression in
mammalian cell, bacteria or yeast.
[0074] FIG. 1B shows the three disulfide bonds (--S--S--) formed by
the six cysteines within the ShK peptide. (Kem et al., U.S. Pat.
No. 6,077,680).
[0075] FIG. 1C shows a space filling stereo model of ShK toxin
peptide. Key residues (D5, 17, R11, S20, M21, K22, Y23, F27)
important for Kv1.3 binding (based on analoging described herein)
are lightly shaded.
[0076] FIG. 1D shows a space filling stereo model of the ShK toxin
peptide. Amino acid residues that when changed to an analog result
in improved Kv1.3 versus Kv1.1 selectivity, are lightly shaded and
include 17, S10, Q16, S20, S26, and R29.
[0077] FIG. 2A-B shows an alignment of the voltage-gated potassium
channel inhibitor Stichodactyla helianthus (ShK) with other closely
related members of the sea anemone toxin family. The sequence of
the 35 amino acid mature ShK toxin (Swiss-Protein Accession
#P29187) isolated from the venom of Stichodactyla helianthus is
shown aligned to other closely related members of the sea anemone
family. The consensus sequence and predicted disulfide linkages are
shown, with highly conserved residues being shaded. The HmK peptide
toxin sequence shown (Swiss-Protein Accession #O16846) is of the
immature precursor from the Magnificent sea anemone (Radianthus
magnifica; Heteractis magnifica). The putative signal peptide and
propeptide regions are single & double underlined,
respectively. The mature HmK peptide toxin would be predicted to be
35 amino acids in length and span residues 40 through 74. The
immature AETX-K toxin precursor (Swiss Protein Accession #Q0EAE5)
from the sea anemone Anemonia erythraea is also shown, with the
mature peptide extending from residues 49-83. The predicted signal
peptide and propeptide regions are single and double underlined,
respectively. AeK is the mature peptide toxin, isolated from the
venom of the sea anemone Actinia equina (Swiss-Protein Accession
#P81897). The sequence of the mature peptide toxins AsKS
(Swiss-Protein Accession #Q9TWG1) and BgK (Swiss-Protein Accession
#P29186) isolated from the venom of the sea anemones Anemonia
sulcata and Bunodosoma granulifera, respectively, are also shown.
FIG. 2A shows the amino acid alignment of ShK (SEQ ID NO:1) to
other members of the sea anemone family of toxins, HmK (SEQ ID NO:2
(Mature Peptide) within the larger SEQ ID NO:276 (Signal,
propeptide and Mature Peptide portions)), AeK (SEQ ID NO:5), AsKs
(SEQ ID NO:6), BgK (SEQ ID NO:7), and AETX-K (SEQ ID NO:3 (Mature
Peptide) within the larger SEQ ID NO:275 (Signal, propeptide and
Mature Peptide portions)). Amino acid residues that are conserved
across all the sequences at a given position are listed beneath the
aligned sequences. Red shading or blocking indicates conserved
cysteine residues which are in bold text. Black shading indicates
residues identical to ShK in given position of aligned sequences.
FIG. 2B shows a disulfide linkage map for members of the family of
mature toxin peptides having 3 disulfide linkages (C1-C6, C2-C4,
C3-C5), including ShK, BgK, HmK, AeKS, AETX-K, AsK, and DTX1.
[0078] FIG. 3 shows a summary of the ShK primary amino acid
sequence and the effect of a single amino acid substitutions at
each position, based on the analog data described herein. Positions
where single substitution analogs tend to reduce Kv1.3 activity
("#") are at residues 5, 7, 11, 20-23, and 27. Positions where
single substitution analogs tend to improve Kv1.3 selectivity ("$")
are at residues 7, 10, 16, 20, 22, 23, 26, 27, and 29. Positions
where single substitution analogs tend to improve Kv1.3 activity
("*") are at residues 2, 4, 10, 15, 18, 30, 31, and 34.
[0079] FIG. 4 shows pharmacokinetic data comparing in vivo half
life in rats for 20 kD-PEG-ShK (SEQ ID NO:8) at 0.3 and 2 mg/kg
with ShK-L5 (SEQ ID NO:17; Beeton et al., Targeting effector memory
T cells with a selective peptide inhibitor of Kv1.3 channels for
therapy of autoimmune diseases, Molec. Pharmacol. 67(4):1369-81
(2005); Chandy et al., Analogs of ShK toxin and their uses in
selective inhibition of Kv1.3 potassium channels, WO 2006/042151
A2). (See, Example 5 and Example 8).
[0080] FIG. 5A shows Coomassie brilliant blue stained Tris-glycine
4-20%, SDS-PAGE of the final pool of 20 kDa PEG-[Lys16]Shk (SEQ ID
NO:16) product. Lanes 1-5 were loaded as follows: SeeBlue.RTM. Plus
2 molecular weight protein standards (10 .mu.L; lanes 1 and 4), 2.0
.mu.g product non-reduced (lane 2), blank (lane 3), 2.0 .mu.g
product reduced (lane 5).
[0081] FIG. 5B shows RP-HPLC chromatograms on final PEG-peptide
pools to demonstrate purity of 20 kDa PEG-[Lys16]Shk (SEQ ID NO:16)
purity >99%.
[0082] FIG. 5C shows Coomassie brilliant blue stained Tris-glycine
4-20%, SDS-PAGE of the final pool of 20 kDa branched PEG-[Lys16]Shk
(SEQ ID NO:315) product. Lanes 1-3 were loaded as follows: 2.0
.mu.g product non-reduced (lane 1), SeeBlue.RTM. Plus 2 molecular
weight protein standards (10 .mu.L; lane 2), 2.0 .mu.g product
reduced (lane 3).
[0083] FIG. 5D shows RP-HPLC chromatograms on final PEG-peptide
pools to demonstrate purity of 20 kDa branched PEG-[Lys16]Shk (SEQ
ID NO:315) purity >98%.
[0084] FIG. 5E shows Coomassie brilliant blue stained Tris-glycine
4-20%, SDS-PAGE of the final pool of 20 kDa PEG-[Lys16]Shk-Ala (SEQ
ID NO:316) product. Lanes 1-3 were loaded as follows: 2.0 .mu.g
product non-reduced (lane 1), SeeBlue.RTM. Plus 2 molecular weight
protein standards (10 .mu.L; lane 2), 2.0 .mu.g product reduced
(lane 3).
[0085] FIG. 5F shows RP-HPLC chromatograms on final PEG-peptide
pools to demonstrate purity of 20 kDa PEG-[Lys16]ShK-Ala (SEQ ID
NO:316) purity >99%.
[0086] FIG. 6A-B demonstrates by PatchXpress.RTM. electrophysiology
that N.alpha.-20 kDa-PEG [Lys16]ShK (SEQ ID NO:16) is more potent
in blocking human Kv1.3 current (FIG. 6A) than human Kv1.1 current
(FIG. 6B), as described in Example 5.
[0087] FIG. 6C-D shows by PatchXpress.RTM. electrophysiology the
impact of various concentrations of ShK-L5 (SEQ ID NO:17) on human
Kv1.3 current (FIG. 6C) or human Kv1.1 current (FIG. 6D), as
described in Example 5.
[0088] FIG. 6E-F demonstrates by PatchXpress.RTM. electrophysiology
that the monovalent aKLH HC-ShK(1-35 Q16K) Ab (SEQ ID NO:338, 339,
342) is more potent in blocking human Kv1.3 current (FIG. 6E) than
human Kv1.1 current (FIG. 6F), as described in Examples 11 and
12.
[0089] FIG. 7 shows AT-EAE data comparing the activity in vivo in
rats of Kv1.3-selective inhibitor 20 kDa-PEG-[Lys16]ShK (SEQ ID
NO:16) and the less Kv1.3-selective 20 kDa-PEG-ShK molecule (SEQ ID
NO:8) as described in Example 9.
[0090] FIG. 8A-D show the AT-EAE data for the individual rats
receiving vehicle or doses of 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16)
or 20 kDa-PEG-ShK molecule (SEQ ID NO:8) as described in Example
9.
[0091] FIG. 9A-B shows that in a 12-week pharmacology study in
cynomolgus monkeys, weekly dosing of cynomolgus monkeys with
N.alpha.-20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16) provided sustained
suppression of T cell responses, as measured using the ex vivo cyno
whole blood PD assay of inflammation that measured production of
IL-4 (FIG. 9A) and IL-17 (FIG. 9B). Arrows indicate the approximate
time when weekly doses were delivered. Further details on the study
are provided in Example 10 and Table 4F.
[0092] FIG. 9C shows predicted versus measured serum concentrations
of N.alpha.-20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16) in cynomolgus
monkeys after weekly subcutaneous (SC) dosing (0.5 mg/kg, n=6), as
described in Example 10. The measured serum trough levels after
weekly dosing (open squares), matched closely those predicted based
on repeat-dose modeling of the single-dose pharmacokinetic data
(solid line).
[0093] FIG. 9D shows animal weight gain during the 12-week cyno
pharmacology study described in Example 10 and FIG. 9A-C; arrows on
x-axis indicate SC dosing with N.alpha.-20 kDa-PEG-[Lys16]ShK (SEQ
ID NO:16).
[0094] FIG. 10A-D shows the stability of 20 kDa-PEG-[Lys16]ShK (SEQ
ID NO:16) in rat, cynomolgus monkey and human plasma, tested by
spiking the peptide conjugate into 100% plasma to a final
concentration of 200 ng/mL and incubating for various periods of
time at 37.degree. C. as described in Example 7.
[0095] FIG. 11A-B shows representative PK profiles of a single
subcutaneous dose (mouse and rat, dose=2 mg/kg; beagle and cyno,
dose=0.5 mg/kg) of 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16), as
described in Example 5 and Example 8.
[0096] FIG. 11C shows a representative cyno PK profile of a single
subcutaneous dose (0.5 mg/kg) of 20 kDa-PEG-[Lys16]ShK (SEQ ID
NO:16) that demonstrates drug levels are above 25 nM for one week,
as described in Example 5 and Example 8.
[0097] FIG. 12A-N shows schematic structures of some embodiments of
a composition of the invention that include one or more units of a
pharmacologically active toxin peptide analog (squiggle) fused, via
an optional peptidyl linker moiety such as but not limited to L5 or
L10 described herein, with one or more domains of an
immunoglobulin. These schematics show a more typical IgG1, although
they are intended to apply as well to IgG2s, which will have 4
disulfide bonds in the hinge and a different arrangement of the
disulfide bond linking the heavy and light chain, and IgG3s and
IgG4s. FIG. 12A represents a monovalent heterodimeric Fc-toxin
peptide analog fusion with the toxin peptide analog fused to the
C-terminal end of one of the immunoglobulin Fc domain monomers.
FIG. 12B represents a bivalent homodimeric Fc-toxin peptide analog
fusion, with toxin peptide analogs fused to the C-terminal ends of
both of the immunoglobulin Fc domain monomers. FIG. 12C represents
a monovalent heterodimeric toxin peptide analog-Fc fusion with the
toxin peptide analog fused to the N-terminal end of one of the
immunoglobulin Fc domain monomers. FIG. 12D represents a bivalent
homodimeric toxin peptide analog-Fc fusion, with toxin peptide
analogs fused to the N-terminal ends of both of the immunoglobulin
Fc domain monomers. FIG. 12E represents a monovalent heterotrimeric
Fc-toxin peptide analog/Ab comprising an immunoglobulin heavy chain
(HC)+immunoglobulin light chain (LC)+an immunoglobulin Fc monomer
with a toxin peptide analog fused to its C-terminal end. FIG. 12 F
represents a monovalent heterotetrameric (HT) antibody HC-toxin
peptide analog fusion, with a toxin peptide analog fused to the
C-terminal end of one of the HC monomers. FIG. 12G represents a
bivalent HT antibody Ab HC-toxin peptide analog fusion having toxin
peptide analogs on the C-terminal ends of both HC monomers. FIG.
12H represents a monovalent HT toxin peptide analog-LC Ab, with the
toxin peptide analog fused to the N-terminal end of one of the LC
monomers. FIG. 12I represents a monovalent HT toxin peptide
analog-HC Ab, with the toxin peptide analog fused to the N-terminal
end of one of the HC monomers. FIG. 12J represents a monovalent HT
Ab LC-toxin peptide analog fusion (i.e., LC-toxin peptide analog
fusion+LC+2(HC)), with the toxin peptide analog fused to the
C-terminal end of one of the LC monomers. FIG. 12K represents a
bivalent HT Ab LC-toxin peptide analog fusion (i.e., 2(LC-toxin
peptide analog fusion)+2(HC)), with toxin peptide analogs fused to
the C-terminal end of both of the LC monomers. FIG. 12 L represents
a trivalent HT Ab LC-toxin peptide analog/HC-toxin peptide analog
(i.e., 2(LC-toxin peptide analog fusion)+HC-toxin peptide analog
fusion+HC), with the toxin peptide analogs fused to the C-terminal
ends of both of the LC monomers and one of the HC monomers. FIG.
12M represents a bivalent antibody with a toxin peptide analog
moiety inserted into an internal loop of the immunoglobulin Fc
domain of each HC monomer. FIG. 12N represents a monovalent
antibody with a toxin peptide analog moiety inserted into an
internal loop of the immunoglobulin Fc domain of one of the HC
monomers. Dimers or trimers will form spontaneously in certain host
cells upon expression of a deoxyribonucleic acid (DNA) construct
encoding a single chain. In other host cells, the cells can be
placed in conditions favoring formation of dimers/trimers or the
dimers/trimers can be formed in vitro. If more than one HC monomer,
LC monomer, or immunoglobulin Fc domain monomer is part of a single
embodiment, the individual monomers can be, if desired, identical
or different from each other.
[0098] FIG. 13A shows a Coomassie brilliant blue stained
Tris-glycine 4-20% SDS-PAGE of the final monovalent
Fc-L10-Shk[1-35, Q16K] product. Lanes 1-12 were loaded as follows:
lane 1: Novex Mark12 wide range protein standards (10 .mu.l); lane
2: 0.5 .mu.g product non-reduced; lane 3: blank; lane 4: 2.0 .mu.g
product, non-reduced; lane 5: blank; lane 6: 10 .mu.g product,
non-reduced; lane 7: Novex Mark12 wide range protein standards (10
.mu.l); lane 8: 0.5 .mu.g product, reduced; lane 9: blank; lane 10:
2.0 .mu.g product, reduced; lane 11: blank; lane 12: 10 .mu.g
product, reduced.
[0099] FIG. 13B shows size exclusion chromatography on 20 .mu.g of
the final monovalent Fc-L10-Shk[1-35, Q16K] product injected onto a
Phenomenex BioSep SEC-3000 column (7.8.times.300 mm) in 50 mM
NaH.sub.2PO.sub.4, 250 mM NaCl, and pH 6.9 at 1 ml/min observing
the absorbance at 280 nm. The deflection observed at 12.5 min is an
injection-related artefact.
[0100] FIG. 13C shows an LC-MS analysis of the final sample of
monovalent Fc-L10-Shk[1-35, Q16K]. The product was chromatographed
through a Waters MassPREP micro desalting column using a Waters
ACQUITY HPLC system. The column was set at 80.degree. C. and the
protein eluted using a linear gradient of increasing acetonitrile
concentration in 0.1% formic acid. Part of the column effluent was
diverted into a Waters LCT Premier ESI-TOF mass spectrometer for
mass analysis. The instrument was run in the positive V mode. The
capillary voltage was set at 3,200 V and the cone voltage at 80 V.
The mass spectrum was acquired from 800 to 3000 m/z and
deconvoluted using the MaxEnt1 software provided by the instrument
manufacturer.
[0101] FIG. 14A shows a Coomassie brilliant blue stained
Tris-glycine 4-20% SDS-PAGE of the final bivalent Fc-L10-Shk[1-35,
Q16K] product. Lanes 1-12 were loaded as follows: lane 1: Novex
Mark12 wide range protein standards (10 .mu.l); lane 2: 0.5 .mu.g
product, non-reduced; lane 3: blank; lane 4: 2.0 .mu.g product,
non-reduced; lane 5: blank; lane 6: 10 .mu.g product, non-reduced;
lane 7: Novex Mark12 wide range protein standards (10 .mu.l); lane
8: 0.5 .mu.g product, reduced; lane 9: blank; lane 10: 2.0 .mu.g
product, reduced; lane 11: blank; lane 12: 10 .mu.g product,
reduced.
[0102] FIG. 14B shows size exclusion chromatography on 25 .mu.g of
the final bivalent Fc-L10-Shk[1-35, Q16K] product injected onto a
Phenomenex BioSep SEC-3000 column (7.8.times.300 mm) in 50 mM
NaH.sub.2PO.sub.4, 500 mM NaCl, and pH 6.9 at 1 mL/min observing
the absorbance at 280 nm. The deflection observed at 12 min is an
injection-related artefact.
[0103] FIG. 14C shows a MALDI mass spectral analysis of the final
sample of bivalent Fc-L10-Shk[1-35, Q16K] analyzed using a
Micromass MALDI micro MX mass spectrometer equipped with a nitrogen
laser. The sample was run at positive linear mode. The instrument's
voltage was set at 12 kV and the high mass detector was set at 5
kV. Each spectrum was produced by accumulating data from about 200
laser shots. External mass calibration was achieved using purified
proteins of known molecular masses.
[0104] FIG. 15A shows a Coomassie brilliant blue stained
Tris-glycine 4-20% SDS-PAGE of the final monovalent
Fc-L10-Shk[1-35, Q16K]/anti-KLH Ab product. Lanes 1-12 were loaded
as follows: lane 1: Novex Mark12 wide range protein standards (10
.mu.l); lane 2: 0.5 .mu.g product, non-reduced; lane 3: blank; lane
4: 2.0 .mu.g product, non-reduced; lane 5:blank; lane 6: 10 .mu.g
product, non-reduced; lane 7: Novex Mark12 wide range protein
standards (10 .mu.l); lane 8: 0.5 .mu.g product, reduced; lane 9:
blank; lane 10: 2.0 .mu.g product, reduced; lane 11: blank; lane
12: 10 .mu.g product, reduced.
[0105] FIG. 15B shows size exclusion chromatography on 50 .mu.g of
the final monovalent Fc-L10-Shk[1-35, Q16K]/anti-KLH Ab product
injected onto a Phenomenex BioSep SEC-3000 column (7.8.times.300
mm) in 50 mM NaH.sub.2PO.sub.4, 250 mM NaCl, and pH 6.9 at 1 mL/min
observing the absorbance at 280 nm.
[0106] FIG. 15C shows an LC-MS analysis of the final sample of
monovalent Fc-L10-Shk[1-35, Q16K]/anti-KLH Ab. The product was
chromatographed through a Waters MassPREP micro desalting column
using a Waters ACQUITY HPLC system. The column was set at
80.degree. C. and the protein eluted using a linear gradient of
increasing acetonitrile concentration in 0.1% formic acid. Part of
the column effluent was diverted into a Waters LCT Premier ESI-TOF
mass spectrometer for mass analysis. The instrument was run in the
positive V mode. The capillary voltage was set at 3,200 V and the
cone voltage at 80V. The mass spectrum was acquired from 800 to
3000 m/z and deconvoluted using the MaxEnt1 software provided by
the instrument manufacturer.
[0107] FIG. 16A shows a Coomassie brilliant blue stained
Tris-glycine 4-20% SDS-PAGE of the final monovalent anti-KLH
HC-L10-Shk[1-35, Q16K] product. Lanes 1-12 were loaded as follows:
lane 1: Novex Mark12 wide range protein standards (10 .mu.l); lane
2: 0.5 .mu.g product, non-reduced; lane 3: blank; lane 4: 2.0 .mu.g
product, non-reduced; lane 5:blank; lane 6: 10 .mu.g product,
non-reduced; lane 7: Novex Mark12 wide range protein standards (10
.mu.l); lane 8: 0.5 .mu.g product, reduced; lane 9: blank; lane 10:
2.0 .mu.g product, reduced; lane 11: blank; lane 12: 10 .mu.g
product, reduced.
[0108] FIG. 16B shows size exclusion chromatography on 25 .mu.g of
the final monovalent anti-KLH HC-L10-Shk[1-35, Q16K] Ab product
injected onto a Phenomenex BioSep SEC-3000 column (7.8.times.300
mm) in 50 mM NaH.sub.2PO.sub.4, 250 mM NaCl, and pH 6.9 at 1 mL/min
observing the absorbance at 280 nm. The deflection observed at 11
min is an injection-related artefact.
[0109] FIG. 16C shows a MALDI mass spectral analysis of the final
sample of monovalent anti-KLH HC-L10-Shk[1-35, Q16K] Ab analyzed
using a Micromass MALDI micro MX mass spectrometer equipped with a
nitrogen laser. The sample was run at positive linear mode. The
instrument's voltage was set at 12 kV and the high mass detector
was set at 5 kV. Each spectrum was produced by accumulating data
from about 200 laser shots. External mass calibration was achieved
using purified proteins of known molecular masses.
[0110] FIG. 17A shows a Coomassie brilliant blue stained
Tris-glycine 4-20% SDS-PAGE of the final bivalent aKLH
HC-L10-Shk[1-35 Q16K] Ab product. Lanes 1-12 were loaded as
follows: lane 1: Novex Mark12 wide range protein standards (10
.mu.l); lane 2: 0.5 .mu.g product, non-reduced; lane 3: blank; lane
4: 2.0 .mu.g product, non-reduced; lane 5:blank; lane 6: 10 .mu.g
product, non-reduced; lane 7: Novex Mark12 wide range protein
standards (10 .mu.l); lane 8: 0.5 .mu.g product, reduced; lane 9:
blank; lane 10: 2.0 .mu.g product, reduced; lane 11: blank; lane
12: 10 .mu.g product, reduced.
[0111] FIG. 17B shows size exclusion chromatography on 25 .mu.g of
the final bivalent anti-KLH HC-L10-Shk[1-35, Q16K] Ab product
injected onto a Phenomenex BioSep SEC-3000 column (7.8.times.300
mm) in 50 mM NaH.sub.2PO.sub.4, 500 mM NaCl, and pH 6.9 at 1 mL/min
observing the absorbance at 280 nm. The deflection observed at 11.5
min is an injection-related artefact.
[0112] FIG. 17C shows a MALDI mass spectral analysis of the final
sample of bivalent anti-KLH HC-L10-Shk[1-35, Q16K] Ab analyzed
using a Micromass MALDI micro MX mass spectrometer equipped with a
nitrogen laser. The sample was run at positive linear mode. The
instrument's voltage was set at 12 kV and the high mass detector
was set at 5 kV. Each spectrum was produced by accumulating data
from about 200 laser shots. External mass calibration was achieved
using purified proteins of known molecular masses.
[0113] FIG. 18A shows a Coomassie brilliant blue stained
Tris-glycine 4-20% SDS-PAGE of the final monovalent aKLH
HC-L10-Shk[2-35, Q16K] Ab product. Lanes 1-12 were loaded as
follows: lane 1: Novex Mark12 wide range protein standards (10
.mu.l); lane 2: 0.5 .mu.g product, non-reduced; lane 3: blank; lane
4: 2.0 .mu.g product, non-reduced; lane 5:blank; lane 6: 10 .mu.g
product, non-reduced; lane 7: Novex Mark12 wide range protein
standards (10 .mu.l); lane 8: 0.5 .mu.g product, reduced; lane 9:
blank; lane 10: 2.0 .mu.g product, reduced; lane 11: blank; lane
12: 10 .mu.g product, reduced.
[0114] FIG. 18B shows size exclusion chromatography on 20 .mu.g of
the final monovalent anti-KLH HC-L10-Shk[2-35, Q16K] Ab product
injected onto a Phenomenex BioSep SEC-3000 column (7.8.times.300
mm) in 50 mM NaH.sub.2PO.sub.4, 250 mM NaCl, and pH 6.9 at 1 mL/min
observing the absorbance at 280 nm. The deflection observed at 11
min is an injection-related artefact.
[0115] FIG. 18C shows an LC-MS mass spectral analysis of the final
sample of monovalent anti-KLH HC-L10-Shk[2-35, Q16K] Ab. The
product was chromatographed through a Waters MassPREP micro
desalting column using a Waters ACQUITY HPLC system. The column was
set at 80.degree. C. and the protein eluted using a linear gradient
of increasing acetonitrile concentration in 0.1% formic acid. Part
of the column effluent was diverted into a Waters LCT Premier
ESI-TOF mass spectrometer for mass analysis. The instrument was run
in the positive V mode. The capillary voltage was set at 3,200 V
and the cone voltage at 80V. The mass spectrum was acquired from
800 to 3000 m/z and deconvoluted using the MaxEnt1 software
provided by the instrument manufacturer.
[0116] FIG. 19A shows results of pharmacokinetic study in SD rats
comparing intravenous administration of CHO-Fc (solid circles; 4
mg/kg) versus bivalent Fc-L10-ShK[2-35] (open squares; 2 mg/kg),
described further in Example 12.
[0117] FIG. 19B shows results from a pharmacokinetic study on the
bivalent dimeric Fc-L10-ShK(2-35) (here designated "FcShK") in SD
rats (see Example 12). Serum samples were added to microtiter
plates coated with an anti-human Fc antibody to enable affinity
capture. Plates were then washed, captured samples were released by
SDS and run on a polyacrylamide gel. Samples were then visualized
by western blot using an anti-human Fc-specific antibody and
secondary-HRP conjugate. At 0.25 hr after IV injection (lane 1),
the molecular weight of the band from the serum sample is roughly
identical to the original purified material (lanes 5 & 6),
suggesting little, if any, degradation. Serum collected at 1 hr
(lane 2), 24 hr (lane 3) and 48 hr (lane 4) after injection of the
bivalent molecule showed two bands, one being consistent with
full-length Fc-L10-ShK(2-35) and the smaller being consistent with
Fc alone, suggesting the presence of a monovalent
Fc/Fc-L10-ShK(2-35) heterodimer in the slow elimination phase 2-48
hours after IV injection.
[0118] FIG. 19C-D shows western blot analysis of serum samples from
a pharmacokinetic study on the bivalent Fc-L10-OSK1[K7S] homodimer
(FIG. 19C; single 2 mg/kg IV dose) and the monovalent Fc/Fc-L10-ShK
heterodimer (FIG. 19D; single 1 mg/kg IV dose) in SD rats. Further
details of this study are provided in Example 12. FIG. 19C or FIG.
19D results are from a single representative animal each. The
bivalent Fc-L10-OSK1[K7S] homodimer (FIG. 19C) showed a rapid &
extensive distribution phase, but a slow elimination phase from
1-168 hours. During the slow elimination phase two bands were
observed, the molecular weight (MW) of the larger being consistent
with the full-length Fc-L10-OSK1[K7S] chain and the smaller being
consistent with Fc alone. Therefore, despite injection of a
bivalent homodimer, a monovalent Fc/Fc-L10-OSK1[K7S] heterodimer
appeared to persist in the slow elimination phase. FIG. 19D
indicates the monovalent Fc/Fc-L10-ShK heterodimer after a single
IV dose remains intact as two chains, and has markedly less
distribution compared to bivalent forms, yet retains a slow
elimination rate. Lanes labeled 5 ng or 20 ng are the purified
monovalent Fc/Fc-L10-ShK heterodimer standard.
[0119] FIG. 20 shows results of pharmacokinetic studies
(single-subcutaneous dose=6 mg/kg) performed in SD rats. Open
squares represent data for monovalent Fc/Fc-L10-ShK(1-35, Q16K)
(heterodimer of SEQ ID NO: 337 and SEQ ID NO:348) closed circles
represent data for monovalent anti-KLH antibody-ShK(1-35, Q16K)
(tetramer of SEQ ID NO: 338, SEQ ID NO:339, SEQ ID NO:338, and SEQ
ID NO:342); and closed triangles represent data for monovalent
anti-KLH antibody (loop)-ShK(1-35, Q16K) (tetramer of SEQ ID NO:
338; SEQ ID NO:344; SEQ ID NO:338; and SEQ ID NO:343), described in
Example 12 and Table 4H.
[0120] FIG. 21 shows results of pharmacokinetic studies
(single-subcutaneous dose=6 mg/kg dose) performed in SD rats for
bivalent (open squares) and monovalent (closed circles) anti-KLH
antibody-ShK(1-35, Q16K) (respectively, tetramers of [SEQ ID NO:
338, SEQ ID NO:342, SEQ ID NO:338, SEQ ID NO:342] and [SEQ ID NO:
338, SEQ ID NO:339, SEQ ID NO:338, SEQ ID NO:342]), as further
described in Example 8, Example 12, and Table 4J.
[0121] FIG. 22 shows results of pharmacokinetic studies
(single-subcutaneous dose=6 mg/kg) performed in SD rats for
bivalent (open squares) and monovalent (closed circles) anti-KLH
antibody (loop)-ShK(1-35, Q16K) (respectively, tetramers of [SEQ ID
NO: 338, SEQ ID NO:344, SEQ ID NO:338, SEQ ID NO:344] and [SEQ ID
NO: 338, SEQ ID NO:343, SEQ ID NO:338, SEQ ID NO:344]), as further
described in Example 8, Example 12, and Table 4L.
[0122] FIG. 23 shows the results of pharmacokinetic studies
(single, 2 mg/kg subcutaneous dose) in SD rats of monovalent
Fc-ShK/Fc heterodimer (open squares), monovalent Fc-ShK/aKLH Ab
(heterotrimer or hemibody)(open triangle) and the bivalent
ShK-Fc/ShK-Fc homodimer (closed circles). The monovalent
heterodimer and heterotrimer provided much greater exposure than
the bivalent homodimer. Further details on this study, are provided
in Example 12.
[0123] FIG. 24 shows a cartoon representation of three monovalent
scFc-Shk toxin peptide analog fusions showing the toxin peptide
analog insertion (crescent) in the first Fc domain with a 25-amino
acid residue peptidyl linker (FcLoop(ShK).L25.Fc; left) or a
construct containing the insertion in the second Fc domain with a
20-amino acid residue peptidyl linker (Fc.L20.FcLoop(ShK); center)
or a construct containing the toxin peptide analog at the
C-terminal of the scFc domain (Fc.L20.Fc.ShK; right). In each of
these embodiments, a peptidyl linker (represented as a wavy line)
is shown extending from the C-terminal of the first immunoglobulin
Fc domain to the N-terminal of the second immunoglobulin Fc domain
as part of a single polypeptide chain.
[0124] FIG. 25A-C shows sequences of 3 different scFc-Shk
constructs. The amino acid sequence used to link the two Fc domains
is underlined and the bioactive Shk peptide is in boldface.
Additional linker used to fuse Shk to the C-terminus of the scFc in
the construct of FIG. 25C is also underlined. FIG.
25A--Fc.L20.FcLoop(ShK) (SEQ ID NO:411), FIG.
25B-FcLoop(ShK).L25.Fc (SEQ ID NO:412) and FIG.
25C--Fc.L20.Fc.[Lys16]ShK (SEQ ID NO:410).
[0125] FIG. 26A shows Coomassie brilliant blue stained Tris-glycine
4-20% SDS-PAGE gel of purified FcLoop(ShK).L25.Fc (SEQ ID NO:412;
"16347") and Fc.L20.FcLoop(ShK) (SEQ ID NO:411; "16369").
[0126] FIG. 26B shows RP-HPLC analyses of scFcLoop(Shk) constructs
FcLoop(Shk).L25.Fc (#16347; SEQ ID NO:412; upper panel) and
Fc.L20.FcLoop(Shk) (#16369; SEQ ID NO:411; lower panel).
[0127] FIG. 27A-B shows exemplary nucleic acid and amino acid
sequences (SEQ ID NO:277 and SEQ ID NO:278, respectively) of human
IgG1 Fc that is optimized for mammalian expression and can be used
in this invention.
[0128] FIG. 28A-B shows exemplary nucleic acid and amino acid
sequences (SEQ ID NO:388 and SEQ ID NO:389, respectively) of human
IgG1 Fc that is optimized for bacterial expression and can be used
in this invention.
[0129] FIG. 29A-B shows by whole cell patch clamp electrophysiology
the impact of various concentrations of ShK-192 (SEQ ID NO:438) on
human Kv1.3 current (FIG. 29A) or human Kv1.1 current (FIG. 29B),
as described in Example 1 and Example 5. By whole cell patch clamp
electrophysiology, ShK-192 (SEQ ID NO:438) inhibited Kv1.3 currents
with an IC.sub.50=0.039.+-.0.005 nM (n=3), whereas it inhibited
Kv1.1 currents with an IC.sub.50=3.39.+-.1.61 nM (n=2). The reduced
potency on Kv1.1 was confirmed by PatchXpress planar patch clamp
electrophysiology where ShK-192 had an IC.sub.50=3.26.+-.0.36 nM
(n=8).
[0130] FIG. 30 shows representative dose-response curves for
cyclosporine A and three lots of 20 kDa-PEG-ShK[Lys16] (SEQ ID
NO:16) in blocking IL-2 (FIG. 30A) and IFN.gamma. (FIG. 30B)
secretion from T cells induced by thapsigargin stimulation of human
whole blood, as described in Examples 2 and 5. The IC.sub.50 for
PEG-ShK[Lys16] in blocking IL-2 and IFN.gamma. production was
0.109.+-.0.081 nM (n=34) and 0.240.+-.0.163 nM (n=34),
respectively. Cyclosporin A was about 2000-3000 times less active,
with IC.sub.50 values for IL-2 and IFN.gamma. that were
334.8.+-.172.2 nM (n=64) and 495.2.+-.307.8 nM (n=64),
respectively. The curves shown in FIG. 30 are normalized to 100
percent of control (POC).
[0131] FIG. 31 shows that 20 kDa-PEG-ShK[Lys16] (SEQ ID NO: 16)
provides potent (average IC.sub.50=0.09 nM) inhibition of IL-17
secretion from T cells in cynomolgus monkey whole blood induced by
thapsigargin stimulation, as described in Examples 5 and 8. The
response of blood collected from seven separate cynomolgus monkeys
(labeled cyno 1-cyno 7), is shown. Error bars represent the
standard error of the mean.
[0132] FIG. 32 shows that the molecules 20 kDa-PEG-ShK[Lys16] (SEQ
ID NO: 16), monovalent aKLH HC-ShK(1-35,Q16K) Ab (SEQ ID NO: 338;
339; 338; 342) and monovalent Fc-L10-ShK(1-35,Q16K) (SEQ ID NO:
337; 348) all provide potent inhibition of antigen
(myelin)-mediated proliferation (.sup.3H-thymidine incorporation)
of the rat T effector memory cell line, PAS, as described in
Examples 5, 9, 11 and 12. The IC.sub.50 values for inhibition by
each molecule are shown. Error bars represent the standard error of
the mean.
[0133] FIG. 33 shows AT-EAE data comparing the activity in vivo of
rats treated with vehicle or the Kv1.3-selective inhibitors
monovalent aKLH HC-ShK(1-35,Q16K) Ab (SEQ ID NO: 338; 339; 338;
342) and 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16) as described in
Examples 5, 9, 11 and 12. The larger monovalent aKLH
HC-ShK(1-35,Q16K) Ab molecule exhibited an ED.sub.50 of 2.4 nmol/kg
(360 .mu.g/kg) for inhibition of encephalomyelitis, which was
similar to the 2.47 nmol/kg (10 .mu.g/kg) ED.sub.50 of the smaller
PEG-ShK[Lys16] molecule. Each molecule was delivered by daily
subcutaneous dosing from day-1 through day 7. The figure legend
shows the mg/kg (mpk) doses delivered.
[0134] FIG. 34A-D show the AT-EAE data for the individual rats
receiving vehicle, 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16) or various
doses of the monovalent aKLH HC-ShK(1-35,Q16K) Ab molecule (SEQ ID
NO: 338; 339; 338; 342) as described in Examples 5, 9, 11 and
12.
[0135] FIG. 35 shows AT-EAE data comparing the activity in vivo of
rats treated with vehicle or the Kv1.3-selective inhibitor
monovalent Fc-L10-ShK(1-35,Q16K) (SEQ ID NO: 337; 348) as described
in Example 9, Example 11, and Example 12. The Fc-L10-ShK(1-35,Q16K)
molecule exhibited an ED.sub.50.ltoreq.2.5 nmol/kg (138 .mu.g/kg)
for inhibition of encephalomyelitis. The mg/kg (mpk) doses are
shown and involved daily subcutaneous dosing from day-1 through day
7.
[0136] FIG. 36A-D show the AT-EAE data for the individual rats
receiving vehicle or various doses of the monovalent
Fc-L10-ShK(1-35,Q16K) molecule (SEQ ID NO: 337; 348) as described
in Example 9, Example 11, and Example 12.
[0137] FIG. 37A-B shows levels of serum histamine observed 0.083,
0.25, 1.0 and 96 hours after a single bolus IV injection of 2.0
(FIG. 37A, rat #1-#3) or 5.0 mg/kg (FIG. 37B, rat #4)
PEG-[Lys16]ShK (SEQ ID NO:16) was given to Sprague-Dawley rats, as
described further in Example 10. Background levels of serum
histamine measured in benchmark Sprague-Dawley serum reference
controls of untreated animals, was 153 ng/ml.
[0138] FIG. 38 shows levels of serum histamine observed 0.5, 2, 24
and 48 hours after a single subcutaneous injection of 0.1 (rat
#1-#3), 2.0 (rat #7-#8) or 5.0 (rat #1-12) mg/kg PEG-[Lys16]ShK
(SEQ ID NO:16) was given to Sprague-Dawley rats, as described in
Example 10. Each panel shows the response at each dose for three
separate animals.
[0139] FIG. 39 shows levels of histamine released from isolated
Sprague-Dawley rat peritoneal mast cells one hour after in vitro
incubation with mast cell degranulating peptide (MCDP), compound
48/80, substance P, the calcium ionophore A23187, 20
kDa-PEG-[Lys16]ShK (SEQ ID NO:16), monovalent aKLH HC-ShK(1-35,
Q16K) Ab, or monovalent Fc-L10-ShK(1-35, Q16K) as described in
Example 10.
[0140] FIG. 40 shows levels of histamine released from human
CD34-derived mast cells one hour after in vitro incubation with
mast cell degranulating peptide (MCDP), compound 48/80, substance
P, the calcium ionophore A23187, PEG-[Lys16]ShK (SEQ ID NO:16), or
monovalent Fc-L10-ShK(1-35, Q16K) as described in Example 10. Total
mast cell histamine content in this experiment was 882 ng/ml.
[0141] FIG. 41 shows that PEG-[Lys16]ShK (SEQ ID NO:16) induces
histamine release from Sprague-Dawley rat (FIG. 41A) and Lewis rat
(FIG. 41B) peritoneal mast cells, but did not induce histamine
release from mouse peritoneal mast cells (FIG. 41C) or human
CD34-derived mast cells (FIG. 41D), as described in Example 10.
Despite not responding to PEG-[Lys16]ShK (SEQ ID NO:16), mouse
peritoneal mast cells and human mast cells did respond to positive
controls and other basic secretagogues (e.g. A23187, compound
48/80) run at the same time (not shown). Shown in FIG. 41 is the
percent histamine release, where level of total histamine is
determined as described in Example 10.
[0142] FIG. 42 shows a Coomassie brilliant blue stained
Tris-glycine 4-20% SDS-PAGE of the final monovalent aKLH 120.6
LC-ShK[1-35, Q16K] Ab product. Lanes 1-12 were loaded as follows:
lane 1: Novex Mark12 wide range protein standards (10 .mu.l); lane
2: 0.5 .mu.g product, non-reduced; lane 3: blank; lane 4: 2.0 .mu.g
product, non-reduced; lane 5:blank; lane 6: 10 .mu.g product,
non-reduced; lane 7: Novex Mark12 wide range protein standards (10
.mu.l); lane 8: 0.5 .mu.g product, reduced; lane 9: blank; lane 10:
2.0 .mu.g product, reduced; lane 11: blank; lane 12: 10 .mu.g
product, reduced.
[0143] FIG. 43 shows size exclusion chromatography on 25 .mu.g of
the final monovalent aKLH 120.6 LC-ShK[1-35, Q16K] Ab product
injected onto a Phenomenex BioSep SEC-3000 column (7.8.times.300
mm) in 50 mM NaH.sub.2PO.sub.4, 250 mM NaCl, pH 6.9, at 1 mL/min
detecting the absorbance at 280 nm.
[0144] FIG. 44A-B shows MALDI-MS mass spectral analysis of the
final sample of monovalent aKLH 120.6 LC-ShK[1-35, Q16K] Ab product
(non-reduced, FIG. 44A; reduced, FIG. 44B) using a Micromass MALDI
micro MX mass spectrometer equipped with a nitrogen laser. The
sample was run at positive linear mode. The instrument's voltage
was set at 12 kV and the high mass detector was set at 5 kV. Each
spectrum was produced by accumulating data from about 200 laser
shots. External mass calibration was achieved using purified
proteins of known molecular masses.
[0145] FIG. 45 shows a Coomassie brilliant blue stained
Tris-glycine 4-20% SDS-PAGE of the final bivalent aKLH 120.6
LC-ShK[1-35, Q16K] Ab product. Lanes 1-12 were loaded as follows:
lane 1: Novex Mark12 wide range protein standards (10 .mu.l); lane
2: 0.5 .mu.g product, non-reduced; lane 3: blank; lane 4: 2.0 .mu.g
product, non-reduced; lane 5:blank; lane 6: 10 .mu.g product,
non-reduced; lane 7: Novex Mark12 wide range protein standards (10
.mu.l); lane 8: 0.5 .mu.g product, reduced; lane 9: blank; lane 10:
2.0 .mu.g product, reduced; lane 11: blank; lane 12: 10 .mu.g
product, reduced.
[0146] FIG. 46 shows size exclusion chromatography on 25 .mu.g of
the final bivalent aKLH 120.6 LC-ShK[1-35, Q16K] Ab product
injected onto a Phenomenex BioSep SEC-3000 column (7.8.times.300
mm) in 50 mM NaH.sub.2PO.sub.4, 250 mM NaCl, pH 6.9, at 1 mL/min
detecting the absorbance at 280 nm.
[0147] FIG. 47A-B shows MALDI-MS mass spectral analysis of the
final sample of bivalent aKLH 120.6 LC-ShK[1-35, Q16K] Ab product
(non-reduced, FIG. 47A; reduced, FIG. 47B) using a Micromass MALDI
micro MX mass spectrometer equipped with a nitrogen laser. The
sample was run at positive linear mode. The instrument's voltage
was set at 12 kV and the high mass detector was set at 5 kV. Each
spectrum was produced by accumulating data from about 200 laser
shots. External mass calibration was achieved using purified
proteins of known molecular masses.
[0148] FIG. 48 shows a Coomassie brilliant blue stained
Tris-glycine 4-20% SDS-PAGE of the final trivalent aKLH 120.6
LC-ShK[1-35, Q16K] Ab product. Lanes 1-12 were loaded as follows:
lane 1: Novex Mark12 wide range protein standards (10 .mu.l); lane
2: 0.5 .mu.g product, non-reduced; lane 3: blank; lane 4: 2.0 .mu.g
product, non-reduced; lane 5:blank; lane 6: 10 .mu.g product,
non-reduced; lane 7: Novex Mark12 wide range protein standards (10
.mu.l); lane 8: 0.5 .mu.g product, reduced; lane 9: blank; lane 10:
2.0 .mu.g product, reduced; lane 11: blank; lane 12: 10 .mu.g
product, reduced.
[0149] FIG. 49 shows size exclusion chromatography on 25 .mu.g of
the final trivalent aKLH 120.6 LC-ShK[1-35, Q16K] Ab product
injected onto a Phenomenex BioSep SEC-3000 column (7.8.times.300
mm) in 50 mM NaH.sub.2PO.sub.4, 250 mM NaCl, pH 6.9, at 1 mL/min
detecting the absorbance at 280 nm.
[0150] FIG. 50A-B shows MALDI-MS mass spectral analysis of the
final sample of trivalent aKLH 120.6 LC-ShK[1-35, Q16K] Ab product
(non-reduced, FIG. 50A; reduced, FIG. 50B) using a Micromass MALDI
micro MX mass spectrometer equipped with a nitrogen laser. The
sample was run at positive linear mode. The instrument's voltage
was set at 12 kV and the high mass detector was set at 5 kV. Each
spectrum was produced by accumulating data from about 200 laser
shots. External mass calibration was achieved using purified
proteins of known molecular masses.
[0151] FIG. 51 shows a Coomassie brilliant blue stained
Tris-glycine 4-20% SDS-PAGE of the final monovalent aKLH 120.6 IgG2
HC-Shk[1-35, R1A, I4A, Q16K] Ab product. Lanes 1-12 were loaded as
follows: lane 1: Novex Mark12 wide range protein standards (10
.mu.l); lane 2: 0.5 .mu.g product, non-reduced; lane 3: blank; lane
4: 2.0 .mu.g product, non-reduced; lane 5:blank; lane 6: 10 .mu.g
product, non-reduced; lane 7: Novex Mark12 wide range protein
standards (10 .mu.l); lane 8: 0.5 .mu.g product, reduced; lane 9:
blank; lane 10: 2.0 .mu.g product, reduced; lane 11: blank; lane
12: 10 .mu.g product, reduced.
[0152] FIG. 52 shows size exclusion chromatography on 25 .mu.g of
the final monovalent aKLH 120.6 IgG2 HC-Shk[1-35, R1A, I4A, Q16K]
Ab product injected onto a Phenomenex BioSep SEC-3000 column
(7.8.times.300 mm) in 50 mM NaH.sub.2PO.sub.4, 250 mM NaCl, pH 6.9,
at 1 mL/min detecting the absorbance at 280 nm.
[0153] FIG. 53 shows reduced LC-MS mass spectral analysis of the
heavy chain in the final sample of monovalent aKLH 120.6 IgG2
HC-ShK[1-35, R1A, I4A, Q16K] Ab. The product was chromatographed
through a Waters MassPREP micro desalting column using a Waters
ACQUITY HPLC system. The column was set at 80.degree. C. and the
protein eluted using a linear gradient of increasing acetonitrile
concentration in 0.1% formic acid. Part of the column effluent was
diverted into a Waters LCT Premier ESI-TOF mass spectrometer for
mass analysis. The instrument was run in the positive V mode. The
capillary voltage was set at 3,200 V and the cone voltage at 80 V.
The mass spectrum was acquired from 800 to 3000 m/z and
deconvoluted using the MaxEnt1 software provided by the instrument
manufacturer.
[0154] FIG. 54A-B shows the results of electrophysiology studies on
rat peritoneal mast cells indicating that the cells do not express
a recognizable Kv1.3 current or a current sensitive to
PEG-[Lys16]ShK as described in Example 10. FIG. 54A, shows a
representative whole-cell current recorded from holding potential
of 0 mV to different potentials between -100 mV and +80 mV in 20 mV
increments for 100 milliseconds every 10 seconds. Similar profiles
were observed in recordings from three separate cells. FIG. 54B
shows the current-voltage profile evoked at holding potential of 0
mV and different ramp potentials from -120 mV to +100 mV for 400
milliseconds. An excess amount (100 nM) of the Kv1.3 inhibitors
PEG-[Lys16]ShK and charybdotoxin (ChTx), showed no significant
effect on the current. For electrophysiology studies on rat
peritoneal mast cells, the cells were bathed in 135 mM NaCl, 5 mM
KCl, 1.8 mM CaCl.sub.2, 5 mM Glucose and 10 mM HEPES buffer (pH
7.4). Cells were patched with an internal solution containing 10 mM
NaCl, 90 mM KCl, 40 mM KF, 10 mM EGTA and 10 mM HEPES (pH 7.2).
DETAILED DESCRIPTION
[0155] As used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural referents unless
the context clearly indicates otherwise. Thus, for example,
reference to "a protein" includes a plurality of proteins;
reference to "a cell" includes populations of a plurality of
cells.
[0156] "Polypeptide" and "protein" are used interchangeably herein
and include a molecular chain of two or more amino acids linked
covalently through peptide bonds. The terms do not refer to a
specific length of the product. Thus, "peptides," and
"oligopeptides," are included within the definition of polypeptide.
The terms include post-translational modifications of the
polypeptide, for example, glycosylations, acetylations,
phosphorylations and the like. In addition, protein fragments,
analogs, mutated or variant proteins, fusion proteins and the like
are included within the meaning of polypeptide. The terms also
include molecules in which one or more amino acid analogs or
non-canonical or unnatural amino acids are included as can be
expressed recombinantly using known protein engineering techniques.
In addition, inventive toxin peptide analogs can be derivatized as
described herein by well-known organic chemistry techniques.
[0157] "Toxin peptides" include peptides and polypeptides having
the same amino acid sequence of a naturally occurring
pharmacologically active peptide or polypeptide that can be
isolated from a venom, and also include modified peptide analogs of
such naturally occurring molecules. (See, e.g., Kalman et al.,
ShK-Dap22, a potent Kv1.3-specific immunosuppressive polypeptide,
J. Biol. Chem. 273(49):32697-707 (1998); Kem et al., U.S. Pat. No.
6,077,680; Mouhat et al., OsK1 derivatives, WO 2006/002850 A2;
Chandy et al., Analogs of SHK toxin and their uses in selective
inhibition of Kv1.3 potassium channels, WO 2006/042151; Sullivan et
al., Toxin Peptide therapeutic agents, WO 2006/116156 A2, all of
which are incorporated herein by reference in their entirety).
Snakes, scorpions, spiders, bees, snails and sea anemone are a few
examples of organisms that produce venom that can serve as a rich
source of small bioactive toxin peptides or "toxins" that potently
and selectively target ion channels and receptors. An example of a
toxin peptide is OSK1 (also known as OsK1), a toxin peptide
isolated from Orthochirus scrobiculosus scorpion venom. (e.g.,
Mouhat et al., K+ channel types targeted by synthetic OSK1, a toxin
from Orthochirus scrobiculosus scorpion venom, Biochem. J.
385:95-104 (2005); Mouhat et al., Pharmacological profiling of
Orthochirus scrobiculosus toxin 1 analogs with a trimmed N-terminal
domain, Molec. Pharmacol. 69:354-62 (2006); Mouhat et al., OsK1
derivatives, WO 2006/002850 A2). Another example is ShK, isolated
from the venom of the sea anemone Stichodactyla helianthus. (E.g.,
Tudor et al., Ionisation behaviour and solution properties of the
potassium-channel blocker ShK toxin, Eur. J. Biochem.
251(1-2):133-41 (1998); Pennington et al., Role of disulfide bonds
in the structure and potassium channel blocking activity of ShK
toxin, Biochem. 38(44): 14549-58 (1999); Kem et al., ShK toxin
compositions and methods of use, U.S. Pat. No. 6,077,680; Lebrun et
al., Neuropeptides originating in scorpion, U.S. Pat. No.
6,689,749; Beeton et al., Targeting effector memory T cells with a
selective peptide inhibitor of Kv1.3 channels for therapy of
autoimmune diseases, Molec. Pharmacol. 67(4):1369-81 (2005)).
[0158] The toxin peptides are usually between about 20 and about 80
amino acids in length, contain 2-5 disulfide linkages and form a
very compact structure. Toxin peptides (e.g., from the venom of
scorpions, sea anemones and cone snails) have been isolated and
characterized for their impact on ion channels. Such peptides
appear to have evolved from a relatively small number of structural
frameworks that are particularly well suited to addressing the
critical issues of potency and stability. The majority of scorpion
and Conus toxin peptides, for example, contain 10-40 amino acids
and up to five disulfide bonds, forming extremely compact and
constrained structure (microproteins) often resistant to
proteolysis. The conotoxin and scorpion toxin peptides can be
divided into a number of superfamilies based on their disulfide
connections and peptide folds. The solution structure of many toxin
peptides has been determined by NMR spectroscopy, illustrating
their compact structure and verifying conservation of family
folding patterns. (E.g., Tudor et al., Ionisation behaviour and
solution properties of the potassium-channel blocker ShK toxin,
Eur. J. Biochem. 251(1-2):133-41 (1998); Pennington et al., Role of
disulfide bonds in the structure and potassium channel blocking
activity of ShK toxin, Biochem. 38(44): 14549-58 (1999); Jaravine
et al., Three-dimensional structure of toxin OSK1 from Orthochirus
scrobiculosus scorpion venom, Biochem. 36(6):1223-32 (1997); del
Rio-Portillo et al.; NMR solution structure of Cn12, a novel
peptide from the Mexican scorpion Centruroides noxius with a
typical beta-toxin sequence but with alpha-like physiological
activity, Eur. J. Biochem. 271(12): 2504-16 (2004);
Prochnicka-Chalufour et al., Solution structure of discrepin, a new
K+-channel blocking peptide from the alpha-KTx15 subfamily,
Biochem. 45(6):1795-1804 (2006)). Other examples are known in the
art, or can be found in Sullivan et al., WO06116156 A2 or U.S.
patent application Ser. No. 11/406,454 (titled: Toxin Peptide
Therapeutic Agents, published as US 2007/0071764); Mouhat et al.,
OsK1 derivatives, WO 2006/002850 A2; Sullivan et al., U.S. patent
application Ser. No. 11/978,076 (titled: Conjugated Toxin Peptide
Therapeutic Agents, filed 25 Oct. 2007), Lebrun et al., U.S. Pat.
No. 6,689,749, which are each incorporated by reference in their
entireties. Another example, the HmK toxin peptide is from the
Magnificent sea anemone (Radianthus magnifica; Heteractis
magnifica). Other examples include members of the family of sea
anemone toxins HmK, ShK, BgK, AsKs, AeK, and AETX-K, as described
above. AETX-K is isolated from the sea anemone Anemonia erythraea,
and has the amino acid sequence
RACKDYLPKSECTQFRCRTSMKYKYTNCKKTCGTC//SEQ ID NO:3 within the larger
amino acid sequence (including putative signal sequence)
MKGQMIICLVLIALCMSVVVMAQNLRAEELEKANPKDERVRSFERNQKR
ACKDYLPKSECTQFRCRTSMKYKYTNCKKTCGTC//SEQ ID NO: 275. (See, Hasegawa
et al., Isolation and DNA cloning of a potassium channel peptide
toxin from the sea anemone Anemonia erythraea, Toxicon 48(5):536-42
(2006)). HmK has SEQ ID NO: 2 ((Table 5; mature peptide) within the
larger MKSQMIAAVLLIAFCLCVVVTARMELQDVEDMENGFQKRRTCKDLIPVS
ECTDIRCRTSMKYRLNLCRKTCGSC//SEQ ID NO: 276 (including signal and
mature peptide portions)).
[0159] The term "peptide analog" refers to a peptide having a
sequence that differs from a peptide sequence existing in nature by
at least one amino acid residue substitution, internal addition, or
internal deletion of at least one amino acid, and/or amino- or
carboxy-terminal end truncations or additions, and/or
carboxy-terminal amidation. An "internal deletion" refers to
absence of an amino acid from a sequence existing in nature at a
position other than the N- or C-terminus. Likewise, an "internal
addition" refers to presence of an amino acid in a sequence
existing in nature at a position other than the N- or
C-terminus.
[0160] Embodiments of the inventive composition of matter includes
a toxin peptide analog, or a pharmaceutically acceptable salt
thereof "Toxin peptide analogs", such as, but not limited to, an
AETX-K peptide analog, an ShK peptide analog, or a HmK peptide
analog, contain modifications of a native toxin peptide sequence of
interest (e.g., amino acid residue substitutions, internal
additions or insertions, internal deletions, and/or amino- or
carboxy-terminal end truncations, or additions as previously
described above) relative to a native toxin peptide sequence of
interest, such as ShK, HmK, or AETX-K. Toxin peptide analogs of the
present invention are 33 to about 100 amino acid residues long and,
in relation to SEQ ID NO:4, have C.sup.1-C.sup.6, C.sup.2-C.sup.4
and C.sup.3-C.sup.5 disulfide bonding in which, C.sup.1, C.sup.2,
C.sup.3, C.sup.4, C.sup.5 and C.sup.6 represent the order of
cysteine residues appearing in the primary sequence of the toxin
peptide stated conventionally with the N-terminus of the peptide(s)
on the left, with the first and sixth cysteines in the amino acid
sequence forming a disulfide bond, the second and fourth cysteines
forming a disulfide bond, and the third and fifth cysteines forming
a disulfide bond. Examples of toxin peptides with such a
C.sup.1-C.sup.6, C.sup.2-C.sup.4, C.sup.3-C.sup.5 disulfide bonding
pattern include, but are not limited to, ShK, BgK, HmK, AeKS, AsK,
AETX-K and DTX1, and analogs of any of the foregoing. As described
herein, the toxin peptide analogs of the present invention can also
have additional amino acid residues at the N-terminal and/or
C-terminal ends, in relation to SEQ ID NO:4.
[0161] By "physiologically acceptable salt" of the composition of
matter, for example a salt of the toxin peptide analog, is meant
any salt or salts that are known or later discovered to be
pharmaceutically acceptable. Some non-limiting examples of
pharmaceutically acceptable salts are: acetate; trifluoroacetate;
hydrohalides, such as hydrochloride and hydrobromide; sulfate;
citrate; maleate; tartrate; glycolate; gluconate; succinate;
mesylate; besylate; salts of gallic acid esters (gallic acid is
also known as 3,4,5 trihydroxybenzoic acid) such as
PentaGalloylGlucose (PGG) and epigallocatechin gallate (EGCG),
salts of cholesteryl sulfate, pamoate, tannate and oxalate
salts.
[0162] The term "fusion protein" indicates that the protein
includes polypeptide components derived from more than one parental
protein or polypeptide. Typically, a fusion protein is expressed
from a fusion gene in which a nucleotide sequence encoding a
polypeptide sequence from one protein is appended in frame with,
and optionally separated by a linker from, a nucleotide sequence
encoding a polypeptide sequence from a different protein. The
fusion gene can then be expressed by a recombinant host cell as a
single protein.
[0163] The terms "-mimetic peptide," "peptide mimetic," and
"-agonist peptide" refer to a peptide or protein having biological
activity comparable to a naturally occurring protein of interest,
for example, but not limited to, a toxin peptide molecule, e.g.,
naturally occurring ShK, HmK, AETX-K, or OSK1 toxin peptide. These
terms further include peptides that indirectly mimic the activity
of a naturally occurring peptide molecule, such as by potentiating
the effects of the naturally occurring molecule.
[0164] The term "-antagonist peptide," "peptide antagonist," and
"inhibitor peptide" refer to a peptide that blocks or in some way
interferes with the biological activity of a receptor of interest,
or has biological activity comparable to a known antagonist or
inhibitor of a receptor of interest, such as, but not limited to,
an ion channel (e.g., Kv1.3) or a G-Protein Coupled Receptor
(GPCR).
[0165] A "domain" of a protein is any portion of the entire
protein, up to and including the complete protein, but typically
comprising less than the complete protein. A domain can, but need
not, fold independently of the rest of the protein chain and/or be
correlated with a particular biological, biochemical, or structural
function or location (e.g., a ligand binding domain, or a
cytosolic, transmembrane or extracellular domain).
[0166] As used herein "soluble" when in reference to a protein
produced by recombinant DNA technology in a host cell is a protein
that exists in aqueous solution; if the protein contains a
twin-arginine signal amino acid sequence the soluble protein is
exported to the periplasmic space in gram negative bacterial hosts,
or is secreted into the culture medium by eukaryotic host cells
capable of secretion, or by bacterial host possessing the
appropriate genes (e.g., the kil gene). Thus, a soluble protein is
a protein which is not found in an inclusion body inside the host
cell. Alternatively, depending on the context, a soluble protein is
a protein which is not found integrated in cellular membranes. In
contrast, an insoluble protein is one which exists in denatured
form inside cytoplasmic granules (called an inclusion body) in the
host cell, or again depending on the context, an insoluble protein
is one which is present in cell membranes, including but not
limited to, cytoplasmic membranes, mitochondrial membranes,
chloroplast membranes, endoplasmic reticulum membranes, etc.
[0167] A distinction is also drawn between proteins which are
"soluble" (i.e., dissolved or capable of being dissolved) in an
aqueous solution devoid of significant amounts of ionic detergents
(e.g., SDS) or denaturants (e.g., urea, guanidine hydrochloride)
and proteins which exist as a suspension of insoluble protein
molecules dispersed within the solution. A "soluble" protein will
not be removed from a solution containing the protein by
centrifugation using conditions sufficient to remove cells present
in a liquid medium (e.g., centrifugation at 5,000.times.g for 4-5
minutes). In some embodiments of the inventive composition, the
toxin peptide analog is synthesized by the host cell and segregated
in an insoluble form within cellular inclusion bodies, which can
then be purified from other cellular components in a cell extract
with relative ease, and the toxin peptide analog can in turn be
solubilized, refolded and/or further purified.
[0168] A distinction is drawn between a "soluble" protein (i.e., a
protein which when expressed in a host cell is produced in a
soluble form) and a "solubilized" protein. An insoluble recombinant
protein found inside an inclusion body or found integrated in a
cell membrane may be solubilized (i.e., rendered into a soluble
form) by treating purified inclusion bodies or cell membranes with
denaturants such as guanidine hydrochloride, urea or sodium dodecyl
sulfate (SDS). These denaturants must then be removed from the
solubilized protein preparation to allow the recovered protein to
renature (refold). Although the inventive compositions can be
refolded in active form, not all proteins will refold into an
active conformation after solubilization in a denaturant and
removal of the denaturant. Many proteins precipitate upon removal
of the denaturant. SDS may be used to solubilize inclusion bodies
and cell membranes and will maintain the proteins in solution at
low concentration. However, dialysis will not always remove all of
the SDS (SDS can form micelles which do not dialyze out);
therefore, SDS-solubilized inclusion body protein and
SDS-solubilized cell membrane protein is soluble but not
refolded.
[0169] A "secreted" protein refers to those proteins capable of
being directed to the ER, secretory vesicles, or the extracellular
space as a result of a secretory signal peptide sequence, as well
as those proteins released into the extracellular space without
necessarily containing a signal sequence. If the secreted protein
is released into the extracellular space, the secreted protein can
undergo extracellular processing to produce a "mature" protein.
Release into the extracellular space can occur by many mechanisms,
including exocytosis and proteolytic cleavage. In some other
embodiments of the inventive composition, the toxin peptide analog
can be synthesized by the host cell as a secreted protein, which
can then be further purified from the extracellular space and/or
medium.
[0170] The term "recombinant" indicates that the material (e.g., a
nucleic acid or a polypeptide) has been artificially or
synthetically (i.e., non-naturally) altered by human intervention.
The alteration can be performed on the material within, or removed
from, its natural environment or state. For example, a "recombinant
nucleic acid" is one that is made by recombining nucleic acids,
e.g., during cloning, DNA shuffling or other well known molecular
biological procedures. A "recombinant DNA molecule," is comprised
of segments of DNA joined together by means of such molecular
biological techniques. The term "recombinant protein" or
"recombinant polypeptide" as used herein refers to a protein
molecule which is expressed using a recombinant DNA molecule. A
"recombinant host cell" is a cell that contains and/or expresses a
recombinant nucleic acid.
[0171] A "polynucleotide sequence" or "nucleotide sequence" or
"nucleic acid sequence," as used interchangeably herein, is a
polymer of nucleotides, including an oligonucleotide, a DNA, and
RNA, a nucleic acid, or a character string representing a
nucleotide polymer, depending on context. From any specified
polynucleotide sequence, either the given nucleic acid or the
complementary polynucleotide sequence can be determined. Included
are DNA or RNA of genomic or synthetic origin which may be single-
or double-stranded, and represent the sense or antisense
strand.
[0172] As used herein, the terms "nucleic acid molecule encoding,"
"DNA sequence encoding," and "DNA encoding" refer to the order or
sequence of deoxyribonucleotides along a strand of deoxyribonucleic
acid. The order of these deoxyribonucleotides determines the order
of ribonucleotides along the mRNA chain, and also determines the
order of amino acids along the polypeptide (protein) chain. The DNA
sequence thus codes for the RNA sequence and for the amino acid
sequence.
[0173] "Expression of a gene" or "expression of a nucleic acid"
means transcription of DNA into RNA (optionally including
modification of the RNA, e.g., splicing), translation of RNA into a
polypeptide (possibly including subsequent post-translational
modification of the polypeptide), or both transcription and
translation, as indicated by the context.
[0174] The term "gene" is used broadly to refer to any nucleic acid
associated with a biological function. Genes typically include
coding sequences and/or the regulatory sequences required for
expression of such coding sequences.
[0175] The term "gene" applies to a specific genomic or recombinant
sequence, as well as to a cDNA or mRNA encoded by that sequence. A
"fusion gene" contains a coding region that encodes a toxin peptide
analog. Genes also include non-expressed nucleic acid segments
that, for example, form recognition sequences for other proteins.
Non-expressed regulatory sequences including transcriptional
control elements to which regulatory proteins, such as
transcription factors, bind, resulting in transcription of adjacent
or nearby sequences.
[0176] As used herein the term "coding region" or "coding sequence"
when used in reference to a structural gene refers to the
nucleotide sequences which encode the amino acids found in the
nascent polypeptide as a result of translation of an mRNA molecule.
The coding region is bounded, in eukaryotes, on the 5' side by the
nucleotide triplet "ATG" which encodes the initiator methionine and
on the 3' side by one of the three triplets which specify stop
codons (i.e., TAA, TAG, TGA).
[0177] Transcriptional control signals in eukaryotes comprise
"promoter" and "enhancer" elements. Promoters and enhancers consist
of short arrays of DNA sequences that interact specifically with
cellular proteins involved in transcription (Maniatis, et al.,
Science 236:1237 (1987)). Promoter and enhancer elements have been
isolated from a variety of eukaryotic sources including genes in
yeast, insect and mammalian cells and viruses (analogous control
elements, i.e., promoters, are also found in prokaryotes). The
selection of a particular promoter and enhancer depends on what
cell type is to be used to express the protein of interest. Some
eukaryotic promoters and enhancers have a broad host range while
others are functional in a limited subset of cell types (for review
see Voss, et al., Trends Biochem. Sci., 11:287 (1986) and Maniatis,
et al., Science 236:1237 (1987)).
[0178] The term "expression vector" as used herein refers to a
recombinant DNA molecule containing a desired coding sequence and
appropriate nucleic acid sequences necessary for the expression of
the operably linked coding sequence in a particular host cell.
Nucleic acid sequences necessary for expression in prokaryotes
include a promoter, optionally an operator sequence, a ribosome
binding site and possibly other sequences. Eukaryotic cells are
known to utilize promoters, enhancers, and termination and
polyadenylation signals. A secretory signal peptide sequence can
also, optionally, be encoded by the expression vector, operably
linked to the coding sequence for the inventive toxin peptide
analog, so that the expressed toxin peptide analog can be secreted
by the recombinant host cell, for more facile isolation of the
toxin peptide analog from the cell, if desired. Such techniques are
well known in the art. (E.g., Goodey, Andrew R.; et al., Peptide
and DNA sequences, U.S. Pat. No. 5,302,697; Weiner et al.,
Compositions and methods for protein secretion, U.S. Pat. No.
6,022,952 and U.S. Pat. No. 6,335,178; Uemura et al., Protein
expression vector and utilization thereof, U.S. Pat. No. 7,029,909;
Ruben et al., 27 human secreted proteins, US 2003/0104400 A1).
[0179] The terms "in operable combination", "in operable order" and
"operably linked" as used herein refer to the linkage of nucleic
acid sequences in such a manner that a nucleic acid molecule
capable of directing the transcription of a given gene and/or the
synthesis of a desired protein molecule is produced. The term also
refers to the linkage of amino acid sequences in such a manner so
that a functional protein is produced.
[0180] Peptides. Recombinant DNA- and/or RNA-mediated protein
expression and protein engineering techniques, or any other methods
of preparing peptides, are applicable to the making of the
inventive toxin peptide analogs and fusion protein conjugates
thereof (e.g., fusion proteins containing a toxin peptide analog
and an immunoglobulin Fc domain, transthyretin, or human serum
albumin). For example, the peptides can be made in transformed host
cells. Briefly, a recombinant DNA molecule, or construct, coding
for the peptide is prepared. Methods of preparing such DNA
molecules are well known in the art. For instance, sequences
encoding the peptides can be excised from DNA using suitable
restriction enzymes. Any of a large number of available and
well-known host cells may be used in the practice of this
invention. The selection of a particular host is dependent upon a
number of factors recognized by the art. These include, for
example, compatibility with the chosen expression vector, toxicity
of the peptides encoded by the DNA molecule, rate of
transformation, ease of recovery of the peptides, expression
characteristics, bio-safety and costs. A balance of these factors
must be struck with the understanding that not all hosts may be
equally effective for the expression of a particular DNA sequence.
Within these general guidelines, useful microbial host cells in
culture include bacteria (such as Escherichia coli sp.), yeast
(such as Saccharomyces sp.) and other fungal cells, insect cells,
plant cells, mammalian (including human) cells, e.g., CHO cells and
HEK293 cells. Modifications can be made at the DNA level, as well.
The peptide-encoding DNA sequence may be changed to codons more
compatible with the chosen host cell. For E. coli, optimized codons
are known in the art. Codons can be substituted to eliminate
restriction sites or to include silent restriction sites, which may
aid in processing of the DNA in the selected host cell. Next, the
transformed host is cultured and purified. Host cells may be
cultured under conventional fermentation conditions so that the
desired compounds are expressed. Such fermentation conditions are
well known in the art. In addition, the DNA optionally further
encodes, 5' to the coding region of a fusion protein, a signal
peptide sequence (e.g., a secretory signal peptide) operably linked
to the expressed toxin peptide analog. For further examples of
appropriate recombinant methods and exemplary DNA constructs useful
for recombinant expression of the inventive compositions by
mammalian cells, including dimeric Fc fusion proteins
("peptibodies") or chimeric immunoglobulin(light chain+heavy
chain)-Fc heterotrimers ("hemibodies"), conjugated to
pharmacologically active toxin peptide analogs of the invention,
see, e.g., Sullivan et al., Toxin Peptide Therapeutic Agents,
US2007/0071764 and Sullivan et al., Toxin Peptide Therapeutic
Agents, PCT/US2007/022831, published as WO 2008/088422, which are
both incorporated herein by reference in their entireties.
[0181] Peptide compositions of the present invention can also be
made by synthetic methods. Solid phase synthesis is the preferred
technique of making individual peptides since it is the most
cost-effective method of making small peptides. For example, well
known solid phase synthesis techniques include the use of
protecting groups, linkers, and solid phase supports, as well as
specific protection and deprotection reaction conditions, linker
cleavage conditions, use of scavengers, and other aspects of solid
phase peptide synthesis. Suitable techniques are well known in the
art. (E.g., Merrifield (1973), Chem. Polypeptides, pp. 335-61
(Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem.
Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414;
Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat.
No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2:
105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2:
257-527; "Protecting Groups in Organic Synthesis," 3rd Edition, T.
W. Greene and P. G. M. Wuts, Eds., John Wiley & Sons, Inc.,
1999; NovaBiochem Catalog, 2000; "Synthetic Peptides, A User's
Guide," G. A. Grant, Ed., W.H. Freeman & Company, New York,
N.Y., 1992; "Advanced Chemtech Handbook of Combinatorial &
Solid Phase Organic Chemistry," W. D. Bennet, J. W. Christensen, L.
K. Hamaker, M. L. Peterson, M. R. Rhodes, and H. H. Saneii, Eds.,
Advanced Chemtech, 1998; "Principles of Peptide Synthesis, 2nd
ed.," M. Bodanszky, Ed., Springer-Verlag, 1993; "The Practice of
Peptide Synthesis, 2nd ed.," M. Bodanszky and A. Bodanszky, Eds.,
Springer-Verlag, 1994; "Protecting Groups," P. J. Kocienski, Ed.,
Georg Thieme Verlag, Stuttgart, Germany, 1994; "Fmoc Solid Phase
Peptide Synthesis, A Practical Approach," W. C. Chan and P. D.
White, Eds., Oxford Press, 2000, G. B. Fields et al., Synthetic
Peptides: A User's Guide, 1990, 77-183). For further examples of
synthetic and purification methods known in the art, which are
applicable to making the inventive compositions of matter, see,
e.g., Sullivan et al., Toxin Peptide Therapeutic Agents,
US2007/0071764 and Sullivan et al., Toxin Peptide Therapeutic
Agents, PCT/US2007/022831, published as WO 2008/088422 A2, which
are both incorporated herein by reference in their entireties.
[0182] In further describing the toxin peptide analogs herein, a
one-letter abbreviation system is frequently applied to designate
the identities of the twenty "canonical" amino acid residues
generally incorporated into naturally occurring peptides and
proteins (Table 1). Such one-letter abbreviations are entirely
interchangeable in meaning with three-letter abbreviations, or
non-abbreviated amino acid names. Within the one-letter
abbreviation system used herein, an upper case letter indicates a
L-amino acid, and a lower case letter indicates a D-amino acid. For
example, the abbreviation "R" designates L-arginine and the
abbreviation "r" designates D-arginine.
TABLE-US-00002 TABLE 1 One-letter abbreviations for the canonical
amino acids. Three-letter abbreviations are in parentheses. Alanine
(Ala) A Glutamine (Gln) Q Leucine (Leu) L Serine (Ser) S Arginine
(Arg) R Glutamic Acid (Glu) E Lysine (Lys) K Threonine (Thr) T
Asparagine (Asn) N Glycine (Gly) G Methionine (Met) M Tryptophan
(Trp) W Aspartic Acid (Asp) D Histidine (His) H Phenylalanine (Phe)
F Tyrosine (Tyr) Y Cysteine (Cys) C Isoleucine (Ile) I Proline
(Pro) P Valine (Val) V
[0183] An amino acid substitution in an amino acid sequence is
typically designated herein with a one-letter abbreviation for the
amino acid residue in a particular position, followed by the
numerical amino acid position relative to a native sequence of
interest, which is then followed by the one-letter symbol for the
amino acid residue substituted in. For example, "T30D" symbolizes a
substitution of a threonine residue by an aspartate residue at
amino acid position 30, relative to the native sequence of
interest.
[0184] Non-canonical amino acid residues can be incorporated into a
peptide within the scope of the invention by employing known
techniques of protein engineering that use recombinantly expressing
cells. (See, e.g., Link et al., Non-canonical amino acids in
protein engineering, Current Opinion in Biotechnology,
14(6):603-609 (2003)). The term "non-canonical amino acid residue"
refers to amino acid residues in D- or L-form that are not among
the 20 canonical amino acids generally incorporated into naturally
occurring proteins, for example, .beta.-amino acids, homoamino
acids, cyclic amino acids and amino acids with derivatized side
chains. Examples include (in the L-form or D-form) .beta.-alanine,
.beta.-aminopropionic acid, piperidinic acid, aminocaprioic acid,
aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic
acid, N.sup..alpha.-ethylglycine, N.sup..alpha.-ethylaspargine,
hydroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine,
w-methylarginine, N.sup..alpha.-methylglycine,
N.sup..alpha.-methylisoleucine, N.sup..alpha.-methylvaline,
.gamma.-carboxyglutamate, .epsilon.-N,N,N-trimethyllysine,
.epsilon.-N-acetyllysine, O-phosphoserine,
N.sup..alpha.-acetylserine, N.sup..alpha.-formylmethionine,
3-methylhistidine, 5-hydroxylysine, and other similar amino acids,
and those listed in Table 2 below, and derivatized forms of any of
these as described herein. Table 2 contains some exemplary
non-canonical amino acid residues that are useful in accordance
with the present invention and associated abbreviations as
typically used herein, although the skilled practitioner will
understand that different abbreviations and nomenclatures may be
applicable to the same substance and appear interchangeably
herein.
TABLE-US-00003 TABLE 2 Useful non-canonical amino acids for amino
acid addition, insertion, or substitution into peptide sequences in
accordance with the present invention. Amino Acid Abbreviation(s)
Acetamidomethyl Acm Acetylarginine acetylarg .alpha.-aminoadipic
acid Aad aminobutyric acid Abu 6-aminohexanoic acid Ahx;
.epsilon.Ahx 3-amino-6-hydroxy-2-piperidone Ahp
2-aminoindane-2-carboxylic acid Aic .alpha.-amino-isobutyric acid
Aib 3-amino-2-naphthoic acid Anc 2-aminotetraline-2-carboxylic acid
Atc aminophenylalanine Aminophe; Amino-Phe 4-amino-phenylalanine
4AmP 4-amidino-phenylalanine 4AmPhe
2-amino-2-(1-carbamimidoylpiperidin-4- 4AmPig yl)acetic acid Arg
.psi.(CH.sub.2NH) -reduced amide bond rArg .beta.-homoarginine
bhArg .beta.-homolysine bhomoK .beta.-homo Tic BhTic
.beta.-homophenylalanine BhPhe .beta.-homoproline BhPro
.beta.-homotryptophan BhTrp 4,4'-biphenylalanine Bip
.beta.,.beta.-diphenyl-alanine BiPhA .beta.-phenylalanine BPhe
p-carboxyl-phenylalanine Cpa citrulline Cit cyclohexylalanine Cha
cyclohexylglycine Chg cyclopentylglycine Cpg
2-amino-3-guanidinopropanoic acid 3G-Dpr
.alpha.,.gamma.-diaminobutyric acid Dab 2,4-diaminobutyric acid Dbu
diaminopropionic acid Dap .alpha.,.beta.-diaminopropionoic acid (or
2,3- Dpr diaminopropionic acid 3,3-diphenylalanine Dip 4-guanidino
phenylalanine Guf 4-guanidino proline 4GuaPr homoarginine hArg; hR
homocitrulline hCit homoglutamine hQ homolysine hLys; hK; homoLys
homophenylalanine hPhe; homoPhe 4-hydroxyproline (or
hydroxyproline) Hyp 2-indanylglycine (or indanylglycine) IgI
indoline-2-carboxylic acid Idc Iodotyrosine I-Tyr Lys
.psi.(CH.sub.2NH)-reduced amide bond rLys methionine oxide Met[O]
methionine sulfone Met[O].sub.2 N.sup..alpha.-methylarginine NMeR
N.alpha.-[(CH.sub.2).sub.3NHCH(NH)NH.sub.2] substituted N-Arg
glycine N.sup..alpha.-methylcitrulline NMeCit
N.sup..alpha.-methylglutamine NMeQ
N.sup..alpha.-methylhomocitrulline N.sup..alpha.-MeHoCit
N.sup..alpha.-methylhomolysine NMeHoK N.sup..alpha.-methylleucine
N.sup..alpha.-MeL; NMeL; NMeLeu; NMe-Leu N.sup..alpha.-methyllysine
NMe-Lys N.epsilon.-methyl-lysine N-eMe-K N.epsilon.-ethyl-lysine
N-eEt-K N.epsilon.-isopropyl-lysine N-eIPr-K
N.sup..alpha.-methylnorleucine NMeNle; NMe-Nle
N.sup..alpha.-methylornithine N.sup..alpha.-MeOrn; NMeOrn
N.sup..alpha.-methylphenylalanine NMe-Phe 4-methyl-phenylalanine
MePhe .alpha.-methylphenyalanine AMeF N.sup..alpha.-methylthreonine
NMe-Thr; NMeThr N.sup..alpha.-methylvaline NMeVal; NMe-Val
N.epsilon.-(O-(aminoethyl)-O'-(2-propanoyl)- K(NPeg11)
undecaethyleneglycol)-Lysine
N.epsilon.-(O-(aminoethyl)-O'-(2-propanoyl)- K(NPeg27)
(ethyleneglycol)27-Lysine 3-(1-naphthyl)alanine 1-Nal; 1Nal
3-(2-naphthyl)alanine 2-Nal; 2Nal nipecotic acid Nip
nitrophenylalanine nitrophe norleucine Nle norvaline Nva or Nvl
O-methyltyrosine Ome-Tyr octahydroindole-2-carboxylic acid Oic
ornithine Orn Orn .psi.(CH2NH)-reduced amide bond rOrn
4-piperidinylalanine 4PipA 4-pyridinylalanine 4Pal
3-pyridinylalanine 3Pal 2-pyridinylalanine 2Pal
para-aminophenylalanine 4AmP; 4-Amino-Phe para-iodophenylalanine
(or 4- pI-Phe iodophenylalanine) phenylglycine Phg
4-phenyl-phenylalanine (or 4Bip biphenylalanine) 4,4'-biphenyl
alanine Bip pipecolic acid Pip 4-amino-1-piperidine-4-carboxylic
acid 4Pip sarcosine Sar 1,2,3,4-tetrahydroisoquinoline Tic
1,2,3,4-tetrahydroisoquinoline-1- Tiq carboxylic acid
1,2,3,4-tetrahydroisoquinoline-7- Hydroxyl-Tic hydroxy-3-carboxylic
acid 1,2,3,4-tetrahydronorharman-3- Tpi carboxylic acid
thiazolidine-4-carboxylic acid Thz 3-thienylalanine Thi In the
event an abbreviation listed in Table 2 differs from another
abbreviation for the same substance disclosed elsewhere herein,
both abbreviations are understood to be applicable. The amino acids
listed in Table 2 can be in the L-form or D-form.
[0185] Nomenclature and Symbolism for Amino Acids and Peptides by
the UPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN)
have been published in the following documents: Biochem. J., 1984,
219, 345-373; Eur. J. Biochem., 1984, 138, 9-37; 1985, 152, 1;
1993, 213, 2; Internat. J. Pept. Prot. Res., 1984, 24, following p
84; J. Biol. Chem., 1985, 260, 14-42; Pure Appl. Chem., 1984, 56,
595-624; Amino Acids and Peptides, 1985, 16, 387-410; Biochemical
Nomenclature and Related Documents, 2nd edition, Portland Press,
1992, pages 39-69.
[0186] The one or more useful modifications to peptide domains of
the inventive compositions can include amino acid additions or
insertions, amino acid deletions, peptide truncations, amino acid
substitutions, and/or chemical derivatization of amino acid
residues, accomplished by known chemical techniques. For example,
the thusly modified amino acid sequence includes at least one amino
acid residue inserted or substituted therein, relative to the amino
acid sequence of the native sequence of interest, in which the
inserted or substituted amino acid residue has a side chain
comprising a nucleophilic or electrophilic reactive functional
group by which the peptide is conjugated to a linker and/or
half-life extending moiety. In accordance with the invention,
useful examples of such a nucleophilic or electrophilic reactive
functional group include, but are not limited to, a thiol, a
primary amine, a seleno, a hydrazide, an aldehyde, a carboxylic
acid, a ketone, an aminooxy, a masked (protected) aldehyde, or a
masked (protected) keto functional group. Examples of amino acid
residues having a side chain comprising a nucleophilic reactive
functional group include, but are not limited to, a lysine residue,
a homolysine, an .alpha.,.beta.-diaminopropionic acid residue, an
.alpha.,.gamma.-diaminobutyric acid residue, an ornithine residue,
a cysteine, a homocysteine, a glutamic acid residue, an aspartic
acid residue, or a selenocysteine residue.
[0187] Amino acid residues are commonly categorized according to
different chemical and/or physical characteristics. The term
"acidic amino acid residue" refers to amino acid residues in D- or
L-form having side chains comprising acidic groups. Exemplary
acidic residues include aspartatic acid and glutamatic acid
residues. The term "alkyl amino acid residue" refers to amino acid
residues in D- or L-form having C.sub.1-6alkyl side chains which
may be linear, branched, or cyclized, including to the amino acid
amine as in proline, wherein the C.sub.1-6alkyl is substituted by
0, 1, 2 or 3 substituents selected from C.sub.1-4haloalkyl, halo,
cyano, nitro, --C(.dbd.O)R.sup.b, --C(.dbd.O)OR.sup.a,
--C(.dbd.O)NR.sup.aR.sup.a, --C(.dbd.NR.sup.a)NR.sup.aR.sup.a,
--NR.sup.aC(.dbd.NR.sup.a)NR.sup.aR.sup.a, --OR.sup.a,
--OC(.dbd.O)R.sup.b, --OC(.dbd.O)NR.sup.aR.sup.a,
--OC.sub.2-6alkylNR.sup.aR.sup.a, --OC.sub.2-6alkylOR.sup.a,
--SR.sup.a, --S(.dbd.O)R.sup.b, --S(.dbd.O).sub.2R.sup.b,
--S(.dbd.O).sub.2NR.sup.aR.sup.a, --NR.sup.aR.sup.a,
--N(R.sup.a)C(.dbd.O)R.sup.b, --N(R.sup.a)C(.dbd.O)OR.sup.b,
--N(R.sup.a)C(.dbd.O)NR.sup.aR.sup.a,
--N(R.sup.a)C(.dbd.NR.sup.a)NR.sup.aR.sup.a,
--N(R.sup.a)S(.dbd.O).sub.2R.sup.b,
--N(R.sup.a)S(.dbd.O).sub.2NR.sup.aR.sup.a,
--NR.sup.aC.sub.2-6alkylNR.sup.aR.sup.a and
--NR.sup.aC.sub.2-6alkylOR.sup.a; wherein R.sup.a is independently,
at each instance, H or R.sup.b; and R.sup.b is independently, at
each instance C.sub.1-6alkyl substituted by 0, 1, 2 or 3
substituents selected from halo, C.sub.1-4alk, C.sub.1-3haloalk,
--OC.sub.1-4alk, --NH.sub.2, --NHC.sub.1-4alk, and
--N(C.sub.1-4alk)C.sub.1-4alk; or any protonated form thereof,
including alanine, valine, leucine, isoleucine, proline, serine,
threonine, lysine, arginine, histidine, aspartate, glutamate,
asparagine, glutamine, cysteine, methionine, hydroxyproline, but
which residues do not contain an aryl or aromatic group. The term
"aromatic amino acid residue" refers to amino acid residues in D-
or L-form having side chains comprising aromatic groups. Exemplary
aromatic residues include tryptophan, tyrosine,
3-(1-naphthyl)alanine, or phenylalanine residues. The term "basic
amino acid residue" refers to amino acid residues in D- or L-form
having side chains comprising basic groups. Exemplary basic amino
acid residues include histidine, lysine, homolysine, ornithine,
arginine, N-methyl-arginine, .omega.-aminoarginine,
.omega.-methyl-arginine, 1-methyl-histidine, 3-methyl-histidine,
and homoarginine (hR) residues. The term "hydrophilic amino acid
residue" refers to amino acid residues in D- or L-form having side
chains comprising polar groups. Exemplary hydrophilic residues
include cysteine, serine, threonine, histidine, lysine, asparagine,
aspartate, glutamate, glutamine, and citrulline (Cit) residues. The
terms "lipophilic amino acid residue" refers to amino acid residues
in D- or L-form having sidechains comprising uncharged, aliphatic
or aromatic groups. Exemplary lipophilic sidechains include
phenylalanine, isoleucine, leucine, methionine, valine, tryptophan,
and tyrosine. Alanine (A) is amphiphilic--it is capable of acting
as a hydrophilic or lipophilic residue. Alanine, therefore, is
included within the definition of both "lipophilic residue" and
"hydrophilic residue." The term "nonfunctional amino acid residue"
refers to amino acid residues in D- or L-form having side chains
that lack acidic, basic, or aromatic groups. Exemplary neutral
amino acid residues include methionine, glycine, alanine, valine,
isoleucine, leucine, and norleucine (Nle) residues.
[0188] Additional useful embodiments of toxin peptide analogs can
result from conservative modifications of the amino acid sequences
of the toxin polypeptides disclosed herein. Conservative
modifications will produce half-life extending moiety-conjugated
peptides having functional, physical, and chemical characteristics
similar to those of the conjugated (e.g., PEG-conjugated) peptide
from which such modifications are made. Such conservatively
modified forms of the conjugated toxin peptide analogs disclosed
herein are also contemplated as being an embodiment of the present
invention.
[0189] In contrast, substantial modifications in the functional
and/or chemical characteristics of peptides may be accomplished by
selecting substitutions in the amino acid sequence that differ
significantly in their effect on maintaining (a) the structure of
the molecular backbone in the region of the substitution, for
example, as an .alpha.-helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the size
of the molecule.
[0190] For example, a "conservative amino acid substitution" may
involve a substitution of a native amino acid residue with a
normative residue such that there is little or no effect on the
polarity or charge of the amino acid residue at that position.
Furthermore, any native residue in the polypeptide may also be
substituted with alanine, as has been previously described for
"alanine scanning mutagenesis" (see, for example, MacLennan et al.,
Acta Physiol. Scand. Suppl., 643:55-67 (1998); Sasaki et al., 1998,
Adv. Biophys. 35:1-24 (1998), which discuss alanine scanning
mutagenesis).
[0191] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. For example, amino acid
substitutions can be used to identify important residues of the
peptide sequence, or to increase or decrease the affinity of the
peptide or vehicle-conjugated peptide molecules described
herein.
[0192] Naturally occurring residues may be divided into classes
based on common side chain properties:
[0193] 1) hydrophobic: norleucine (Nor or Nle), Met, Ala, Val, Leu,
Ile;
[0194] 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0195] 3) acidic: Asp, Glu;
[0196] 4) basic: His, Lys, Arg;
[0197] 5) residues that influence chain orientation: Gly, Pro;
and
[0198] 6) aromatic: Tip, Tyr, Phe.
[0199] Conservative amino acid substitutions may involve exchange
of a member of one of these classes with another member of the same
class. Conservative amino acid substitutions may encompass
non-naturally occurring amino acid residues, which are typically
incorporated by chemical peptide synthesis rather than by synthesis
in biological systems. These include peptidomimetics and other
reversed or inverted forms of amino acid moieties.
[0200] Non-conservative substitutions may involve the exchange of a
member of one of these classes for a member from another class.
Such substituted residues may be introduced into regions of the
toxin peptide analog.
[0201] In making such changes, according to certain embodiments,
the hydropathic index of amino acids may be considered. Each amino
acid has been assigned a hydropathic index on the basis of its
hydrophobicity and charge characteristics. They are: isoleucine
(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);
cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine
(-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9);
tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate
(-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5);
lysine (-3.9); and arginine (-4.5).
[0202] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
understood in the art (see, for example, Kyte et al., 1982, J. Mol.
Biol. 157:105-131). It is known that certain amino acids may be
substituted for other amino acids having a similar hydropathic
index or score and still retain a similar biological activity. In
making changes based upon the hydropathic index, in certain
embodiments, the substitution of amino acids whose hydropathic
indices are within .+-.2 is included. In certain embodiments, those
that are within .+-.1 are included, and in certain embodiments,
those within .+-.0.5 are included.
[0203] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the biologically functional
protein or peptide thereby created is intended for use in
immunological embodiments, as disclosed herein. In certain
embodiments, the greatest local average hydrophilicity of a
protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and antigenicity, i.e.,
with a biological property of the protein.
[0204] The following hydrophilicity values have been assigned to
these amino acid residues: arginine (+3.0); lysine (+3.0);
aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3);
asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine
(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and
tryptophan (-3.4). In making changes based upon similar
hydrophilicity values, in certain embodiments, the substitution of
amino acids whose hydrophilicity values are within .+-.2 is
included, in certain embodiments, those that are within .+-.1 are
included, and in certain embodiments, those within .+-.0.5 are
included. One may also identify epitopes from primary amino acid
sequences on the basis of hydrophilicity. These regions are also
referred to as "epitopic core regions."
[0205] Examples of conservative substitutions include the
substitution of one non-polar (hydrophobic) amino acid residue such
as isoleucine, valine, leucine norleucine, alanine, or methionine
for another, the substitution of one polar (hydrophilic) amino acid
residue for another such as between arginine and lysine, between
glutamine and asparagine, between glycine and serine, the
substitution of one basic amino acid residue such as lysine,
arginine or histidine for another, or the substitution of one
acidic residue, such as aspartic acid or glutamic acid for another.
The phrase "conservative amino acid substitution" also includes the
use of a chemically derivatized residue in place of a
non-derivatized residue, provided that such polypeptide displays
the requisite bioactivity. Other exemplary amino acid substitutions
that can be useful in accordance with the present invention are set
forth in Table 3 below.
TABLE-US-00004 TABLE 3 Some Useful Amino Acid Substitutions.
Original Exemplary Residues Substitutions Ala Val, Leu, Ile Arg
Lys, Gln, Asn Asn Gln Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro,
Ala His Asn, Gln, Lys, Arg Ile Leu, Val, Met, Ala, Phe, Norleucine
Leu Norleucine, Ile, Val, Met, Ala, Phe Lys Arg, 1,4-Diaminobutyric
Acid, Gln, Asn Met Leu, Phe, Ile Phe Leu, Val, Ile, Ala, Tyr Pro
Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr, Phe Tyr Trp, Phe, Thr, Ser
Val Ile, Met, Leu, Phe, Ala, Norleucine
[0206] In exemplary embodiments of the inventive composition, in
relation to SEQ ID NO: 4:
[0207] The alkyl, basic, or acidic amino acid residue of
X.sub.aa.sup.4 is selected from Ser, Thr, Ala, Gly, Leu, Ile, Val,
Met, Cit, Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf,
and 4-Amino-Phe,Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp,
Arg, N.sup..alpha. Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn,
D-Orn, Asn, Gln, Glu, Asp, .alpha.-aminoadipic acid, and
para-carboxyl-phenylalanine; or more particularly X.sub.aa.sup.4 is
selected from Ala, Ile, Lys, Orn, Glu and Asp; and/or
[0208] The acidic amino acid residue of X.sub.aa.sup.9 and
X.sub.aa.sup.14 is each independently selected from Glu, Asp, and
.alpha.-aminoadipic acid;
[0209] and/or
[0210] the alkyl or aromatic amino acid residue of X.sub.aa.sup.15
is selected from Ala, 1-Nal, 2-Nal, Phe, Tyr, Val, Ile, and Leu, or
more particularly the amino acid residue of X.sub.aa.sup.15 is
selected from Phe, Ala, and Ile;
[0211] and/or
[0212] the basic, alkyl, or aromatic amino acid residue of
X.sub.aa.sup.16 is selected from Lys, Orn, Dab, Dap 1-Nal, 2-Nal,
Tyr, Phe, Pip, 2Pal, 3Pal, N-Me-Lys, N-Me-Orn, alpha-methyl-lysine,
Lys(N.sup..epsilon.-Me), Lys(N.sup..epsilon.-Me).sub.2,
Lys(N.sup..epsilon.-Me).sub.3, para-Methyl-Phe, AMeF
(alpha-methyl-phenylalanine), and homoPhe;
[0213] and/or
[0214] the basic or acidic amino acid residue of X.sub.aa.sup.18
and X.sub.aa.sup.30 is each independently selected from Lys, Arg,
Orn, Glu, Asp, His, Trp, and 2-phenylacetic acid (Pac);
[0215] and/or
[0216] the basic amino acid residue of X.sub.aa.sup.19,
X.sub.aa.sup.20 and X.sub.aa.sup.29 is each independently selected
from Lys, Arg, His, Orn, D-Orn, Dab, Dap, 1Pip, 2Pal, 3Pal,
N-Me-Lys, Na Methyl-Arg; homoarginine, Cit, N.alpha.-Methyl-Cit,
Homocitrulline, Guf, and 4-Amino-Phe, and N-Me-Orn; and/or
[0217] the alkyl or aromatic amino acid residue of X.sub.aa.sup.21
is selected from Nle, Nva, Abu, Phe,Tyr, Asn, Gln, Met[O], Val,
Ile, Leu, Met[O.sub.2], Cha, Chg, Asn, Trp, para-Methyl-Phe,
alpha-methyl-Phe, and homoPhe;
[0218] and/or
[0219] the aromatic amino acid residue of X.sub.aa.sup.26,
X.sub.aa.sup.27, X.sub.aa.sup.31, and X.sub.aa.sup.34 is each
independently selected from 1-Nal, 2-Nal, Phe, Trp, and Tyr.
[0220] and/or
[0221] the amino acid residue of X.sub.aa.sup.36, X.sub.aa.sup.37,
and X.sub.aa.sup.38, if present, is each independently selected
from Ala, Leu, Lys, Glu, Asp, Phe, Arg, Phe, Asp-amide, Aib-amide,
Tyr, Ser-amide, Thr-amide, Glu, Glu-amide, beta-Ala, and
N-Me-Ala.
[0222] In some embodiments the carboxy-terminal residue is
amidated. For example, some C-terminally amidated embodiments are
set forth in Table 11, Table 12, Table 14, Table 16, and Table
17.
[0223] Some embodiments of the composition of matter include
C-terminal extensions beyond position X.sub.aa.sup.35. Some
examples are set forth in Table 15 and Table 16.
[0224] Certain embodiments of the inventive composition of matter
that have particular utility in improving the potency, stability,
selectivity, and/or ease of synthesis of the toxin peptide analogs
involve substitutions as summarized in Table 4 below, relative to
SEQ ID NO: 4 and the native ShK sequence.
TABLE-US-00005 TABLE 4 Toxin Peptide Analogs with Improved
Therapeutic Potential SEQ ID NO: 4 Native ShK Improved Description
of Residue # Amino Acid Analog Improvement 2 Ser Glu Potency 4 Ile
Glu, Lys, Ala Potency 7 Ile Lys Selectivity 10 Ser Glu, Arg, Ala
Potency, selectivity 15 Phe Ala Potency 16 Gln Lys, Nal Selectivity
18 Lys Arg, Ala Potency 20 Ser Lys, Arg Selectivity 21 Met Gln, Nva
Limit oxidation 22 Lys Ala Selectivity 23 Tyr Ala Selectivity 26
Ser Nal Selectivity 27 Phe Nal Selectivity 29 Arg Lys, Nal Potency,
selectivity 30 Lys Glu, Arg Potency 31 Thr Nal Potency 34 Thr Nal
Potency C-terminus carboxy Amide, Ala Potency, selectivity, added
& synthesis
[0225] In some embodiments, the inventive composition of matter
comprises a toxin peptide analog comprising an amino acid sequence
selected from SEQ ID NOS: 10, 11, 12, 14, 15, 16, 19 through 29, 31
through 34, 36 through 50, 52, 54, 55, 56, 59, 60, 61, 63, 65
through 100, 130 through 140, 142 through 174, 176 through 254, and
257 through 274.
[0226] As stated herein above, in accordance with the present
invention, the peptide portions of the inventive composition of
matter can also be chemically derivatized at one or more amino acid
residues by known organic chemistry techniques. "Chemical
derivative" or "chemically derivatized" refers to a subject peptide
having one or more residues chemically derivatized by reaction of a
functional side group. Such derivatized molecules include, for
example, those molecules in which free amino groups have been
derivatized to form amine hydrochlorides, p-toluene sulfonyl
groups, carbobenzoxy groups, t-butyloxycarbonyl groups,
chloroacetyl groups or formyl groups. Free carboxyl groups may be
derivatized to form salts, methyl and ethyl esters or other types
of esters or hydrazides. Free hydroxyl groups may be derivatized to
form O-acyl or O-alkyl derivatives. The imidazole nitrogen of
histidine may be derivatized to form N-im-benzylhistidine. Also
included as chemical derivatives are those peptides which contain
one or more naturally occurring amino acid derivatives of the
twenty canonical amino acids, whether in L- or D-form. For example,
4-hydroxyproline may be substituted for proline; 5-hydroxylysine
maybe substituted for lysine; 3-methylhistidine may be substituted
for histidine; homoserine may be substituted for serine; and
ornithine may be substituted for lysine.
[0227] Useful derivatizations include, in some embodiments, those
in which the amino terminal of the peptide is chemically blocked so
that conjugation with the vehicle will be prevented from taking
place at an N-terminal free amino group. There may also be other
beneficial effects of such a modification, for example a reduction
in the toxin peptide analog's susceptibility to enzymatic
proteolysis. The N-terminus can be acylated or modified to a
substituted amine, or derivatized with another functional group,
such as an aromatic moiety (e.g., an indole acid, benzyl (Bzl or
Bn), dibenzyl (DiBz1 or Bn.sub.2), or benzyloxycarbonyl (Cbz or
Z)), N,N-dimethylglycine or creatine. For example, in some
embodiments, an acyl moiety, such as, but not limited to, a formyl,
acetyl (Ac), propanoyl, butanyl, heptanyl, hexanoyl, octanoyl, or
nonanoyl, can be covalently linked to the N-terminal end of the
peptide, which can prevent undesired side reactions during
conjugation of the vehicle to the peptide. Other exemplary
N-terminal derivative groups include --NRR.sup.1 (other than
--NH.sub.2), --NRC(O)R.sup.1, --NRC(O)OR.sup.1,
--NRS(O).sub.2R.sup.1, --NHC(O)NHR.sup.1, succinimide, or
benzyloxycarbonyl-NH-- (Cbz-NH--), wherein R and R.sup.1 are each
independently hydrogen or lower alkyl and wherein the phenyl ring
may be substituted with 1 to 3 substituents selected from
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, chloro, and
bromo.
[0228] In some embodiments, one or more peptidyl [--C(O)NR--]
linkages (bonds) between amino acid residues can be replaced by a
non-peptidyl linkage. Exemplary non-peptidyl linkages are
--CH.sub.2-carbamate [--CH.sub.2--OC(O)NR--], phosphonate,
--CH.sub.2-sulfonamide [--CH.sub.2--S(O).sub.2NR--], urea
[--NHC(O)NH--], --CH.sub.2-secondary amine, and alkylated peptide
[--C(O)NR.sup.6-- wherein R.sup.6 is lower alkyl].
[0229] In some embodiments, one or more individual amino acid
residues can be derivatized. Various derivatizing agents are known
to react specifically with selected sidechains or terminal
residues, as described in detail below by way of example.
[0230] Lysinyl residues and amino terminal residues may be reacted
with succinic or other carboxylic acid anhydrides, which reverse
the charge of the lysinyl residues. Other suitable reagents for
derivatizing alpha-amino-containing residues include imidoesters
such as methyl picolinimidate; pyridoxal phosphate; pyridoxal;
chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea;
2,4 pentanedione; and transaminase-catalyzed reaction with
glyoxylate.
[0231] Arginyl residues may be modified by reaction with any one or
combination of several conventional reagents, including
phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and
ninhydrin. Derivatization of arginyl residues requires that the
reaction be performed in alkaline conditions because of the high
pKa of the guanidine functional group. Furthermore, these reagents
may react with the groups of lysine as well as the arginine
epsilon-amino group.
[0232] Specific modification of tyrosyl residues has been studied
extensively, with particular interest in introducing spectral
labels into tyrosyl residues by reaction with aromatic diazonium
compounds or tetranitromethane. Most commonly, N-acetylimidizole
and tetranitromethane are used to form O-acetyl tyrosyl species and
3-nitro derivatives, respectively.
[0233] Carboxyl sidechain groups (aspartyl or glutamyl) may be
selectively modified by reaction with carbodiimides
(R'--N.dbd.C.dbd.N--R') such as
1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,
aspartyl and glutamyl residues may be converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
[0234] Glutaminyl and asparaginyl residues may be deamidated to the
corresponding glutamyl and aspartyl residues. Alternatively, these
residues are deamidated under mildly acidic conditions. Either form
of these residues falls within the scope of this invention.
[0235] Cysteinyl residues can be replaced by amino acid residues or
other moieties either to eliminate disulfide bonding or,
conversely, to stabilize cross-linking (See, e.g., Bhatnagar et
al., J. Med. Chem., 39:3814-3819 (1996)).
[0236] Derivatization with bifunctional agents is useful for
cross-linking the peptides or their functional derivatives to a
water-insoluble support matrix, if desired, or to other
macromolecular vehicles. Commonly used cross-linking agents
include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides
such as bis-N-maleimido-1,8-octane. Derivatizing agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate yield
photoactivatable intermediates that are capable of forming
crosslinks in the presence of light. Alternatively, reactive
water-insoluble matrices such as cyanogen bromide-activated
carbohydrates and the reactive substrates, e.g., as described in
U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642;
4,229,537; and 4,330,440, are employed for protein
immobilization.
[0237] Other possible modifications include hydroxylation of
proline and lysine, phosphorylation of hydroxyl groups of seryl or
threonyl residues, oxidation of the sulfur atom in Cys, methylation
of the alpha-amino groups of lysine, arginine, and histidine side
chains. Creighton, Proteins: Structure and Molecule Properties (W.
H. Freeman & Co., San Francisco), 79-86 (1983).
[0238] The above examples of derivatizations are not intended to be
an exhaustive treatment, but merely illustrative.
[0239] The production of the composition of matter can also involve
suitable protein purification techniques, when applicable. In some
embodiments of the composition of matter of the invention, the
molecule can be prepared to include a suitable isotopic label
(e.g., .sup.125I, .sup.14C, .sup.13C, .sup.35S, .sup.3H, .sup.2H,
.sup.13N, .sup.15N, .sup.18O, .sup.17O, etc.), for ease of
quantification or detection.
[0240] Half-life extending moieties. Optionally, for modulation of
the pharmacokinetic profile of the molecule to fit the therapeutic
need, the composition of the present invention can include one or
more half-life extending moieties of various masses and
configurations, which half-life extending moiety, or moieties, can
be covalently fused, attached, linked or conjugated to the toxin
peptide analog. A "half-life extending moiety" refers to a molecule
that prevents or mitigates in vivo degradation by proteolysis or
other activity-diminishing chemical modification, increases in vivo
half-life or other pharmacokinetic properties such as but not
limited to increasing the rate of absorption, reduces toxicity,
reduces immunogenicity, improves solubility, increases biological
activity and/or target selectivity of the toxin peptide analog with
respect to a target of interest, and/or increases
manufacturability, compared to an unconjugated form of the toxin
peptide analog. In accordance with the invention, the half-life
extending moiety is one that is pharmaceutically acceptable.
[0241] The half-life extending moiety can be selected such that the
inventive composition achieves a sufficient hydrodynamic size to
prevent clearance by renal filtration in vivo. For example, a
half-life extending moiety can be selected that is a polymeric
macromolecule, which is substantially straight chain,
branched-chain (br), or dendritic in form. Alternatively, a
half-life extending moiety can be selected such that, in vivo, the
inventive composition of matter will bind to a serum protein to
form a complex, such that the complex thus formed avoids
substantial renal clearance. The half-life extending moiety can be,
for example, a lipid; a cholesterol group (such as a steroid); a
carbohydrate or oligosaccharide; or any natural or synthetic
protein, polypeptide or peptide that binds to a salvage
receptor.
[0242] Exemplary half-life extending moieties that can be used, in
accordance with the present invention, include an immunoglobulin Fc
domain, or a portion thereof, or a biologically suitable polymer or
copolymer, for example, a polyalkylene glycol compound, such as a
polyethylene glycol (PEG) or a polypropylene glycol. Other
appropriate polyalkylene glycol compounds include, but are not
limited to, charged or neutral polymers of the following types:
dextran, polylysine, colominic acids or other carbohydrate based
polymers, polymers of amino acids, and biotin derivatives. In some
monomeric fusion protein embodiments an immunoglobulin (including
light and heavy chains) or a portion thereof, can be used as a
half-life-extending moiety, preferably an immunoglobulin of human
origin, and including any of the immunoglobulins, such as, but not
limited to, IgG1, IgG2, IgG3 or IgG4.
[0243] Other examples of the half-life extending moiety, in
accordance with the invention, include a copolymer of ethylene
glycol, a copolymer of propylene glycol, a carboxymethylcellulose,
a polyvinyl pyrrolidone, a poly-1,3-dioxolane, a
poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a
polyaminoacid (e.g., polylysine or polyornithine), a dextran
n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol
homopolymer, a propylene oxide polymer, an ethylene oxide polymer,
a polyoxyethylated polyol, a polyvinyl alcohol, a linear or
branched glycosylated chain, a polyacetal, a long chain fatty acid,
a long chain hydrophobic aliphatic group, or a polysialic acid
(e.g., PolyXen.TM. technology; Gregoriadis et al., Improving the
therapeutic efficacy of peptides and proteins: a role for
polysialic acids, Intl. J. Pharmaceutics, 300:125-30 (2005),
incorporated herein by reference in its entirety).
[0244] In other embodiments of the composition of matter, the
half-life extending moiety is an anionically charged chemical
entity, covalently linked to the N-terminus of the toxin peptide
analog, which anionically charged chemical entities include, but
are not limited to, phosphotyrosine, phosphoserine,
p-phosphono(difluoro-methyl)-phenylalanine (Pfp),
p-phosphono-methyl-phenylalanine (Pmp),
p-phosphatidyl-phenylalanine (Ppa), or
p-phosphono-methylketo-phenylalanine (Pkp), which can be covalently
linked to the N-terminal of the toxin peptide analog, optionally
indirectly, via an AEEA linker or other linker as described herein.
(See, Chandy et al., Analogs of ShK toxin and their uses in
selective inhibition of Kv1.3 potassium channels, WO 2006/042151
A2; Beeton et al., Targeting effector memory T cells with a
selective peptide inhibitor of Kv1.3 channels for therapy of
autoimmune diseases, Molec. Pharmacol. 67(4):1369-81 (2005);
Pennington et al., Engineering a stable and selective peptide
blocker of the Kv1.3 channel in T lymphocytes, Molecular
Pharmacology Fast Forward, published Jan. 2, 2009 as
doi:10.1124/mol.108.052704 (2009), all of which references are
incorporated herein by reference in their entireties). AEEA is
2-(2-(2-aminoethoxy)ethoxy)acetic acid (also known as
8-Amino-3,6-Dioxaoctanoic Acid). (See, e.g., Beeton et al.,
Targeting effector memory T cells with a selective peptide
inhibitor of Kv1.3 channels for therapy of autoimmune diseases,
Molec. Pharmacol. 67(4):1369-81 (2005)).
[0245] Other embodiments of the half-life extending moiety, in
accordance with the invention, include peptide ligands or small
(organic) molecule ligands that have binding affinity for a long
half-life serum protein under physiological conditions of
temperature, pH, and ionic strength. Examples include an
albumin-binding peptide or small molecule ligand, a
transthyretin-binding peptide or small molecule ligand, a
thyroxine-binding globulin-binding peptide or small molecule
ligand, an antibody-binding peptide or small molecule ligand, or
another peptide or small molecule that has an affinity for a long
half-life serum protein. (See, e.g., Blaney et al., Method and
compositions for increasing the serum half-life of
pharmacologically active agents by binding to
transthyretin-selective ligands, U.S. Pat. No. 5,714,142; Sato et
al., Serum albumin binding moieties, US 2003/0069395 A1; Jones et
al., Pharmaceutical active conjugates, U.S. Pat. No. 6,342,225). A
"long half-life serum protein" is one of the hundreds of different
proteins dissolved in mammalian blood plasma, including so-called
"carrier proteins" (such as albumin, transferrin and haptoglobin),
fibrinogen and other blood coagulation factors, complement
components, immunoglobulins, enzyme inhibitors, precursors of
substances such as angiotensin and bradykinin and many other types
of proteins. The invention encompasses the use of any single
species of pharmaceutically acceptable half-life extending moiety,
such as, but not limited to, those described herein, or the use of
a combination of two or more different half-life extending
moieties, such as PEG and immunoglobulin Fc domain or a portion
thereof (see, e.g., Feige et al., Modified peptides as therapeutic
agents, U.S. Pat. No. 6,660,843), such as a CH2 domain of Fc,
albumin (e.g., human serum albumin (HSA); see, e.g., Rosen et al.,
Albumin fusion proteins, U.S. Pat. No. 6,926,898 and US
2005/0054051; Bridon et al., Protection of endogenous therapeutic
peptides from peptidase activity through conjugation to blood
components, U.S. Pat. No. 6,887,470), a transthyretin (TTR; see,
e.g., Walker et al., Use of transthyretin peptide/protein fusions
to increase the serum half-life of pharmacologically active
peptides/proteins, US 2003/0195154 A1; 2003/0191056 A1), or a
thyroxine-binding globulin (TBG), or a combination such as
immunoglobulin(light chain+heavy chain) and Fc domain (the
heterotrimeric combination a so-called "hemibody"), for example as
described in Sullivan et al., Toxin Peptide Therapeutic Agents,
PCT/US2007/022831, published as WO 2008/088422, which is
incorporated herein by reference in its entirety.
[0246] Conjugation of the toxin peptide analogs(s) to the half-life
extending moiety, or moieties, can be via the N-terminal and/or
C-terminal of the toxin peptide, or can be intercalary as to its
primary amino acid sequence, F1 being linked closer to the toxin
peptide analog's N-terminus.
[0247] Particularly useful half-life extending moieties include
immunoglobulins (e.g., human immunoglobulin, including IgG1, IgG2,
IgG3 or IgG4). The term "immunoglobulin" encompasses full
antibodies comprising two dimerized heavy chains (HC), each
covalently linked to a light chain (LC); a single undimerized
immunoglobulin heavy chain and covalently linked light chain
(HC+LC); or a chimeric immunoglobulin (light chain+heavy chain)-Fc
heterotrimer (a so-called "hemibody").
[0248] An "antibody", or interchangeably "Ab", is a tetrameric
glycoprotein. In a naturally-occurring antibody, each tetramer is
composed of two identical pairs of polypeptide chains, each pair
having one "light" chain of about 220 amino acids (about 25 kDa)
and one "heavy" chain of about 440 amino acids (about 50-70 kDa).
The amino-terminal portion of each chain includes a "variable"
("V") region of about 100 to 110 or more amino acids primarily
responsible for antigen recognition. The carboxy-terminal portion
of each chain defines a constant region primarily responsible for
effector function. The variable region differs among different
antibodies. The constant region is the same among different
antibodies. Within the variable region of each heavy or light
chain, there are three hypervariable subregions that help determine
the antibody's specificity for antigen. The variable domain
residues between the hypervariable regions are called the framework
residues and generally are somewhat homologous among different
antibodies. Immunoglobulins can be assigned to different classes
depending on the amino acid sequence of the constant domain of
their heavy chains. Human light chains are classified as kappa
(.kappa.) and lambda (.lamda.) light chains. Within light and heavy
chains, the variable and constant regions are joined by a "J"
region of about 12 or more amino acids, with the heavy chain also
including a "D" region of about 10 more amino acids. See generally,
Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press,
N.Y. (1989)). Within the scope of the invention, an "antibody" also
encompasses a recombinantly made antibody, and antibodies that are
lacking glycosylation.
[0249] The term "light chain" or "immunoglobulin light chain"
includes a full-length light chain and fragments thereof having
sufficient variable region sequence to confer binding specificity.
A full-length light chain includes a variable region domain,
V.sub.L, and a constant region domain, C.sub.L. The variable region
domain of the light chain is at the amino-terminus of the
polypeptide. Light chains include kappa chains and lambda
chains.
[0250] The term "heavy chain" or "immunoglobulin heavy chain"
includes a full-length heavy chain and fragments thereof having
sufficient variable region sequence to confer binding specificity.
A full-length heavy chain includes a variable region domain,
V.sub.H, and three constant region domains, C.sub.H1, C.sub.H2, and
C.sub.H3. The V.sub.H domain is at the amino-terminus of the
polypeptide, and the C.sub.H domains are at the carboxyl-terminus,
with the C.sub.H3 being closest to the carboxy-terminus of the
polypeptide. Heavy chains are classified as mu (.mu.), delta
(.DELTA.), gamma (.gamma.), alpha (.alpha.), and epsilon
(.epsilon.), and define the antibody's isotype as IgM, IgD, IgG,
IgA, and IgE, respectively. In separate embodiments of the
invention, heavy chains may be of any isotype, including IgG
(including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1
and IgA2 subtypes), IgM and IgE. Several of these may be further
divided into subclasses or isotypes, e.g. IgG1, IgG2, IgG3, IgG4,
IgA1 and IgA2. Different IgG isotypes may have different effector
functions (mediated by the Fc region), such as antibody-dependent
cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity
(CDC). In ADCC, the Fc region of an antibody binds to Fc receptors
(Fc.gamma.Rs) on the surface of immune effector cells such as
natural killers and macrophages, leading to the phagocytosis or
lysis of the targeted cells. In CDC, the antibodies kill the
targeted cells by triggering the complement cascade at the cell
surface.
[0251] An "Fc region", or used interchangeably herein, "Fc domain"
or "immunoglobulin Fc domain", contains two heavy chain fragments,
which in a full antibody comprise the C.sub.H1 and C.sub.H2 domains
of the antibody. The two heavy chain fragments are held together by
two or more disulfide bonds and by hydrophobic interactions of the
C.sub.H3 domains.
[0252] The term "salvage receptor binding epitope" refers to an
epitope of the Fc region of an IgG molecule (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, or IgG.sub.4) that is responsible for
increasing the in vivo serum half-life of the IgG molecule.
[0253] "Allotypes" are variations in antibody sequence, often in
the constant region, that can be immunogenic and are encoded by
specific alleles in humans. Allotypes have been identified for five
of the human IGHC genes, the IGHG1, IGHG2, IGHG3, IGHA2 and IGHE
genes, and are designated as G1m, G2m, G3m, A2m, and Em allotypes,
respectively. At least 18 Gm allotypes are known: nG1m(1), nG1m(2),
G1m (1, 2, 3, 17) or G1m (a, x, f, z), G2m (23) or G2m (n), G3m (5,
6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (b1, c3, b5,
b0, b3, b4, s, t, g1, c5, u, v, g5). There are two A2m allotypes
A2m(1) and A2m(2).
[0254] For a detailed description of the structure and generation
of antibodies, see Roth, D. B., and Craig, N. L., Cell, 94:411-414
(1998), herein incorporated by reference in its entirety. Briefly,
the process for generating DNA encoding the heavy and light chain
immunoglobulin sequences occurs primarily in developing B-cells.
Prior to the rearranging and joining of various immunoglobulin gene
segments, the V, D, J and constant (C) gene segments are found
generally in relatively close proximity on a single chromosome.
During B-cell-differentiation, one of each of the appropriate
family members of the V, D, J (or only V and J in the case of light
chain genes) gene segments are recombined to form functionally
rearranged variable regions of the heavy and light immunoglobulin
genes. This gene segment rearrangement process appears to be
sequential. First, heavy chain D-to-J joints are made, followed by
heavy chain V-to-DJ joints and light chain V-to-J joints. In
addition to the rearrangement of V, D and J segments, further
diversity is generated in the primary repertoire of immunoglobulin
heavy and light chains by way of variable recombination at the
locations where the V and J segments in the light chain are joined
and where the D and J segments of the heavy chain are joined. Such
variation in the light chain typically occurs within the last codon
of the V gene segment and the first codon of the J segment. Similar
imprecision in joining occurs on the heavy chain chromosome between
the D and J.sub.H segments and may extend over as many as 10
nucleotides. Furthermore, several nucleotides may be inserted
between the D and J.sub.H and between the V.sub.H and D gene
segments which are not encoded by genomic DNA. The addition of
these nucleotides is known as N-region diversity. The net effect of
such rearrangements in the variable region gene segments and the
variable recombination which may occur during such joining is the
production of a primary antibody repertoire.
[0255] The term "hypervariable" region refers to the amino acid
residues of an antibody which are responsible for antigen-binding.
The hypervariable region comprises amino acid residues from a
complementarity determining region or CDR [i.e., residues 24-34
(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain
and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain
variable domain as described by Kabat et al., Sequences of Proteins
of Immunological Interest, 5.sup.th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)]. Even a single
CDR may recognize and bind antigen, although with a lower affinity
than the entire antigen binding site containing all of the
CDRs.
[0256] An alternative definition of residues from a hypervariable
"loop" is described by Chothia et al., J. Mol. Biol. 196: 901-917
(1987) as residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the
light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101
(H3) in the heavy chain variable domain.
[0257] "Framework" or "FR" residues are those variable region
residues other than the hypervariable region residues.
[0258] "Antibody fragments" comprise a portion of an intact full
length antibody, preferably the antigen binding or variable region
of the intact antibody. Examples of antibody fragments include Fab,
Fab', F(ab').sub.2, and Fv fragments; diabodies; linear antibodies
(Zapata et al., Protein Eng., 8(10):1057-1062 (1995)); single-chain
antibody molecules; and multispecific antibodies formed from
antibody fragments.
[0259] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment which
contains the constant region. The Fab fragment contains all of the
variable domain, as well as the constant domain of the light chain
and the first constant domain (CH1) of the heavy chain. The Fc
fragment displays carbohydrates and is responsible for many
antibody effector functions (such as binding complement and cell
receptors), that distinguish one class of antibody from
another.
[0260] Pepsin treatment yields an F(ab').sub.2 fragment that has
two "Single-chain Fv" or "scFv" antibody fragments comprising the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Fab fragments differ from Fab'
fragments by the inclusion of a few additional residues at the
carboxy terminus of the heavy chain CH1 domain including one or
more cysteines from the antibody hinge region. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the VH
and VL domains that enables the Fv to form the desired structure
for antigen binding. For a review of scFv see Pluckthun in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
[0261] A "Fab fragment" is comprised of one light chain and the
C.sub.H1 and variable regions of one heavy chain. The heavy chain
of a Fab molecule cannot form a disulfide bond with another heavy
chain molecule.
[0262] A "Fab' fragment" contains one light chain and a portion of
one heavy chain that contains the V.sub.H domain and the C.sub.H1
domain and also the region between the C.sub.H1 and C.sub.H2
domains, such that an interchain disulfide bond can be formed
between the two heavy chains of two Fab' fragments to form an
F(ab').sub.2 molecule.
[0263] A "F(ab').sub.2 fragment" contains two light chains and two
heavy chains containing a portion of the constant region between
the C.sub.H1 and C.sub.H2 domains, such that an interchain
disulfide bond is formed between the two heavy chains. A
F(ab').sub.2 fragment thus is composed of two Fab' fragments that
are held together by a disulfide bond between the two heavy
chains.
[0264] "Fv" is the minimum antibody fragment that contains a
complete antigen recognition and binding site. This region consists
of a dimer of one heavy- and one light-chain variable domain in
tight, non-covalent association. It is in this configuration that
the three CDRs of each variable domain interact to define an
antigen binding site on the surface of the VH VL dimer. A single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
[0265] "Single-chain antibodies" are Fv molecules in which the
heavy and light chain variable regions have been connected by a
flexible linker to form a single polypeptide chain, which forms an
antigen-binding region. Single chain antibodies are discussed in
detail in International Patent Application Publication No. WO
88/01649 and U.S. Pat. No. 4,946,778 and No. 5,260,203, the
disclosures of which are incorporated by reference in their
entireties.
[0266] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain, and optionally comprising a
polypeptide linker between the V.sub.H and V.sub.L domains that
enables the Fv to form the desired structure for antigen binding
(Bird et al., Science 242:423-426, 1988, and Huston et al., Proc.
Natl. Acad. Sci. USA 85:5879-5883, 1988). An "Fd" fragment consists
of the V.sub.H and C.sub.H1 domains.
[0267] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO
93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993).
[0268] A "domain antibody" is an immunologically functional
immunoglobulin fragment containing only the variable region of a
heavy chain or the variable region of a light chain. In some
instances, two or more V.sub.H regions are covalently joined with a
peptide linker to create a bivalent domain antibody. The two
V.sub.H regions of a bivalent domain antibody may target the same
or different antigens.
[0269] The terms "DNP" or "dinitrophenol" are used interchangeably
herein and denote the antigen 2,4-dinitrophenol. "Anti-DNP" or
".alpha.DNP" or ".alpha.DNP" are used interchangeably herein to
refer to an antigen binding protein, e.g., an antibody or antibody
fragment, that specifically binds DNP.
[0270] The terms "KLH" or "keyhole limpet hemocyanin" are used
interchangeably herein and denote the Imject.RTM. Mariculture
Keyhole Limpet hemocyanin (mcKLH; Pierce Biotechnology, Rockford,
Ill.). According to the manufacturer, mcKLH is harvested from
select populations of the mollusk Megathura crenulata (keyhole
limpet) that are grown in mariculture, rather than being extracted
from wild populations; KLH has a high molecular mass
(4.5.times.10.sup.5-1.3.times.10.sup.7 Daltons of mixed aggregates
of 350 and 390 kDa subunits) and elicits a stronger immune response
than BSA or ovalbumin. "Anti-KLH" or ".alpha.KLH" or "aKLH" are
used interchangeably herein to refer to an antigen binding protein,
e.g., an antibody or antibody fragment, that specifically binds
KLH.
[0271] The term "epitope" is the portion of a molecule that is
bound by an antigen binding protein (for example, an antibody). The
term includes any determinant capable of specifically binding to an
antigen binding protein, such as an antibody or to a T-cell
receptor. An epitope can be contiguous or non-contiguous (e.g., in
a single-chain polypeptide, amino acid residues that are not
contiguous to one another in the polypeptide sequence but that
within the context of the molecule are bound by the antigen binding
protein). In certain embodiments, epitopes may be mimetic in that
they comprise a three dimensional structure that is similar to an
epitope used to generate the antigen binding protein, yet comprise
none or only some of the amino acid residues found in that epitope
used to generate the antigen binding protein. Most often, epitopes
reside on proteins, but in some instances may reside on other kinds
of molecules, such as nucleic acids. Epitope determinants may
include chemically active surface groupings of molecules such as
amino acids, sugar side chains, phosphoryl or sulfonyl groups, and
may have specific three dimensional structural characteristics,
and/or specific charge characteristics. Generally, antibodies
specific for a particular target antigen will preferentially
recognize an epitope on the target antigen in a complex mixture of
proteins and/or macromolecules.
[0272] The term "identity" refers to a relationship between the
sequences of two or more polypeptide molecules or two or more
nucleic acid molecules, as determined by aligning and comparing the
sequences. "Percent identity" means the percent of identical
residues between the amino acids or nucleotides in the compared
molecules and is calculated based on the size of the smallest of
the molecules being compared. For these calculations, gaps in
alignments (if any) must be addressed by a particular mathematical
model or computer program (i.e., an "algorithm"). Methods that can
be used to calculate the identity of the aligned nucleic acids or
polypeptides include those described in Computational Molecular
Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University
Press; Biocomputing Informatics and Genome Projects, (Smith, D. W.,
ed.), 1993, New York: Academic Press; Computer Analysis of Sequence
Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New
Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in
Molecular Biology, New York: Academic Press; Sequence Analysis
Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M.
Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math.
48:1073. For example, sequence identity can be determined by
standard methods that are commonly used to compare the similarity
in position of the amino acids of two polypeptides. Using a
computer program such as BLAST or FASTA, two polypeptide or two
polynucleotide sequences are aligned for optimal matching of their
respective residues (either along the full length of one or both
sequences, or along a pre-determined portion of one or both
sequences). The programs provide a default opening penalty and a
default gap penalty, and a scoring matrix such as PAM 250 [a
standard scoring matrix; see Dayhoff et al., in Atlas of Protein
Sequence and Structure, vol. 5, supp. 3 (1978)] can be used in
conjunction with the computer program. For example, the percent
identity can then be calculated as: the total number of identical
matches multiplied by 100 and then divided by the sum of the length
of the longer sequence within the matched span and the number of
gaps introduced into the longer sequences in order to align the two
sequences. In calculating percent identity, the sequences being
compared are aligned in a way that gives the largest match between
the sequences.
[0273] The GCG program package is a computer program that can be
used to determine percent identity, which package includes GAP
(Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer
Group, University of Wisconsin, Madison, Wis.). The computer
algorithm GAP is used to align the two polypeptides or two
polynucleotides for which the percent sequence identity is to be
determined. The sequences are aligned for optimal matching of their
respective amino acid or nucleotide (the "matched span", as
determined by the algorithm). A gap opening penalty (which is
calculated as 3.times. the average diagonal, wherein the "average
diagonal" is the average of the diagonal of the comparison matrix
being used; the "diagonal" is the score or number assigned to each
perfect amino acid match by the particular comparison matrix) and a
gap extension penalty (which is usually 1/10 times the gap opening
penalty), as well as a comparison matrix such as PAM 250 or BLOSUM
62 are used in conjunction with the algorithm. In certain
embodiments, a standard comparison matrix (see, Dayhoff et al.,
1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM
250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad.
Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) is
also used by the algorithm.
[0274] Recommended parameters for determining percent identity for
polypeptides or nucleotide sequences using the GAP program include
the following:
[0275] Algorithm: Needleman et al., 1970, J. Mol. Biol.
48:443-453;
[0276] Comparison matrix: BLOSUM 62 from Henikoff et al., 1992,
supra;
[0277] Gap Penalty: 12 (but with no penalty for end gaps)
[0278] Gap Length Penalty: 4
[0279] Threshold of Similarity: 0
[0280] Certain alignment schemes for aligning two amino acid
sequences may result in matching of only a short region of the two
sequences, and this small aligned region may have very high
sequence identity even though there is no significant relationship
between the two full-length sequences. Accordingly, the selected
alignment method (GAP program) can be adjusted if so desired to
result in an alignment that spans at least 50 contiguous amino
acids of the target polypeptide.
[0281] The term "modification" when used in connection with
immmunoglobulins, including antibodies and antibody fragments, of
the invention, include, but are not limited to, one or more amino
acid changes (including substitutions, insertions or deletions);
chemical modifications; covalent modification by conjugation to
therapeutic or diagnostic agents; labeling (e.g., with
radionuclides or various enzymes); covalent polymer attachment such
as PEGylation (derivatization with polyethylene glycol) and
insertion or substitution by chemical synthesis of non-natural
amino acids.
[0282] The term "derivative" when used in connection with an
immunoglobulin (including antibodies and antibody fragments) within
the scope of the invention refers to immunoglobulin proteins that
are covalently modified by conjugation to therapeutic or diagnostic
agents, labeling (e.g., with radionuclides or various enzymes),
covalent polymer attachment such as PEGylation (derivatization with
polyethylene glycol) and insertion or substitution by chemical
synthesis of non-natural amino acids. Derivatives of the invention
will retain the binding properties of underivatized molecules of
the invention.
[0283] In some embodiments of the invention, the half-life
extending moiety is an immunoglobulin Fc domain (e.g., a human
immunoglobulin Fc domain, including Fc of allotype IgG1, IgG2, IgG3
or IgG4) or a portion thereof (e.g., CH2 domain of the Fc domain),
human serum albumin (HSA), or poly(ethylene glycol) (PEG), in
particular PEG of molecular weight of about 1000 Da to about 100000
Da.
[0284] Monovalent dimeric or bivalent dimeric Fc-toxin peptide
analog fusions are useful embodiments of the inventive composition
of matter. A "monovalent dimeric" Fc-toxin peptide analog fusion,
or interchangeably, "monovalent dimer", or interchangeably,
"monovalent heterodimer", is a Fc-toxin peptide analog fusion that
includes a toxin peptide analog conjugated with only one of the
dimerized Fc domains (e.g., as represented schematically in FIG. 2B
of Sullivan et al., Toxin Peptide Therapeutic Agents,
US2007/0071764 and Sullivan et al., Toxin Peptide Therapeutic
Agents, PCT/US2007/022831, published as WO 2008/088422, which are
both incorporated herein by reference in their entireties). A
"bivalent dimeric" Fc-toxin peptide analog fusion, or
interchangeably, "bivalent dimer" or "bivalent homodimer", is a
Fc-toxin peptide analog fusion having both of the dimerized Fc
domains each conjugated separately with a toxin peptide analog
(e.g., as represented schematically in FIG. 2C of Sullivan et al.,
Toxin Peptide Therapeutic Agents, US2007/0071764 and Sullivan et
al., Toxin Peptide Therapeutic Agents, PCT/US2007/022831, published
as WO 2008/088422).
[0285] Immunoglobulin Fc domains include Fc variants, which are
suitable half-life extending moieties within the scope of this
invention. A native Fc can be extensively modified to form an Fc
variant in accordance with this invention, provided binding to the
salvage receptor is maintained; see, for example WO 97/34631, WO
96/32478, and WO 04/110 472. In such Fc variants, one can remove
one or more sites of a native Fc that provide structural features
or functional activity not required by the fusion molecules of this
invention. One can remove these sites by, for example, substituting
or deleting residues, inserting residues into the site, or
truncating portions containing the site. The inserted or
substituted residues can also be altered amino acids, such as
peptidomimetics or D-amino acids. Fc variants can be desirable for
a number of reasons, several of which are described below.
Exemplary Fc variants include molecules and sequences in which:
[0286] 1. Sites involved in disulfide bond formation are removed.
Such removal can avoid reaction with other cysteine-containing
proteins present in the host cell used to produce the molecules of
the invention. For this purpose, the cysteine-containing segment at
the N-terminus can be truncated or cysteine residues can be deleted
or substituted with other amino acids (e.g., alanyl, seryl). In
particular, one can truncate the N-terminal 20-amino acid segment
of SEQ ID NO: 278 or delete or substitute the cysteine residues at
positions 7 and 10 of SEQ ID NO: 278. Even when cysteine residues
are removed, the single chain Fc domains can still form a dimeric
Fc domain that is held together non-covalently. [0287] 2. A native
Fc is modified to make it more compatible with a selected host
cell. For example, one can remove the PA dipeptide sequence near
the N-terminus of a typical native Fc, which can be recognized by a
digestive enzyme in E. coli such as proline iminopeptidase. One can
also add an N-terminal methionine residue, especially when the
molecule is expressed recombinantly in a bacterial cell such as E.
coli. The Fc domain of SEQ ID NO: 278 (FIG. 27A-B) is one such Fc
variant. [0288] 3. A portion of the N-terminus of a native Fc is
removed to prevent N-terminal heterogeneity when expressed in a
selected host cell. For this purpose, one can delete any of the
first 20 amino acid residues at the N-terminus, particularly those
at positions 1, 2, 3, 4 and 5. [0289] 4. One or more glycosylation
sites are removed. Residues that are typically glycosylated (e.g.,
asparagine) can confer cytolytic response. Such residues can be
deleted or substituted with unglycosylated residues (e.g.,
alanine). [0290] 5. Sites involved in interaction with complement,
such as the C1q binding site, are removed. For example, one can
delete or substitute the EKK tripeptide sequence of human IgG1.
Complement recruitment may not be advantageous for the molecules of
this invention and so can be avoided with such an Fc variant.
[0291] 6. Sites are removed that affect binding to Fc receptors
other than a salvage receptor. A native Fc can have sites for
interaction with certain white blood cells that are not required
for the fusion molecules of the present invention and so can be
removed. [0292] 7. The ADCC site is removed to decrease or
eliminate ADCC effector function, or alternatively, modified for
enhanced ADCC effector function by non-fucosylation or
de-fucosylation. ADCC sites are known in the art; see, for example,
Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in
IgG1. These sites, as well, are not required for the fusion
molecules of the present invention and so can be removed, or
enhanced for ADCC effector function, as may be desired. (See, Iida
et al., Two mechanisms of the enhanced antibody-dependent cellular
cytotoxicity (ADCC) efficacy of non-fucosylated therapeutic
antibodies in human blood, BMC Cancer 9:58
doi:10.1186/1471-2407-9-58 (2009)). [0293] 8. When the native Fc is
derived from a non-human antibody, the native Fc can be humanized.
Typically, to humanize a native Fc, one will substitute selected
residues in the non-human native Fc with residues that are normally
found in human native Fc. Techniques for antibody humanization are
well known in the art. [0294] 9. One or more toxin peptide analog
sequences can be inserted into an internal conjugation site, or
sites, within a loop region of an immunoglobulin Fc domain, as
disclosed in U.S. Pat. No. 7,442,778 B2. The term "loop" region or
"Fc-loop" region refers to a primary sequence of amino acid
residues which connects two regions comprising secondary structure,
such as an .alpha.-helix or a .beta.-sheet, in the immediate
N-terminal and C-terminal directions of primary structure from the
loop region. Examples include, but are not limited to, CH2 or CH3
loop regions. One of skill in the art understands that a loop
region, while not itself comprising secondary structure, may
influence or contribute to secondary or higher order protein
structure. The term "internal" conjugation site means that the
toxin peptide analog moiety, or moieties, is non-terminal, i.e.,
not through the .alpha.-amino site or the .alpha.-carboxy site of
the Fc domain, although there optionally can also be additional
moieties conjugated terminally at the N-terminal and/or C-terminal
of the Fc domain. (See, e.g., Example 11, Example 12, Table 4H and
Table 4L herein). [0295] 10. A linker of suitable length and
neutral charge, such as "L25"(GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID
NO:293) or "L20" (GGGGSGGGGSGGGGSGGGGS; SEQ ID NO:86), can be
covalently fused between the C-terminal of one monomer of an Fc
domain and the N-terminal of a second Fc domain monomer, with a
toxin peptide analog fused to the N-terminal of the first Fc domain
monomer or the C-terminal of the second Fc domain monomer, or
within a loop region of the first and/or second Fc domain monomer.
Such a molecule can be recombinantly expressed in bacterial or
mammalian cells to produce a variant "monovalent dimeric" Fc-toxin
peptide analog fusion with the typical disulfide bond formation
between the Fc monomers. (See, e.g., Example 13 herein).
[0296] Other examples of Fc variants include the following: In SEQ
ID NO: 278, the leucine at position 15 can be substituted with
glutamate; the glutamate at position 99, with alanine; and the
lysines at positions 101 and 103, with alanines In addition,
phenyalanine residues can replace one or more tyrosine residues.
For purposes of the invention, a variant Fc domain can also be part
of a monomeric immunoglobulin heavy chain, an antibody, or a
heterotrimeric hemibody (LC+HC+Fc).
[0297] An alternative half-life extending moiety would be a
protein, polypeptide, peptide, antibody, antibody fragment, or
small molecule (e.g., a peptidomimetic compound) capable of binding
to a salvage receptor. For example, one could use as a half-life
extending moiety a polypeptide as described in U.S. Pat. No.
5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could
also be selected by phage display for binding to the FcRn salvage
receptor. Such salvage receptor-binding compounds are also included
within the meaning of "half-life extending moiety" and are within
the scope of this invention. Such half-life extending moieties
should be selected for increased half-life (e.g., by avoiding
sequences recognized by proteases) and decreased immunogenicity
(e.g., by favoring non-immunogenic sequences, as discovered in
antibody humanization).
[0298] As noted above, polymer half-life extending moieties can
also be used. Various means for attaching chemical moieties useful
as half-life extending moieties are currently available, see, e.g.,
Patent Cooperation Treaty ("PCT") International Publication No. WO
96/11953, entitled "N-Terminally Chemically Modified Protein
Compositions and Methods," herein incorporated by reference in its
entirety. This PCT publication discloses, among other things, the
selective attachment of water-soluble polymers to the N-terminus of
proteins.
[0299] In some embodiments of the inventive compositions, the
polymer half-life extending moiety is polyethylene glycol (PEG),
covalently linked at the N-terminal, C-terminal or at one or more
intercalary side chains of toxin peptide analog. Some embodiments
of the inventive composition of matter further include one or more
PEG moieties conjugated to a non-PEG half-life extending moiety or
to the toxin peptide analog, or to any combination of any of these.
For example, an Fc domain or portion thereof in the inventive
composition can be made mono-PEGylated, di-PEGylated, or otherwise
multi-PEGylated, by the process of reductive alkylation.
[0300] Covalent conjugation of proteins and peptides with
poly(ethylene glycol) (PEG) has been widely recognized as an
approach to significantly extend the in vivo circulating half-lives
of therapeutic proteins. PEGylation achieves this effect
predominately by retarding renal clearance, since the PEG moiety
adds considerable hydrodynamic radius to the protein. (Zalipsky,
S., et al., Use of functionalized poly(ethylene glycol)s for
modification of polypeptides, in poly(ethylene glycol) chemistry:
Biotechnical and biomedical applications, J. M. Harris, Ed., Plenum
Press: New York., 347-370 (1992)). Additional benefits often
conferred by PEGylation of proteins and peptides include increased
solubility, resistance to proteolytic degradation, and reduced
immunogenicity of the therapeutic polypeptide. The merits of
protein PEGylation are evidenced by the commercialization of
several PEGylated proteins including PEG-Adenosine deaminase
(Adagen.TM./Enzon Corp.), PEG-L-asparaginase (Oncaspar.TM./Enzon
Corp.), PEG-Interferon .alpha.-2b (PEG-Intron.TM./Schering/Enzon),
PEG-Interferon .alpha.-2a (PEGASYS.TM./Roche) and PEG-G-CSF
(Neulasta.TM./Amgen) as well as many others in clinical trials.
[0301] By "PEGylated peptide" or "PEGylated protein" is meant a
peptide having a polyethylene glycol (PEG) moiety covalently bound
to an amino acid residue of the peptide itself or to a peptidyl or
non-peptidyl linker that is covalently bound to a residue of the
peptide.
[0302] By "polyethylene glycol" or "PEG" is meant a polyalkylene
glycol compound or a derivative thereof, with or without coupling
agents or derivatization with coupling or activating moieties
(e.g., with aldehyde, hydroxysuccinimidyl, hydrazide, thiol,
triflate, tresylate, azirdine, oxirane, orthopyridyl disulphide,
vinylsulfone, iodoacetamide or a maleimide moiety). In accordance
with the present invention, useful PEG includes substantially
linear, straight chain PEG, branched PEG (brPEG), or dendritic PEG.
(See, e.g., Merrill, U.S. Pat. No. 5,171,264; Harris et al.,
Multiarmed, monofunctional, polymer for coupling to molecules and
surfaces, U.S. Pat. No. 5,932,462; Shen, N-maleimidyl polymer
derivatives, U.S. Pat. No. 6,602,498).
[0303] Briefly, the PEG groups are generally attached to the
peptide portion of the composition of the invention via acylation
or reductive alkylation (or reductive amination) through a reactive
group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester
group) to a reactive group on the inventive compound (e.g., an
aldehyde, amino, or ester group).
[0304] A useful strategy for the PEGylation of synthetic peptides
consists of combining, through forming a conjugate linkage in
solution, a peptide and a PEG moiety, each bearing a special
functionality that is mutually reactive toward the other. The
peptides can be easily prepared with conventional solid phase
synthesis (see, for example, FIGS. 5 and 6 and the accompanying
text herein). The peptides are "preactivated" with an appropriate
functional group at a specific site. The precursors are purified
and fully characterized prior to reacting with the PEG moiety.
Ligation of the peptide with PEG usually takes place in aqueous
phase and can be easily monitored by reverse phase analytical HPLC.
The PEGylated peptides can be easily purified by preparative HPLC
and characterized by analytical HPLC, amino acid analysis and laser
desorption mass spectrometry.
[0305] PEG is a well-known, water soluble polymer that is
commercially available or can be prepared by ring-opening
polymerization of ethylene glycol according to methods well known
in the art (Sandler and Karo, Polymer Synthesis, Academic Press,
New York, Vol. 3, pages 138-161). In the present application, the
term "PEG" is used broadly to encompass any polyethylene glycol
molecule, in mono-, bi-, or poly-functional form, without regard to
size or to modification at an end of the PEG, and can be
represented by the formula:
X--O(CH.sub.2CH.sub.2O).sub.n-1CH.sub.2CH.sub.2OH, (I)
[0306] where n is 20 to 2300 and X is H or a terminal modification,
e.g., a C.sub.1-4 alkyl.
[0307] In some useful embodiments, a PEG used in the invention
terminates on one end with hydroxy or methoxy, i.e., X is H or
CH.sub.3 ("methoxy PEG"). It is noted that the other end of the
PEG, which is shown in formula (I) terminating in OH, covalently
attaches to an activating moiety via an ether oxygen bond, an amine
linkage, or amide linkage. When used in a chemical structure, the
term "PEG" includes the formula (I) above without the hydrogen of
the hydroxyl group shown, leaving the oxygen available to react
with a free carbon atom of a linker to form an ether bond. More
specifically, in order to conjugate PEG to a peptide, the peptide
must be reacted with PEG in an "activated" form. Activated PEG can
be represented by the formula:
(PEG)-(A) (II)
[0308] where PEG (defined supra) covalently attaches to a carbon
atom of the activation moiety (A) to form an ether bond, an amine
linkage, or amide linkage, and (A) contains a reactive group which
can react with an amino, azido, alkyne, imino, maleimido,
N-succinimidyl, carboxyl, aminooxy, seleno, or thiol group on an
amino acid residue of a peptide or a linker moiety covalently
attached to the peptide, e.g., the toxin peptide analog.
[0309] Techniques for the preparation of activated PEG and its
conjugation to biologically active peptides are well known in the
art. (E.g., see U.S. Pat. Nos. 5,643,575, 5,919,455, 5,932,462, and
5,990,237; Kinstler et al., N-terminally chemically modified
protein compositions and methods, U.S. Pat. Nos. 5,985,265, and
5,824,784; Thompson et al., PEGylation of polypeptides, EP 0575545
B1; Petit, Site specific protein modification, U.S. Pat. Nos.
6,451,986, and 6,548,644; S. Herman et al., Poly(ethylene glycol)
with reactive endgroups: I. Modification of proteins, J. Bioactive
Compatible Polymers, 10:145-187 (1995); Y. Lu et al., Pegylated
peptides III: Solid-phase synthesis with PEGylating reagents of
varying molecular weight: synthesis of multiply PEGylated peptides,
Reactive Polymers, 22:221-229 (1994); A. M. Felix et al., PEGylated
Peptides IV: Enhanced biological activity of site-directed
PEGylated GRF analogs, Int. J. Peptide Protein Res., 46:253-264
(1995); A.M. Felix, Site-specific poly(ethylene glycol)ylation of
peptides, ACS Symposium Series 680(poly(ethylene glycol)): 218-238
(1997); Y. Ikeda et al., Polyethylene glycol derivatives, their
modified peptides, methods for producing them and use of the
modified peptides, EP 0473084 B1; G. E. Means et al., Selected
techniques for the modification of protein side chains, in:
Chemical modification of proteins, Holden Day, Inc., 219
(1971)).
[0310] Activated PEG, such as PEG-aldehydes or PEG-aldehyde
hydrates, can be chemically synthesized by known means or obtained
from commercial sources, e.g., Shearwater Polymers, (Huntsville,
Ala.) or Enzon, Inc. (Piscataway, N.J.).
[0311] An example of a useful activated PEG for purposes of the
present invention is a PEG-aldehyde compound (e.g., a methoxy
PEG-aldehyde), such as PEG-propionaldehyde, which is commercially
available from Shearwater Polymers (Huntsville, Ala.).
PEG-propionaldehyde is represented by the formula
PEG-CH.sub.2CH.sub.2CHO. (See, e.g., U.S. Pat. No. 5,252,714). Also
included within the meaning of "PEG aldehyde compound" are PEG
aldehyde hydrates, e.g., PEG acetaldehyde hydrate and PEG bis
aldehyde hydrate, which latter yields a bifunctionally activated
structure. (See., e.g., Bentley et al., Poly(ethylene glycol)
aldehyde hydrates and related polymers and applications in
modifying amines, U.S. Pat. No. 5,990,237) (See., e.g., Bentley et
al., Poly(ethylene glycol) aldehyde hydrates and related polymers
and applications in modifying amines, U.S. Pat. No. 5,990,237). An
activated multi-branched PEG-aldehyde compound can be used (PEG
derivatives comprising multiple arms to give divalent, trivalent,
tetravalent, octavalent constructs). Using a 4-arm PEG derivative
four (4) toxin peptide analogs are attached to each PEG molecule.
For example, in accordance with the present invention, the toxin
peptide analog can be conjugated to a polyethylene glycol (PEG) at
1, 2, 3 or 4 amino functionalized sites of the PEG.
[0312] In being conjugated in accordance with the inventive method,
the polyethylene glycol (PEG), as described herein, is covalently
bound by reductive amination directly to at least one
solvent-exposed free amine moiety of an amino acid residue of the
toxin peptide analog itself. In some embodiments of the inventive
method, the toxin peptide analog is conjugated to a PEG at one or
more primary or secondary amines on the toxin peptide analog, or to
two PEG groups at a single primary amine site on the toxin peptide
analog (e.g., this can occur when the reductive amination reaction
involves the presence of excess PEG-aldehyde compound). We have
observed that when PEGylation by reductive amination is at a
primary amine on the peptide, it is not uncommon to have amounts (1
to 100% range) of reaction product that have two or more PEGs
present per molecule, and if the desired PEGylation product is one
with only one PEG per molecule, then this "over-PEGylation" may be
undesirable. When PEGylated product with a single PEG per
PEGylation product molecule is desired, an embodiment of the
inventive method can be employed that involves PEGylation using
secondary amines of the pharmacologically active peptide, because
only one PEG group per molecule will be transferred in the
reductive amination reaction.
[0313] Amino acid residues that can provide a primary amine moiety
include residues of lysine, homolysine, ornithine,
.alpha.,.beta.-diaminopropionic acid (Dap),
.alpha.,.beta.-diaminopropionoic acid (Dpr), and
.alpha.,.gamma.-diaminobutyric acid (Dab), aminobutyric acid (Abu),
and .alpha.-amino-isobutyric acid (Aib). The polypeptide N-terminus
also provides a useful .alpha.-amino group for PEGylation. Amino
acid residues that can provide a secondary amine moiety include
8-N-alkyl lysine, .alpha.-N-alkyl lysine, 6-N-alkyl ornithine,
.alpha.-N-alkyl ornithine, or an N-terminal proline, where the
alkyl is C.sub.1 to C.sub.6.
[0314] Another useful activated PEG for generating the PEGylated
toxin peptide analogs of the present invention is a PEG-maleimide
compound, such as, but not limited to, a methoxy PEG-maleimide,
such as maleimido monomethoxy PEG, are particularly useful for
generating the PEG-conjugated peptides of the invention. (E.g.,
Shen, N-maleimidyl polymer derivatives, U.S. Pat. No. 6,602,498; C.
Delgado et al., The uses and properties of PEG-linked proteins,
Crit. Rev. Therap. Drug Carrier Systems, 9:249-304 (1992); S.
Zalipsky et al., Use of functionalized poly(ethylene glycol)s for
modification of polypeptides, in: Poly(ethylene glycol) chemistry:
Biotechnical and biomedical applications (J. M. Harris, Editor,
Plenum Press: New York, 347-370 (1992); S. Herman et al.,
Poly(ethylene glycol) with reactive endgroups: I. Modification of
proteins, J. Bioactive Compatible Polymers, 10:145-187 (1995); P.
J. Shadle et al., Conjugation of polymer to colony stimulating
factor-1, U.S. Pat. No. 4,847,325; G. Shaw et al., Cysteine added
variants IL-3 and chemical modifications thereof, U.S. Pat. No.
5,166,322 and EP 0469074 B1; G. Shaw et al., Cysteine added
variants of EPO and chemical modifications thereof, EP 0668353 A1;
G. Shaw et al., Cysteine added variants G-CSF and chemical
modifications thereof, EP 0668354 A1; N. V. Katre et al.,
Interleukin-2 muteins and polymer conjugation thereof, U.S. Pat.
No. 5,206,344; R. J. Goodson and N. V. Katre, Site-directed
pegylation of recombinant interleukin-2 at its glycosylation site,
Biotechnology, 8:343-346 (1990)).
[0315] A poly(ethylene glycol) vinyl sulfone is another useful
activated PEG for generating the PEG-conjugated toxin peptide
analogs of the present invention by conjugation at thiolated amino
acid residues, e.g., at C residues. (E.g., M. Morpurgo et al.,
Preparation and characterization of poly(ethylene glycol) vinyl
sulfone, Bioconj. Chem., 7:363-368 (1996); see also Harris,
Functionalization of polyethylene glycol for formation of active
sulfone-terminated PEG derivatives for binding to proteins and
biologically compatible materials, U.S. Pat. Nos. 5,446,090;
5,739,208; 5,900,461; 6,610,281 and 6,894,025; and Harris, Water
soluble active sulfones of poly(ethylene glycol), WO 95/13312
A1).
[0316] Another activated form of PEG that is useful in accordance
with the present invention, is a PEG-N-hydroxysuccinimide ester
compound, for example, methoxy PEG-N-hydroxysuccinimidyl (NHS)
ester.
[0317] Heterobifunctionally activated forms of PEG are also useful.
(See, e.g., Thompson et al., PEGylation reagents and biologically
active compounds formed therewith, U.S. Pat. No. 6,552,170).
[0318] In still other embodiments of the inventive method of
producing a composition of matter, the toxin peptide analog is
reacted by known chemical techniques with an activated PEG
compound, such as but not limited to, a thiol-activated PEG
compound, a diol-activated PEG compound, a PEG-hydrazide compound,
a PEG-oxyamine compound, or a PEG-bromoacetyl compound. (See, e.g.,
S. Herman, Poly(ethylene glycol) with Reactive Endgroups: I.
Modification of Proteins, J. Bioactive and Compatible Polymers,
10:145-187 (1995); S. Zalipsky, Chemistry of Polyethylene Glycol
Conjugates with Biologically Active Molecules, Advanced Drug
Delivery Reviews, 16:157-182 (1995); R. Greenwald et al.,
Poly(ethylene glycol) conjugated drugs and prodrugs: a
comprehensive review, Critical Reviews in Therapeutic Drug Carrier
Systems, 17:101-161 (2000)).
[0319] An even more preferred activated PEG for generating the
PEG-conjugated toxin peptide analogs of the present invention is a
multivalent PEG having more than one activated residues. Preferred
multivalent PEG moieties include, but are not limited to, those
shown below:
##STR00001## ##STR00002##
[0320] In still other embodiments of making the composition of
matter, the inventive toxin peptide analog is reacted by known
chemical techniques with an activated multi-branched PEG compound
(PEG derivatives comprising multiple arms to give divalent,
trivalent, tetravalent, octavalent constructs), such as but not
limited to, pentaerythritol tetra-polyethyleneglycol ether.
Functionalization and activated derivatives, such as, but not
limited to, N-succinimidyloxycarbonyl)propyl,
p-nitrophenyloxycarbonyl, (--CO.sub.2-p-C.sub.6H.sub.4NO.sub.2),
3-(N-maleimido)propanamido, 2-sulfanylethyl, and 3-aminopropyl.
Using a 4-arm PEG derivative, four toxin peptide analogs are
attached to each PEG molecule. For example, in accordance with the
present invention, the toxin peptide analog can be conjugated to a
polyethylene glycol (PEG) at:
(a) 1, 2, 3 or 4 amino functionalized sites of the PEG; (b) 1, 2, 3
or 4 thiol functionalized sites of the PEG; (c) 1, 2, 3 or 4
maleimido functionalized sites of the PEG; (d) 1, 2, 3 or 4
N-succinimidyl functionalized sites of the PEG; (e) 1, 2, 3 or 4
carboxyl functionalized sites of the PEG; or (f) 1, 2, 3 or 4
p-nitrophenyloxycarbonyl functionalized sites of the PEG.
[0321] The smallest practical size of PEG is about 500 Daltons
(Da), below which PEG becomes toxic. Above about 500 Da, any
molecular mass for a PEG can be used as practically desired, e.g.,
from about 1,000 Daltons (Da) to 100,000 Da (n is 20 to 2300). The
number of PEG monomers (n) is approximated from the average
molecular mass using a MW=44 Da for each monomer. It is preferred
that the combined molecular mass of PEG on an activated linker is
suitable for pharmaceutical use. Thus, the combined molecular mass
of the PEG molecule should not exceed about 100,000 Da.
[0322] In some embodiments, the combined or total average molecular
mass of PEG used in a PEG-conjugated toxin peptide analog of the
present invention is from about 3,000 Da to 60,000 Da (total n is
from 70 to 1,400), more preferably from about 10,000 Da to 40,000
Da (total n is about 230 to about 910). The most preferred combined
mass for PEG is from about 20,000 Da to 30,000 Da (total n is about
450 to about 680).
[0323] It will be appreciated that "multimers" of the composition
of matter can be made, since the half-life extending moiety
employed for conjugation to the toxin peptide analog (with or
without an intervening linker moiety) can be multivalent (e.g.,
bivalent, trivalent, tetravalent or a higher order valency) as to
the number of amino acid residues at which the half-life extending
moiety can be conjugated. In some embodiments the peptide portion
of the inventive composition of matter can be multivalent (e.g.,
bivalent, trivalent, tetravalent or a higher order valency), and,
thus, some "multimers" of the inventive composition of matter may
have more that one half life extending moiety. Consequently, it is
possible by the inventive method of producing a composition of
matter to produce a variety of conjugated half-life extending
moiety:peptide structures. By way of example, a univalent half-life
extending moiety and a univalent peptide will produce a 1:1
conjugate; a bivalent peptide and a univalent half-life extending
moiety may form conjugates wherein the peptide conjugates bear two
half-life extending moiety moieties, whereas a bivalent half-life
extending moiety and a univalent peptide may produce species where
two peptide entities are linked to a single half-life extending
moiety; use of higher-valence half-life extending moiety can lead
to the formation of clusters of peptide entities bound to a single
half-life extending moiety, whereas higher-valence peptides may
become encrusted with a plurality of half-life extending moiety
moieties. By way of further example, if the site of conjugation of
a multivalent half-life extending moiety to the toxin peptide
analog is a cysteine or other aminothiol the methods disclosed by
D'Amico et al. may be employed (D'Amico et al., Method of
conjugating aminothiol containing molecules to vehicles, published
as US 2006/0199812, which application is incorporated herein by
reference in its entirety).
[0324] The peptide moieties may have more than one reactive group
which will react with the activated half-life extending moiety and
the possibility of forming complex structures must always be
considered; when it is desired to form simple structures such as
1:1 adducts of half-life extending moiety and peptide, or to use
bivalent half-life extending moiety to form peptide:half-life
extending moiety:peptide adducts, it will be beneficial to use
predetermined ratios of activated half-life extending moiety and
peptide material, predetermined concentrations thereof and to
conduct the reaction under predetermined conditions (such as
duration, temperature, pH, etc.) so as to form a proportion of the
described product and then to separate the described product from
the other reaction products. The reaction conditions, proportions
and concentrations of the reagents can be obtained by relatively
simple trial-and-error experiments which are within the ability of
an ordinarily skilled artisan with appropriate scaling-up as
necessary. Purification and separation of the products is similarly
achieved by conventional techniques well known to those skilled in
the art.
[0325] Additionally, physiologically acceptable salts of the
half-life extending moiety-fused or conjugated to the toxin peptide
analogs of this invention are also encompassed within the
composition of matter of the present invention.
[0326] The above-described half-life extending moieties and other
half-life extending moieties described herein are useful, either
individually or in combination, and as further described in the
art, for example, in Sullivan et al., Toxin Peptide Therapeutic
Agents, US2007/0071764 and Sullivan et al., Toxin Peptide
Therapeutic Agents, PCT/US2007/022831, published as WO 2008/088422,
which are both incorporated herein by reference in their
entireties. The invention encompasses the use of any single species
of pharmaceutically acceptable half-life extending moiety, such as,
but not limited to, those described herein, in conjugation with the
toxin peptide analog, or the use of a combination of two or more
like or different half-life extending moieties.
[0327] Linkers. A "linker moiety" as used herein refers to a
biologically acceptable peptidyl or non-peptidyl organic group that
is covalently bound to an amino acid residue of a toxin peptide
analog or other polypeptide chain (e.g., an immunoglobulin HC or LC
or immunoglobulin Fc domain) contained in the inventive
composition, which linker moiety covalently joins or conjugates the
toxin peptide analog or other polypeptide chain to another peptide
or polypeptide chain in the composition, or to a half-life
extending moiety. In some embodiments of the composition, a
half-life extending moiety, as described herein, is conjugated,
i.e., covalently bound directly to an amino acid residue of the
toxin peptide analog itself, or optionally, to a peptidyl or
non-peptidyl linker moiety (including but not limited to aromatic
or aryl linkers) that is covalently bound to an amino acid residue
of the toxin peptide analog. The presence of any linker moiety is
optional. When present, its chemical structure is not critical,
since it serves primarily as a spacer to position, join, connect,
or optimize presentation or position of one functional moiety in
relation to one or more other functional moieties of a molecule of
the inventive composition. The presence of a linker moiety can be
useful in optimizing pharamcologial activity of some embodiments of
the inventive composition. The linker is preferably made up of
amino acids linked together by peptide bonds. The linker moiety, if
present, can be independently the same or different from any other
linker, or linkers, that may be present in the inventive
composition.
[0328] As stated above, the linker moiety, if present (whether
within the primary amino acid sequence of the toxin peptide analog,
or as a linker for attaching a half-life extending moiety to the
toxin peptide analog), can be "peptidyl" in nature (i.e., made up
of amino acids linked together by peptide bonds) and made up in
length, preferably, of from 1 up to about 40 amino acid residues,
more preferably, of from 1 up to about 20 amino acid residues, and
most preferably of from 1 to about 10 amino acid residues.
Preferably, but not necessarily, the amino acid residues in the
linker are from among the twenty canonical amino acids, more
preferably, cysteine, glycine, alanine, proline, asparagine,
glutamine, and/or serine. Even more preferably, a peptidyl linker
is made up of a majority of amino acids that are sterically
unhindered, such as glycine, serine, and alanine linked by a
peptide bond. It is also desirable that, if present, a peptidyl
linker be selected that avoids rapid proteolytic turnover in
circulation in vivo. Some of these amino acids may be glycosylated,
as is well understood by those in the art. For example, a useful
linker sequence constituting a sialylation site is
X.sub.1X.sub.2NX.sub.4X.sub.5G (SEQ ID NO:279), wherein X.sub.1,
X.sub.2,X.sub.4 and X.sub.5 are each independently any amino acid
residue.
[0329] In other embodiments, the 1 to 40 amino acids of the
peptidyl linker moiety are selected from glycine, alanine, proline,
asparagine, glutamine, and lysine. Preferably, a linker is made up
of a majority of amino acids that are sterically unhindered, such
as glycine and alanine Thus, preferred linkers include
polyglycines, polyserines, and polyalanines, or combinations of any
of these. Some exemplary peptidyl linkers are poly(Gly).sub.1-8,
particularly (Gly).sub.3, (Gly).sub.4 (SEQ ID NO:280), (Gly).sub.5
(SEQ ID NO:281) and (Gly) (SEQ ID NO:282), as well as,
poly(Gly).sub.4Ser (SEQ ID NO:283), poly(Gly-Ala).sub.2-4 and
poly(Ala).sub.1-8. Other specific examples of peptidyl linkers
include (Gly).sub.5Lys (SEQ ID NO:284), and (Gly).sub.5LysArg (SEQ
ID NO:285). Other examples of useful peptidyl linkers are: Other
examples of useful peptidyl linkers are:
TABLE-US-00006 (Gly).sub.3Lys(Gly).sub.4; (SEQ ID NO: 286)
(Gly).sub.3AsnGlySer(Gly).sub.2; (SEQ ID NO: 287)
(Gly).sub.3Cys(Gly).sub.4; (SEQ ID NO: 288) and GlyProAsnGlyGly.
(SEQ ID NO: 289)
[0330] To explain the above nomenclature, for example,
(Gly).sub.3Lys(Gly).sub.4 means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly
(SEQ ID NO:290). Other combinations of Gly and Ala are also
useful.
[0331] Other preferred linkers are those identified herein as "L5"
(GGGGS; or "G.sub.45"; SEQ ID NO:291), "L10" (GGGGSGGGGS; SEQ ID
NO:292), "L25" (GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO:293) and any
linkers used in the working examples hereinafter.
[0332] In some embodiments of the compositions of this invention,
which comprise a peptide linker moiety, acidic residues, for
example, glutamate or aspartate residues, are placed in the amino
acid sequence of the linker moiety. Examples include the following
peptide linker sequences
TABLE-US-00007 GGEGGG; (SEQ ID NO: 294) GGEEEGGG; (SEQ ID NO: 295)
GEEEG; (SEQ ID NO: 296) GEEE; (SEQ ID NO: 297) GGDGGG; (SEQ ID NO:
298) GGDDDGG; (SEQ ID NO: 299) GDDDG; (SEQ ID NO: 300) GDDD; (SEQ
ID NO: 301) GGGGSDDSDEGSDGEDGGGGS; (SEQ ID NO: 302) WEWEW; (SEQ ID
NO: 303) FEFEF; (SEQ ID NO: 304) EEEWWW; (SEQ ID NO: 305) EEEFFF;
(SEQ ID NO: 306) WWEEEWW; (SEQ ID NO: 307) or FFEEEFF. (SEQ ID NO:
308)
[0333] In other embodiments, the linker constitutes a
phosphorylation site, e.g., X.sub.1X.sub.2YX.sub.4X.sub.5G (SEQ ID
NO:309), wherein X.sub.1, X.sub.2, X.sub.4, and X.sub.5 are each
independently any amino acid residue;
X.sub.1X.sub.2SX.sub.4X.sub.5G (SEQ ID NO:310), wherein X.sub.1,
X.sub.2,X.sub.4 and X.sub.5 are each independently any amino acid
residue; or X.sub.1X.sub.2TX.sub.4X.sub.5G (SEQ ID NO:311), wherein
X.sub.1, X.sub.2, X.sub.4 and X.sub.5 are each independently any
amino acid residue.
[0334] The linkers shown here are exemplary; peptidyl linkers
within the scope of this invention may be much longer and may
include other residues. A peptidyl linker can contain, e.g., a
cysteine, another thiol, or nucleophile for conjugation with a
half-life extending moiety. In another embodiment, the linker
contains a cysteine or homocysteine residue, or other
2-amino-ethanethiol or 3-amino-propanethiol moiety for conjugation
to maleimide, iodoacetaamide or thioester, functionalized half-life
extending moiety.
[0335] Another useful peptidyl linker is a large, flexible linker
comprising a random Gly/Ser/Thr sequence, for example:
GSGSATGGSGSTASSGSGSATH (SEQ ID NO:312) or HGSGSATGGSGSTASSGSGSAT
(SEQ ID NO:313), that is estimated to be about the size of a 1 kDa
PEG molecule. Alternatively, a useful peptidyl linker may be
comprised of amino acid sequences known in the art to form rigid
helical structures (e.g., Rigid linker: -AEAAAKEAAAKEAAAKAGG-//SEQ
ID NO:314). Additionally, a peptidyl linker can also comprise a
non-peptidyl segment such as a 6 carbon aliphatic molecule of the
formula
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--. The
peptidyl linkers can be altered to form derivatives as described
herein.
[0336] Optionally, a non-peptidyl linker moiety is also useful for
conjugating the half-life extending moiety to the peptide portion
of the half-life extending moiety-conjugated toxin peptide analog.
For example, alkyl linkers such as --NH--(CH.sub.2).sub.s--C(O)--,
wherein s=2-20 can be used. These alkyl linkers may further be
substituted by any non-sterically hindering group such as lower
alkyl (e.g., C.sub.1-C.sub.6) lower acyl, halogen (e.g., Cl, Br),
CN, NH.sub.2, phenyl, etc. Exemplary non-peptidyl linkers are PEG
linkers (e.g., shown below):
##STR00003##
wherein n is such that the linker has a molecular weight of about
100 to about 5000 Daltons (Da), preferably about 100 to about 500
Da.
[0337] In one embodiment, the non-peptidyl linker is aryl. The
linkers may be altered to form derivatives in the same manner as
described herein. In addition, PEG moieties may be attached to the
N-terminal amine or selected side chain amines by either reductive
alkylation using PEG aldehydes or acylation using
hydroxysuccinimido or carbonate esters of PEG, or by thiol
conjugation.
[0338] "Aryl" is phenyl or phenyl vicinally-fused with a saturated,
partially-saturated, or unsaturated 3-, 4-, or 5 membered carbon
bridge, the phenyl or bridge being substituted by 0, 1, 2 or 3
substituents selected from C.sub.1-8 alkyl, C.sub.1-4 haloalkyl or
halo.
[0339] "Heteroaryl" is an unsaturated 5, 6 or 7 membered monocyclic
or partially-saturated or unsaturated 6-, 7-, 8-, 9-, 10- or 11
membered bicyclic ring, wherein at least one ring is unsaturated,
the monocyclic and the bicyclic rings containing 1, 2, 3 or 4 atoms
selected from N, O and S, wherein the ring is substituted by 0, 1,
2 or 3 substituents selected from C.sub.1-8 alkyl, C.sub.1-4
haloalkyl and halo.
[0340] Non-peptide portions of the inventive composition of matter,
such as non-peptidyl linkers or non-peptide half-life extending
moieties can be synthesized by conventional organic chemistry
reactions.
[0341] The above is merely illustrative and not an exhaustive
treatment of the kinds of linkers that can optionally be employed
in accordance with the present invention.
[0342] Compositions of this invention incorporating peptide
antagonists of the voltage-gated potassium channel Kv1.3, in
particular toxin peptide analogs of the present invention, whether
or not conjugated to a half-life extending moiety, are useful as
immunosuppressive agents with therapeutic value for autoimmune
diseases. For example, such molecules are useful in treating
multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel
disease, and rheumatoid arthritis. (See, e.g., H. Wulff et al.
(2003) J. Clin. Invest. 111, 1703-1713 and H. Rus et al. (2005)
PNAS102, 11094-11099; Beeton et al., Targeting effector memory T
cells with a selective inhibitor peptide of Kv1.3 channels for
therapy of autoimmune diseases, Molec. Pharmacol. 67(4):1369-81
(2005);1 Beeton et al. (2006), Kv1.3: therapeutic target for
cell-mediated autoimmune disease, electronic preprint at
//webfiles.uci.edu/xythoswfs/webui/2670029.1). Inhibitors of the
voltage-gated potassium channel Kv1.3 have been examined in a
variety of preclinical animal models of inflammation. Small
molecule and peptide inhibitors of Kv1.3 have been shown to block
delayed type hypersensitivity responses to ovalbumin [C. Beeton et
al. (2005) Mol. Pharmacol. 67, 1369] and tetanus toxoid [G. C. Koo
et al. (1999) Clin. Immunol. 197, 99]. In addition to suppressing
inflammation in the skin, inhibitors also reduced antibody
production [G. C. Koo et al. (1997) J. Immunol. 158, 5120]. Kv1.3
antagonists have shown efficacy in a rat adoptive-transfer
experimental autoimmune encephalomyelitis (AT-EAE) model of
multiple sclerosis (MS), which model can be employed in assessing
the therapeutic efficacy of the inventive compositions of matter in
practicing the inventive method of preventing or mitigating a
relapse of a symptom of multiple sclerosis or method of treating an
autoimmune disorder. (See, C. Beeton et al., J. Immunol. 166:936
(2001); C. Beeton et al., PNAS 98: 13942 (2001); Sullivan et al.,
Toxin Peptide Therapeutic Agents, US2007/0071764, FIG. 61, all of
which are incorporated herein by reference in their entireties).
The Kv1.3 channel is overexpressed on myelin-specific T cells from
MS patients, lending further support to the utility Kv1.3
inhibitors may provide in treating MS. Inflammatory bone resorption
was also suppressed by Kv1.3 inhibitors in a preclinical
adoptive-transfer model of periodontal disease [P. Valverde et al.
(2004) J. Bone Mineral Res. 19, 155]. In this study, inhibitors
additionally blocked antibody production to a bacterial outer
membrane protein,--one component of the bacteria used to induce
gingival inflammation. Recently in preclinical rat models, efficacy
of Kv1.3 inhibitors was shown in treating pristane-induced
arthritis and diabetes [C. Beeton et al. (2006) preprint available
at //webfiles.uci.edu/xythoswfs/webui/_xy-2670029.sub.--1.]. The
Kv1.3 channel is expressed on all subsets of T cells and B cells,
but effector memory T cells and class-switched memory B cells are
particularly dependent on Kv1.3 [H. Wulff et al. (2004) J. Immunol.
173, 776]. GadS/insulin-specific T cells from patients with new
onset type 1 diabetes, myelin-specific T cells from MS patients and
T cells from the synovium of rheumatoid arthritis patients all
overexpress Kv1.3 [C. Beeton et al. (2006) preprint at
//webfiles.uci.edu/xythoswfs/webui/_xy-2670029.sub.--1.]. Because
mice deficient in Kv1.3 gained less weight when placed on a high
fat diet [J. Xu et al. (2003) Human Mol. Genet. 12, 551] and showed
altered glucose utilization [J. Xu et al. (2004) Proc. Natl. Acad.
Sci. 101, 3112], Kv1.3 is also being investigated for the treatment
of obesity and diabetes. Breast cancer specimens [M. Abdul et al.
(2003) Anticancer Res. 23, 3347] and prostate cancer cell lines [S.
P. Fraser et al. (2003) Pflugers Arch. 446, 559] have also been
shown to express Kv1.3, and Kv1.3 blockade may be of utility for
treatment of cancer. Disorders that can be treated with the
inventive compositions of matter, involving Kv1.3 inhibitor toxin
peptide analog(s), include multiple sclerosis, type 1 diabetes,
psoriasis, inflammatory bowel disease, contact-mediated dermatitis,
rheumatoid arthritis, psoriatic arthritis, asthma, allergy,
restinosis, systemic sclerosis, fibrosis, scleroderma,
glomerulonephritis, Sjogren syndrome, inflammatory bone resorption,
transplant rejection, graft-versus-host disease, and systemic lupus
erythematosus (SLE) and other forms of lupus.
[0343] The practice of the inventive method of treating an
autoimmune disorder involves administering to a patient, e.g., one
who has been diagnosed with an autoimmune disorder, such as, but
not limited to, multiple sclerosis, type 1 diabetes, psoriasis,
inflammatory bowel disease (IBD, including Crohn's Disease and
ulcerative colitis), contact-mediated dermatitis, rheumatoid
arthritis, psoriatic arthritis, asthma, allergy, restinosis,
systemic sclerosis, fibrosis, scleroderma, glomerulonephritis,
Sjogren syndrome, inflammatory bone resorption, transplant
rejection, graft-versus-host disease, or lupus, a therapeutically
effective amount of the inventive composition of matter, such that
at least one symptom of the disorder is alleviated in the
patient.
[0344] The practice of the inventive method of preventing or
mitigating a relapse of a symptom of multiple sclerosis involves
administering to a patient, e.g., one who has previously
experienced at least one symptom of multiple sclerosis, a
prophylactically effective amount of the inventive composition of
matter, such that the at least one symptom of multiple sclerosis is
prevented from recurring or is mitigated.
[0345] The diseases and pharmacologically active compositions
described herein are merely exemplary and in no way limit the range
of inventive pharmacologically active compounds, compositions, and
medicaments that can be prepared using the inventive compositions
of matter or the diseases and disorders that can be treated with
the benefit of the present invention.
[0346] Accordingly, the present invention also relates to the use
of one or more of the inventive compositions of matter in the
manufacture of a medicament for the treatment or prevention of a
disease, disorder, or other medical condition described herein.
[0347] Such pharmaceutical compositions can be configured for
administration to a patient by a wide variety of delivery routes,
e.g., an intravascular delivery route such as by injection or
infusion, subcutaneous, intramuscular, intraperitoneal, epidural,
or intrathecal delivery routes, or for oral, enteral, pulmonary
(e.g., inhalant), intranasal, transmucosal (e.g., sublingual
administration), transdermal or other delivery routes and/or forms
of administration known in the art. The inventive pharmaceutical
compositions may be prepared in liquid form, or may be in dried
powder form, such as lyophilized form. For oral or enteral use, the
pharmaceutical compositions can be configured, for example, as
tablets, troches, lozenges, aqueous or oily suspensions,
dispersible powders or granules, emulsions, hard or soft capsules,
syrups, elixirs or enteral formulas.
[0348] Pharmaceutical Compositions
[0349] In General. The present invention also provides
pharmaceutical compositions comprising the inventive composition of
matter and a pharmaceutically acceptable carrier. Such
pharmaceutical compositions can be configured for administration to
a patient by a wide variety of delivery routes, e.g., an
intravascular delivery route such as by injection or infusion,
subcutaneous, intramuscular, intraperitoneal, epidural, or
intrathecal delivery routes, or for oral, enteral, pulmonary (e.g.,
inhalant), intranasal, transmucosal (e.g., sublingual
administration), transdermal or other delivery routes and/or forms
of administration known in the art. The inventive pharmaceutical
compositions may be prepared in liquid form, or may be in dried
powder form, such as lyophilized form. For oral or enteral use, the
pharmaceutical compositions can be configured, for example, as
tablets, troches, lozenges, aqueous or oily suspensions,
dispersible powders or granules, emulsions, hard or soft capsules,
syrups, elixirs or enteral formulas.
[0350] In the practice of this invention the "pharmaceutically
acceptable carrier" is any physiologically tolerated substance
known to those of ordinary skill in the art useful in formulating
pharmaceutical compositions, including, any pharmaceutically
acceptable diluents, excipients, dispersants, binders, fillers,
glidants, anti-frictional agents, compression aids,
tablet-disintegrating agents (disintegrants), suspending agents,
lubricants, flavorants, odorants, sweeteners, permeation or
penetration enhancers, preservatives, surfactants, solubilizers,
emulsifiers, thickeners, adjuvants, dyes, coatings, encapsulating
material(s), and/or other additives singly or in combination. Such
pharmaceutical compositions can include diluents of various buffer
content (e.g., Tris-HCl, acetate, phosphate), pH and ionic
strength; additives such as detergents and solubilizing agents
(e.g., Tween.RTM. 80, Polysorbate 80), anti-oxidants (e.g.,
ascorbic acid, sodium metabisulfite), preservatives (e.g.,
Thimersol.RTM., benzyl alcohol) and bulking substances (e.g.,
lactose, mannitol); incorporation of the material into particulate
preparations of polymeric compounds such as polylactic acid,
polyglycolic acid, etc. or into liposomes. Hyaluronic acid can also
be used, and this can have the effect of promoting sustained
duration in the circulation. Such compositions can influence the
physical state, stability, rate of in vivo release, and rate of in
vivo clearance of the present proteins and derivatives. See, e.g.,
Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack
Publishing Co., Easton, Pa. 18042) pages 1435-1712, which are
herein incorporated by reference in their entirety. The
compositions can be prepared in liquid form, or can be in dried
powder, such as lyophilized form. Implantable sustained release
formulations are also useful, as are transdermal or transmucosal
formulations. Additionally (or alternatively), the present
invention provides compositions for use in any of the various slow
or sustained release formulations or microparticle formulations
known to the skilled artisan, for example, sustained release
microparticle formulations, which can be administered via
pulmonary, intranasal, or subcutaneous delivery routes. (See, e.g.,
Murthy et al., Injectable compositions for the controlled delivery
of pharmacologically active compound, U.S. Pat. No. 6,887,487;
Manning et al., Solubilization of pharmaceutical substances in an
organic solvent and preparation of pharmaceutical powders using the
same, U.S. Pat. Nos. 5,770,559 and 5,981,474; Lieberman et al.,
Lipophilic complexes of pharmacologically active inorganic mineral
acid esters of organic compounds, U.S. Pat. No. 5,002,936; Gen,
Formative agent of protein complex, US 2002/0119946 A1; Goldenberg
et al., Sustained release formulations, WO 2005/105057 A1).
[0351] One can dilute the inventive compositions or increase the
volume of the pharmaceutical compositions of the invention with an
inert material. Such diluents can include carbohydrates,
especially, mannitol, .alpha.-lactose, anhydrous lactose,
cellulose, sucrose, modified dextrans and starch. Certain inorganic
salts may also be used as fillers, including calcium triphosphate,
magnesium carbonate and sodium chloride. Some commercially
available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and
Avicell.
[0352] A variety of conventional thickeners are useful in creams,
ointments, suppository and gel configurations of the pharmaceutical
composition, such as, but not limited to, alginate, xanthan gum, or
petrolatum, may also be employed in such configurations of the
pharmaceutical composition of the present invention. A permeation
or penetration enhancer, such as polyethylene glycol monolaurate,
dimethyl sulfoxide, N-vinyl-2-pyrrolidone,
N-(2-hydroxyethyl)-pyrrolidone, or 3-hydroxy-N-methyl-2-pyrrolidone
can also be employed. Useful techniques for producing hydrogel
matrices are known. (E.g., Feijen, Biodegradable hydrogel matrices
for the controlled release of pharmacologically active agents, U.S.
Pat. No. 4,925,677; Shah et al., Biodegradable pH/thermosensitive
hydrogels for sustained delivery of biologically active agents, WO
00/38651 A1). Such biodegradable gel matrices can be formed, for
example, by crosslinking a proteinaceous component and a
polysaccharide or mucopolysaccharide component, then loading with
the inventive composition of matter to be delivered.
[0353] Liquid pharmaceutical compositions of the present invention
that are sterile solutions or suspensions can be administered to a
patient by injection, for example, intramuscularly, intrathecally,
epidurally, intravascularly (e.g., intravenously or
intraarterially), intraperitoneally or subcutaneously. (See, e.g.,
Goldenberg et al., Suspensions for the sustained release of
proteins, U.S. Pat. No. 6,245,740 and WO 00/38652 A1). Sterile
solutions can also be administered by intravenous infusion. The
inventive composition can be included in a sterile solid
pharmaceutical composition, such as a lyophilized powder, which can
be dissolved or suspended at a convenient time before
administration to a patient using sterile water, saline, buffered
saline or other appropriate sterile injectable medium.
[0354] Implantable sustained release formulations are also useful
embodiments of the inventive pharmaceutical compositions. For
example, the pharmaceutically acceptable carrier, being a
biodegradable matrix implanted within the body or under the skin of
a human or non-human vertebrate, can be a hydrogel similar to those
described above. Alternatively, it may be formed from a
poly-alpha-amino acid component. (Sidman, Biodegradable,
implantable drug delivery device, and process for preparing and
using same, U.S. Pat. No. 4,351,337). Other techniques for making
implants for delivery of drugs are also known and useful in
accordance with the present invention.
[0355] In powder forms, the pharmaceutically acceptable carrier is
a finely divided solid, which is in admixture with finely divided
active ingredient(s), including the inventive composition. For
example, in some embodiments, a powder form is useful when the
pharmaceutical composition is configured as an inhalant. (See,
e.g., Zeng et al., Method of preparing dry powder inhalation
compositions, WO 2004/017918; Trunk et al., Salts of the CGRP
antagonist BIBN4096 and inhalable powdered medicaments containing
them, U.S. Pat. No. 6,900,317).
[0356] One can dilute or increase the volume of the compound of the
invention with an inert material. These diluents could include
carbohydrates, especially mannitol, .alpha.-lactose, anhydrous
lactose, cellulose, sucrose, modified dextrans and starch. Certain
inorganic salts can also be used as fillers including calcium
triphosphate, magnesium carbonate and sodium chloride. Some
commercially available diluents are Fast-Flo.TM., Emdex.TM.,
STA-Rx.TM.1500, Emcompress.TM. and Avicell.TM.
[0357] Disintegrants can be included in the formulation of the
pharmaceutical composition into a solid dosage form. Materials used
as disintegrants include but are not limited to starch including
the commercial disintegrant based on starch, Explotab.TM.. Sodium
starch glycolate, Amberlite.TM., sodium carboxymethylcellulose,
ultramylopectin, sodium alginate, gelatin, orange peel, acid
carboxymethyl cellulose, natural sponge and bentonite can all be
used. Insoluble cationic exchange resin is another form of
disintegrant. Powdered gums can be used as disintegrants and as
binders and these can include powdered gums such as agar, Karaya or
tragacanth. Alginic acid and its sodium salt are also useful as
disintegrants.
[0358] Binders can be used to hold the therapeutic agent together
to form a hard tablet and include materials from natural products
such as acacia, tragacanth, starch and gelatin. Others include
methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl
cellulose (CMC). Polyvinyl pyrrolidone (PVP) and
hydroxypropylmethyl cellulose (HPMC) could both be used in
alcoholic solutions to granulate the therapeutic.
[0359] An antifrictional agent can be included in the formulation
of the therapeutic to prevent sticking during the formulation
process. Lubricants can be used as a layer between the therapeutic
and the die wall, and these can include but are not limited to;
stearic acid including its magnesium and calcium salts,
polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and
waxes. Soluble lubricants can also be used such as sodium lauryl
sulfate, magnesium lauryl sulfate, polyethylene glycol of various
molecular weights, Carbowax 4000 and 6000.
[0360] Glidants that might improve the flow properties of the drug
during formulation and to aid rearrangement during compression
might be added. The glidants can include starch, talc, pyrogenic
silica and hydrated silicoaluminate.
[0361] To aid dissolution of the compound of this invention into
the aqueous environment a surfactant might be added as a wetting
agent. Surfactants can include anionic detergents such as sodium
lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium
sulfonate. Cationic detergents might be used and could include
benzalkonium chloride or benzethonium chloride. The list of
potential nonionic detergents that could be included in the
formulation as surfactants are lauromacrogol 400, polyoxyl 40
stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,
glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty
acid ester, methyl cellulose and carboxymethyl cellulose. These
surfactants could be present in the formulation of the protein or
derivative either alone or as a mixture in different ratios.
[0362] Oral dosage forms. Also useful are oral dosage forms of the
inventive compositionss. If necessary, the composition can be
chemically modified so that oral delivery is efficacious.
Generally, the chemical modification contemplated is the attachment
of at least one moiety to the molecule itself, where said moiety
permits (a) inhibition of proteolysis; and (b) uptake into the
blood stream from the stomach or intestine. Also desired is the
increase in overall stability of the compound and increase in
circulation time in the body. Moieties useful as covalently
attached half-life extending moieties in this invention can also be
used for this purpose. Examples of such moieties include: PEG,
copolymers of ethylene glycol and propylene glycol, carboxymethyl
cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and
polyproline. See, for example, Abuchowski and Davis (1981), Soluble
Polymer-Enzyme Adducts, Enzymes as Drugs (Hocenberg and Roberts,
eds.), Wiley-Interscience, New York, N.Y., pp 367-83; Newmark, et
al. (1982), J. Appl. Biochem. 4:185-9. Other polymers that could be
used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for
pharmaceutical usage, as indicated above, are PEG moieties.
[0363] For oral delivery dosage forms, it is also possible to use a
salt of a modified aliphatic amino acid, such as sodium
N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to
enhance absorption of the therapeutic compounds of this invention.
The clinical efficacy of a heparin formulation using SNAC has been
demonstrated in a Phase II trial conducted by Emisphere
Technologies. See U.S. Pat. No. 5,792,451, "Oral drug delivery
composition and methods."
[0364] In one embodiment, the pharmaceutically acceptable carrier
can be a liquid and the pharmaceutical composition is prepared in
the form of a solution, suspension, emulsion, syrup, elixir or
pressurized composition. The active ingredient(s) (e.g., the
inventive composition of matter) can be dissolved, diluted or
suspended in a pharmaceutically acceptable liquid carrier such as
water, an organic solvent, a mixture of both, or pharmaceutically
acceptable oils or fats. The liquid carrier can contain other
suitable pharmaceutical additives such as detergents and/or
solubilizers (e.g., Tween 80, Polysorbate 80), emulsifiers, buffers
at appropriate pH (e.g., Tris-HCl, acetate, phosphate), adjuvants,
anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),
preservatives (e.g., Thimersol, benzyl alcohol), sweeteners,
flavoring agents, suspending agents, thickening agents, bulking
substances (e.g., lactose, mannitol), colors, viscosity regulators,
stabilizers, electrolytes, osmolutes or osmo-regulators. Additives
can also be included in the formulation to enhance uptake of the
inventive composition. Additives potentially having this property
are for instance the fatty acids oleic acid, linoleic acid and
linolenic acid.
[0365] Useful are oral solid dosage forms, which are described
generally in Remington's Pharmaceutical Sciences (1990), supra, in
Chapter 89, which is hereby incorporated by reference in its
entirety. Solid dosage forms include tablets, capsules, pills,
troches or lozenges, cachets or pellets. Also, liposomal or
proteinoid encapsulation can be used to formulate the present
compositions (as, for example, proteinoid microspheres reported in
U.S. Pat. No. 4,925,673). Liposomal encapsulation can be used and
the liposomes can be derivatized with various polymers (e.g., U.S.
Pat. No. 5,013,556). A description of possible solid dosage forms
for the therapeutic is given in Marshall, K., Modern Pharmaceutics
(1979), edited by G. S. Banker and C. T. Rhodes, in Chapter 10,
which is hereby incorporated by reference in its entirety. In
general, the formulation will include the inventive compound, and
inert ingredients that allow for protection against the stomach
environment, and release of the biologically active material in the
intestine.
[0366] The composition of this invention can be included in the
formulation as fine multiparticulates in the form of granules or
pellets of particle size about 1 mm. The formulation of the
material for capsule administration could also be as a powder,
lightly compressed plugs or even as tablets. The therapeutic could
be prepared by compression.
[0367] Colorants and flavoring agents can all be included. For
example, the protein (or derivative) can be formulated (such as by
liposome or microsphere encapsulation) and then further contained
within an edible product, such as a refrigerated beverage
containing colorants and flavoring agents.
[0368] In tablet form, the active ingredient(s) are mixed with a
pharmaceutically acceptable carrier having the necessary
compression properties in suitable proportions and compacted in the
shape and size desired.
[0369] The powders and tablets preferably contain up to 99% of the
active ingredient(s). Suitable solid carriers include, for example,
calcium phosphate, magnesium stearate, talc, sugars, lactose,
dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low
melting waxes and ion exchange resins.
[0370] Controlled release formulation can be desirable. The
composition of this invention can be incorporated into an inert
matrix that permits release by either diffusion or leaching
mechanisms e.g., gums. Slowly degenerating matrices can also be
incorporated into the formulation, e.g., alginates,
polysaccharides. Another form of a controlled release of the
compositions of this invention is by a method based on the Oros.TM.
therapeutic system (Alza Corp.), i.e., the drug is enclosed in a
semipermeable membrane which allows water to enter and push drug
out through a single small opening due to osmotic effects. Some
enteric coatings also have a delayed release effect.
[0371] Other coatings can be used for the formulation. These
include a variety of sugars that could be applied in a coating pan.
The therapeutic agent could also be given in a film-coated tablet
and the materials used in this instance are divided into 2 groups.
The first are the nonenteric materials and include methylcellulose,
ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose,
sodium carboxymethyl cellulose, providone and the polyethylene
glycols. The second group consists of the enteric materials that
are commonly esters of phthalic acid.
[0372] A mix of materials might be used to provide the optimum film
coating. Film coating can be carried out in a pan coater or in a
fluidized bed or by compression coating.
[0373] Pulmonary delivery forms. Pulmonary delivery of the
inventive compositions is also useful. The protein (or derivative)
is delivered to the lungs of a mammal while inhaling and traverses
across the lung epithelial lining to the blood stream. (Other
reports of this include Adjei et al., Pharma. Res. (1990) 7: 565-9;
Adjei et al. (1990), Internatl. J. Pharmaceutics 63: 135-44
(leuprolide acetate); Braquet et al. (1989), J. Cardiovasc.
Pharmacol. 13 (suppl.5): s.143-146 (endothelin-1); Hubbard et al.
(1989), Annals Int. Med. 3: 206-12 (a 1-antitrypsin); Smith et al.
(1989), J. Clin. Invest. 84: 1145-6 (.alpha.1-proteinase); Oswein
et al. (March 1990), "Aerosolization of Proteins," Proc. Symp.
Resp. Drug Delivery II, Keystone, Colo. (recombinant human growth
hormone); Debs et al. (1988), J. Immunol. 140: 3482-8
(interferon-.gamma. and tumor necrosis factor a) and Platz et al.,
U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).
Useful in the practice of this invention are a wide range of
mechanical devices designed for pulmonary delivery of therapeutic
products, including but not limited to nebulizers, metered dose
inhalers, and powder inhalers, all of which are familiar to those
skilled in the art. Some specific examples of commercially
available devices suitable for the practice of this invention are
the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St.
Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest
Medical Products, Englewood, Colo.; the Ventolin metered dose
inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.;
and the Spinhaler powder inhaler, manufactured by Fisons Corp.,
Bedford, Mass. (See, e.g., Helgesson et al., Inhalation device,
U.S. Pat. No. 6,892,728; McDerment et al., Dry powder inhaler, WO
02/11801 A1; Ohki et al., Inhalant medicator, U.S. Pat. No.
6,273,086).
[0374] All such devices require the use of formulations suitable
for the dispensing of the inventive compound. Typically, each
formulation is specific to the type of device employed and can
involve the use of an appropriate propellant material, in addition
to diluents, adjuvants and/or carriers useful in therapy.
[0375] The inventive compound should most advantageously be
prepared in particulate form with an average particle size of less
than 10 .mu.m (or microns), most preferably 0.5 to 5 .mu.m, for
most effective delivery to the distal lung.
[0376] Pharmaceutically acceptable excipients include carbohydrates
such as trehalose, mannitol, xylitol, sucrose, lactose, and
sorbitol. Other ingredients for use in formulations can include
DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants can be
used. PEG can be used (even apart from its use in derivatizing the
protein or analog). Dextrans, such as cyclodextran, can be used.
Bile salts and other related enhancers can be used. Cellulose and
cellulose derivatives can be used. Amino acids can be used, such as
use in a buffer formulation.
[0377] Also, the use of liposomes, microcapsules or microspheres,
inclusion complexes, or other types of carriers is
contemplated.
[0378] Formulations suitable for use with a nebulizer, either jet
or ultrasonic, will typically comprise the inventive compound
dissolved in water at a concentration of about 0.1 to 25 mg of
biologically active protein per mL of solution. The formulation can
also include a buffer and a simple sugar (e.g., for protein
stabilization and regulation of osmotic pressure). The nebulizer
formulation can also contain a surfactant, to reduce or prevent
surface induced aggregation of the protein caused by atomization of
the solution in forming the aerosol.
[0379] Formulations for use with a metered-dose inhaler device will
generally comprise a finely divided powder containing the inventive
compound suspended in a propellant with the aid of a surfactant.
The propellant can be any conventional material employed for this
purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a
hydrofluorocarbon, or a hydrocarbon, including
trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or
combinations thereof. Suitable surfactants include sorbitan
trioleate and soya lecithin. Oleic acid can also be useful as a
surfactant. (See, e.g., Backstrom et al., Aerosol drug formulations
containing hydrofluoroalkanes and alkyl saccharides, U.S. Pat. No.
6,932,962).
[0380] Formulations for dispensing from a powder inhaler device
will comprise a finely divided dry powder containing the inventive
compound and can also include a bulking agent, such as lactose,
sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which
facilitate dispersal of the powder from the device, e.g., 50 to 90%
by weight of the formulation.
[0381] Nasal delivery forms. In accordance with the present
invention, intranasal delivery of the inventive composition of
matter and/or pharmaceutical compositions is also useful, which
allows passage thereof to the blood stream directly after
administration to the inside of the nose, without the necessity for
deposition of the product in the lung. Formulations suitable for
intransal administration include those with dextran or
cyclodextran, and intranasal delivery devices are known. (See, e.g,
Freezer, Inhaler, U.S. Pat. No. 4,083,368).
[0382] Transdermal and transmucosal (e.g., buccal) delivery forms).
In some embodiments, the inventive composition is configured as a
part of a pharmaceutically acceptable transdermal or transmucosal
patch or a troche. Transdermal patch drug delivery systems, for
example, matrix type transdermal patches, are known and useful for
practicing some embodiments of the present pharmaceutical
compositions. (E.g., Chien et al., Transdermal estrogen/progestin
dosage unit, system and process, U.S. Pat. Nos. 4,906,169 and
5,023,084; Cleary et al., Diffusion matrix for transdermal drug
administration and transdermal drug delivery devices including
same, U.S. Pat. No. 4,911,916; Teillaud et al., EVA-based
transdermal matrix system for the administration of an estrogen
and/or a progestogen, U.S. Pat. No. 5,605,702; Venkateshwaran et
al., Transdermal drug delivery matrix for coadministering estradiol
and another steroid, U.S. Pat. No. 5,783,208; Ebert et al., Methods
for providing testosterone and optionally estrogen replacement
therapy to women, U.S. Pat. No. 5,460,820). A variety of
pharmaceutically acceptable systems for transmucosal delivery of
therapeutic agents are also known in the art and are compatible
with the practice of the present invention. (E.g., Heiber et al.,
Transmucosal delivery of macromolecular drugs, U.S. Pat. Nos.
5,346,701 and 5,516,523; Longenecker et al., Transmembrane
formulations for drug administration, U.S. Pat. No. 4,994,439).
[0383] Buccal delivery of the inventive compositions is also
useful. Buccal delivery formulations are known in the art for use
with peptides. For example, known tablet or patch systems
configured for drug delivery through the oral mucosa (e.g.,
sublingual mucosa), include some embodiments that comprise an inner
layer containing the drug, a permeation enhancer, such as a bile
salt or fusidate, and a hydrophilic polymer, such as hydroxypropyl
cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose,
dextran, pectin, polyvinyl pyrrolidone, starch, gelatin, or any
number of other polymers known to be useful for this purpose. This
inner layer can have one surface adapted to contact and adhere to
the moist mucosal tissue of the oral cavity and can have an
opposing surface adhering to an overlying non-adhesive inert layer.
Optionally, such a transmucosal delivery system can be in the form
of a bilayer tablet, in which the inner layer also contains
additional binding agents, flavoring agents, or fillers. Some
useful systems employ a non-ionic detergent along with a permeation
enhancer. Transmucosal delivery devices may be in free form, such
as a cream, gel, or ointment, or may comprise a determinate form
such as a tablet, patch or troche. For example, delivery of the
inventive composition can be via a transmucosal delivery system
comprising a laminated composite of, for example, an adhesive
layer, a backing layer, a permeable membrane defining a reservoir
containing the inventive composition, a peel seal disc underlying
the membrane, one or more heat seals, and a removable release
liner. (E.g., Ebert et al., Transdermal delivery system with
adhesive overlay and peel seal disc, U.S. Pat. No. 5,662,925; Chang
et al., Device for administering an active agent to the skin or
mucosa, U.S. Pat. Nos. 4,849,224 and 4,983,395). These examples are
merely illustrative of available transmucosal drug delivery
technology and are not limiting of the present invention.
[0384] Dosages. The dosage regimen involved in a method for
treating the above-described conditions will be determined by the
attending physician, considering various factors which modify the
action of drugs, e.g. the age, condition, body weight, sex and diet
of the patient, the severity of any infection, time of
administration and other clinical factors. Generally, the daily
regimen should be in the range of 0.1-1000 micrograms of the
inventive compound per kilogram of body weight, preferably 0.1-150
micrograms per kilogram.
[0385] The following working examples are illustrative and not to
be construed in any way as limiting the scope of the present
invention.
EXAMPLES
Example 1
Kv1.3 and Kv1.1 Electrophysiology
[0386] Cell lines expressing Kv1.1 through Kv1.7. CHO-K1 cells were
stably transfected with human Kv1.3, or for counterscreens (see,
Example 6), with hKv1.4, hKv1.6, or hKv1.7; HEK293 cells were
stably expressing human Kv1.3 or with human Kv1.1. Cell lines were
from Amgen or BioFocus DPI (A Galapagos Company). CHO K1 cells
stably expressing hKv1.2, for counterscreens, were purchased from
Millipore (Cat#.CYL3015).
[0387] Whole cell patch clamp electrophysiology. Whole-cell
currents were recorded at room temperature using MultiClamp 700B
amplifier from Molecular Devices Corp. (Sunnyvale, Calif.), with
3-5M.OMEGA. pipettes pulled from borosilicate glass (World
Precision Instruments, Inc). During data acquisition, capacitive
currents were canceled by analogue subtraction, no series
resistance compensation was used, and all currents were filtered at
2 kHz. The cells were bathed in an extracellular solution
containing 1.8 mM CaCl.sub.2, 5 mM KCl, 135 mM NaCl, 5 mM Glucose,
10 mM HEPES, pH 7.4, 290-300 mOsm. The internal solution containing
90 mM KCl, 40 mM KF, 10 mM NaCl, 1 mM MgCl.sub.2, 10 mM EGTA, 10 mM
HEPES, pH 7.2, 290-300 mOsm. The currents were evoked by applying
depolarizing voltage steps from -80 mV to +30 mV every 30 s (Kv1.3)
or 10 s (Kv1.1) for 200 ms intervals at holding potential of -80
mV. To determine IC50, 5-6 peptide or peptide conjugate
concentration at 1:3 dilutions were made in extracellular solution
with 0.1% BSA and delivered locally to cells with Rapid Solution
Changer RSc-160 (BioLogic Science Instruments). Currents were
achieved to steady state for each concentration. Data analysis was
performed using pCLAMP (version 9.2) and OriginPro (version 7), and
peak currents before and after each test article application were
used to calculate the percentage of current inhibition at each
concentration.
[0388] PatchXpress.RTM., planar patch-clamp electrophysiology.
Cells were bathed in an extracellular solution containing 1.8 mM
CaCl.sub.2, 5 mM KCl, 135 mM NaCl, 5 mM Glucose, 10 mM HEPES, pH
7.4, 290-300 mOsm. The internal solution contained 90 mM KCl, 40 mM
KF, 10 mM NaCl, 1 mM MgCl.sub.2, 10 mM EGTA, 10 mM HEPES, pH 7.2,
290-300 mOsm. Usually 5 peptide or peptide conjugate concentrations
at 1:3 dilutions are made to determine the IC50s. The peptide or
peptide conjugates are prepared in extracellular solution
containing 0.1% BSA. Dendrotoxin-k and Margatoxin were purchased
from Alomone Labs Ltd. (Jerusalem, Israel); ShK toxin was purchased
from Bachem Bioscience, Inc. (King of Prussia, Pa.); 4-AP was
purchased from Sigma-Aldrich Corp. (St. Louis, Mo.). Currents were
recorded at room temperature using a PatchXpress.RTM. 7000A
electrophysiology system from Molecular Devices Corp. (Sunnyvale,
Calif.). The voltage protocols for hKv1.3 and hKv1.1 are shown in
Table 4E in Example 6 below. An extracellular solution with 0.1%
BSA was applied first to obtain 100% percent of control (POC), then
followed by 5 different concentrations of 1:3 peptide or peptide
conjugate dilutions for every 400 ms incubation time. At the end,
excess of a specific benchmark ion channel inhibitor (Table 4E in
Example 6) was added to define full or 100% blockage. The residual
current present after addition of benchmark inhibitor, was used in
some cases for calculation of zero percent of control. The
benchmark inhibitors for Kv1.3 and Kv1.1 are described in Table 4E
in Example 6. Each individual set of traces or trial were visually
inspected and either accepted or rejected. The general criteria for
acceptance were:
[0389] 1. Baseline current must be stable
[0390] 2. Initial peak current must be >300 pA
[0391] 3. Intitial Rm and final Rm must >300 Ohm
[0392] 4. Peak current must achieve a steady-state prior to first
compound addition.
[0393] The POC was calculated from the average peak current of the
last 5 sweeps before the next concentration compound addition and
exported to Excel for IC50 calculation.
[0394] IonWorks, high-throughput, planar patch-clamp
electrophysiology. Electrophysiology was performed on CHO cells
stably expressing hKv1.3 and HEK293 cells stably expressing hKv1.1.
The procedure for preparation of the "Assay Plate" containing ShK
analogues and conjugates for IWQ electrophysiology was as follows:
all analogues were dissolved in extracellular buffer (PBS, with 0.9
mM Ca.sup.2+ and 0.5 mM Mg.sup.2+) with 0.3% BSA and dispensed in
the row H of 96-well polypropylene plates at the concentration of
100 nM from column 1 to column 10. Column 11 and 12 were reserved
for negative and positive controls, then serial diluted at 1:3
ratio to row A. IonWorks Quattro (IWQ) electrophysiology and data
analysis were accomplished as follows: re-suspended cells (in
extracellular buffer), the Assay Plate, a Population Patch Clamp
(PPC) PatchPlate as well as appropriate intracellular (90 mM
potassium gluconate, 20 mM KF, 2 mM NaCl, 1 mM MgCl.sub.2, 10 mM
EGTA, 10 mM HEPES, pH 7.35) and extracellular buffers were
positioned on IonWorks Quattro. When the analogues were added to
patch plates, they were further diluted 3-fold from the assay plate
to achieve a final test concentration range from 33.3 nM to 15
.mu.M with 0.1% BSA. Electrophysiology recordings were made from
the CHO-Kv1.3 and HEK-Kv1.1 cells using an amphotericin-based
perforated patch-clamp method. Using the voltage-clamp circuitry of
the IonWorks Quattro, cells were held at a membrane potential of
-80 mV and voltage-activated K+ currents were evoked by stepping
the membrane potential to +30 mV for 400 ms. K+ currents were
evoked under control conditions i.e., in the absence of inhibitor
at the beginning of the experiment and after 10-minute incubation
in the presence of the analogues and controls. The mean K+ current
amplitude was measured between 430 and 440 ms and the data were
exported to a Microsoft Excel spreadsheet. The amplitude of the K+
current in the presence of each concentration of the analogues and
controls was expressed as a percentage of the K+ current of the
pre-compound current amplitude in the same well. When these % of
control values were plotted as a function of concentration, the
IC50 value for each compound could be calculated using the
dose-response fit model 201 in Excel fit program which utilizes the
following equation:
% of control = y min + ( y max - y min 1 + ( conc . IC 50 ) n )
##EQU00001##
where ymin is the minimum y-value of the curve, ymax is the maximum
y-value of the curve, conc. is the test concentration and n is the
Hill slope of the curve.
Example 2
Measuring Bioactivity in Human Whole Blood
[0395] Ex vivo assay to examine impact of toxin peptide analog
Kv1.3 inhibitors on secretion of IL-2 and IFN-g. The potency of ShK
analogs and conjugates in blocking T cell inflammation in human
whole blood was examined using an ex vivo assay that has been
described earlier (see Example 46 of WO 2008/088422 A2,
incorporated herein by reference in its entirety). In brief, 50%
human whole blood is stimulated with thapsigargin to induce store
depletion, calcium mobilization and cytokine secretion. To assess
the potency of molecules in blocking T cell cytokine secretion,
various concentrations of Kv1.3 blocking peptides and
peptide-conjugates were pre-incubated with the human whole blood
sample for 30-60 min prior to addition of the thapsigargin
stimulus. After 48 hours at 37.degree. C., 5% CO.sub.2, conditioned
medium was collected and the level of cytokine secretion was
determined using a 4-spot electrochemilluminescent immunoassay from
MesoScale Discovery. Using the thapsigargin stimulus, the cytokines
IL-2 and IFN-g were secreted robustly from blood isolated from
multiple donors. The IL-2 and IFN-g produced in human whole blood
following thapsigargin stimulation were produced from T cells, as
revealed by intracellular cytokine staining and
fluorescence-activated cell sorting (FACS) analysis.
[0396] Kv1.3 is the major voltage-gated potassium channel present
on T cells. Allowing for K.sup.+ efflux, Kv1.3 provides the driving
force for continued Cat.sup.2+ influx which is necessary for the
sustained elevation in intracellular calcium needed for efficient T
cell activation and cytokine secretion. Kv1.3 inhibitors have been
shown earlier to suppress this calcium flux induced by TCR ligation
(G. C. Koo et al., 1999, Cell. Immunol. 197, 99-107).
Thapsigargin-induced store-depletion and TCR ligation elicits
similar patterns of Ca.sup.2+ mobilization in isolated T cells (E.
Donnadieu et al., 1991, J. Biol. Chem. 267, 25864-25872), but we
have found thapsigargin gives a more robust response in whole
blood. Therefore, we developed a bioassay whereby the bioactivity
of Kv1.3 inhibitors is assessed by examining their ability to block
thapsigargin-induced cytokine secretion from T cells in human whole
blood. Since whole blood is a complex fluid containing high protein
levels, the activity of peptides and peptide conjugates in this
whole blood assay has an additional advantage in assessing the
molecules stability over 48 hours in a biologically relevant fluid.
The whole blood assay provides important confirmation of the Kv1.3
potency of molecules determined by electrophysiology (ePhys), since
ePhys assays are generally of short duration (<1-2 hours) and
use physiological saline containing no protein. The longer duration
of the whole blood assay may allow for more effective determination
of equilibrium binding kinetics relative to ePhys studies which are
of short duration.
[0397] For example, ShK-Dap22 (SEQ ID NO:317) is a Lys22 analog of
ShK, reported earlier to have improved Kv1.3 versus Kv1.1
selectivity (Kalman et al., ShK-Dap22, a potent Kv1.3-specific
immunosuppressive polypeptide, J. Biol. Chem. 273(49):32697-707
(1998)). The ShK-Dap22 molecule was reported to have similar
potency to native ShK and to provide potent blockade of Kv1.3 with
an IC50 of about 23 pM as measured by whole cell patch clamp
electrophysiology (K. Kalman et al., 1998, ibid). We also found by
whole cell patch clamp electrophysiology that ShK-Dap22 (purchased
from Bachem) potently blocks Kv1.3 with an IC50 of about 12 pM
(see, Example 3, Example 5, and Table 4E, below), but find it is
about 300 times less potent in blocking IL-2 production from human
whole blood (IC50 of about 3763 pM). In contrast, native ShK is
about equipotent in blocking Kv1.3 and IL-2 whole blood responses.
Consistent with our whole blood findings that ShK-Dap22 has reduced
potency, R. E. Middleton et al. (Biochemistry 42, 13698-13707,
2004) report by radioligand binding competition that ShK-Dap22 has
a about 700-fold weaker equilibrium binding affinity for Kv1.3
compared to native ShK. Therefore, it appears that patch-clamp
electrophysiology alone sometimes fails to reveal a toxin peptide
analog's true potency or affinity, and alternative methods should
be employed to decipher whether any significant changes have
occurred in the toxin peptide analog's equilibrium binding
affinity.
[0398] For this reason, we developed a parallel screening approach
that tested all toxin peptide analogs and conjugates for potency in
two distinct assays: (a) an electrophysiology assay to measure
impact on Kv1.3 or Kv1.1, and (b) the 48 hour human whole blood
assay described herein, which we believe measures equilibrium
binding. Using the later assay, a toxin peptide analog PEGylated at
its N-terminus with linear PEG, 20 kDa-PEG-[Lys16]ShK (SEQ ID
NO:16; see, Example 4, below) was found to provide potent blockade
of whole blood IL-2 secretion with an IC50 of 0.092 nM (n=14) and
an IC95 of 1.013 nM (n=14). The bioactivity of toxin peptide
analogs and conjugates in the whole blood assay of inflammation is
provided in other examples and tables disclosed herein.
Example 3
Toxin Peptide Analogs
[0399] All ShK amino acid residue positions except Cys residues
were each substituted at a single position with either an Ala, an
Arg, a Lys, a Glu, or a 1-Nal residue. Conventional solid phase
synthetic methods were employed to make and purify the toxin
peptides and toxin peptide analogs. (See, e.g., Example 4
hereunder). Kv1.3 and Kv1.1 activity were measured using
electrophysiology (IWQ) and an ex vivo whole blood assay (WB) to
examine impact of Kv1.3 inhibitor toxin peptide analogs on
secretion of IL-2 and IFN-.gamma.. Electrophysiology (IWQ) data and
human whole blood (WB) functional assay data were collected as
described in Example 1 and Example 2 and were analyzed.
Electrophysiology and whole blood assay results for the Ala, Arg,
Lys, Glu, or 1-Nal scans are described in Tables 6-10 below. We
found in the IWQ and WB assays that single residue changes of
specific ShK residues, in addition to the previously identified
position 22, can uniquely modify the Kv1.3 inhibition potency
and/or Kv1.3 selectivity (i.e., inhibition of Kv1.3 versus Kv1.1)
of the toxin peptide analogs. General conclusions are represented
in FIG. 3, which are supported by (a)-(c) below.
[0400] (a) Certain single substitution toxin peptide analogs at
various positions in relation to SEQ ID NO:4 were found to
significantly reduce Kv1.3 inhibition activity (i.e., IC50
increased) compared to the native ShK sequence (SEQ ID NO:1). These
included the following positions, with the amino acid residues that
reduced Kv1.3 activity in parentheses: Asp5 (K,R,E, Nal, [D5A not
done]), Ile7(K,R,E, Nal), Arg11 (A,E, Nal), Ser20(R,E, Nal),
Met21(K,R,E, Nal), Lys22(R,E, Nal), Tyr23(R,E, Nal, [Y23K not
done]), Phe27(K,R,A, Nal, [F27E not done]). These ShK residues (D5,
I7, R11, S20, M21, K22, Y23, F27) can be considered key binding
sites for Kv1.3 and were found to cluster to a single face of the
3-dimensional solution structure of ShK (FIG. 1C).
[0401] (b) Certain single substitution toxin peptide analogs at
various positions in relation to SEQ ID NO:4 were found to improve
Kv1.3 inhibition activity (i.e., IC50 decreased) compared to the
native ShK sequence (SEQ ID NO:1). These included the following
positions, with the amino acid residues that improved Kv1.3
activity in parentheses: Ser2(E), Ile4(K,E,A), Ser10(R,E),
Phe15(A), Lys18(R,A), Lys30(R,E), Thr31(Nal), Thr34(Nal).
[0402] (c) Certain single substitution toxin peptide analogs at
various positions in relation to SEQ ID NO:4 were found to improve
selectivity for Kv1.3 (versus Kv1.1; i.e. IC50 Kv1.1/IC50 Kv1.3
increased) compared to the native ShK sequence (SEQ ID NO:1), while
having no substantial effect on Kv1.3 inhibition activity. These
included the following positions, with the amino acid residues that
improved selectivity for Kv1.3 in parentheses: Ile7(K), Ser10(A),
Gln16(K, Nal), Ser20(K,R), Lys22(A), Tyr23(A, [Y23K not done]),
Ser26(Nal), Phe27(Nal [F27E not done]), Arg29(K, Nal). Analysis of
the atomic structure of ShK revealed that several key residues
improving Kv1.3 selectivity upon analoging, were located at the
periphery and surrounding those ShK residues critical for Kv1.3
binding, described above in part (a). These residues (17, S10, Q16,
S20, S26, R29) that, when converted to an analog, result in
improved Kv1.3 selectivity, are light shaded in FIG. 1D which shows
a space filling stereo model of the NMR structure of ShK. Analogs
of some residues critical for Kv1.3 binding, also improve
selectivity, such as Ile7(K), Lys22(A), Tyr23(A) and Phe27(1-Nal).
However for several of these analogs, this gain in selectivity is
sometimes accompanied by an unacceptably large loss in potency that
makes them undesirable.
[0403] The data indicated that amino acid residues at positions 8,
13, and 33, relative to SEQ ID NO:4 can be substituted with the
scanning residues (Arg, Lys, Ala, 1-Nal, and Glu) without a major
loss in Kv1.3 inhibition potency.
[0404] The same cannot be said for residue AspS, where Arg, Glu,
1-Nal, and lysine substitutions were not well tolerated. We
observed a major loss of Kv1.3 inhibition activity. (Substitution
of Ala at position 5 has yet to be assayed since this molecule is
very difficult to prepare.) Position 5 is quite sensitive, even the
change from Asp (short side chain acidic) to Glu (long side chain
acidic residue) was not well tolerated. From the entire multipass
ShK scan, we found the most intolerant residues of substitutions
were AspS (most intolerant), Ser20, Lys22 and Tyr23. ShK residues
Lys22 and Tyr23 have been reported earlier as important binding
sites for Kv1.3. Whereas Ala analogs of Lys22 and Tyr23 showed
reduced activity, all other analogs of these residues were
inactive. The Lys22(A) and Tyr23(A) analogs were 7-10 times less
active against Kv1.3 and 35-56 times less active in the human whole
blood assay of T cell inflammation compared to native ShK. The
ShK-Dap22 analog reported earlier to have improved Kv1.3
selectivity (K. Kalman et al., 1998, ibid), was 56 times less
active in this whole blood assay. Therefore, despite the slight
improvement in Kv1.3 selectivity conferred by Ala and
diaminoproprionic acid (Dap) analogs of Lys22 of ShK, the large
reduction in potency negated much of the benefit of the slight
selectivity improvement.
[0405] We confirmed that, in accordance with literature
observations, alanine substitution at position 16 was not found to
confer selectivity. (See, e.g., Kem et al., ShK toxin compositions
and methods of use, U.S. Pat. No. 6,077,680). However, when
position 16 was changed from the Gln residue in the native ShK
sequence (SEQ ID NO:1) to a basic lysine residue, high potency and
very good selectivity were obtained (Table 5 and Table 10).
Interestingly, the same result was not obtained with arginine
(Table 6), another basic residue, but compared to lysine the Arg
side chain is nearly 1 .ANG. longer. Furthermore, through its
guanido functionality, arginine imparts a bulky planar
configuration to the positive charge compared with the pyramidal
configuration in the primary amine. Good Kv1.3 selectivity was also
observed with the introduction of a large bulky neutral group, such
as 1-Naphthylalanine (1-Nal), at position 16 (Table 8). However,
the 1-Nal modification at position 16 did reduce the potency of the
molecule for Kv1.3 inhibition (>5-fold reduction) and in the
whole blood assay (about 325-500-fold reduction in activity). Kv1.3
selectivity was also observed from the introduction of 1-Nal at
positions 26 or 27 of ShK, although again like with 1-Nal at
position 16, these substitutions also led to a reduction in potency
in the whole blood assay.
[0406] Another observation we made based on the data in Table 11
(Position 16 toxin peptide analogs), was that improved Kv1.3
selectivity was obtained, without a substantial change in Kv1.3
activity measured by IWQ or WB assays, by changing the polar by
neutral Gln side chain in the native ShK sequence to a long basic
residue (e.g., Lys). Selectivity for Kv1.3 (compared to Kv1.1) was
gradually lost as the side chain was shortened by 1 methylene group
at a time (Lys>Orn>Dab>Dap). Interestingly, a His16
substitution (basic, aryl residue) failed to yield a selective
molecule. Improved Kv1.3 selectivity over Kv1.1 was obtained by
changing the polar by neutral Gln side chain to a large aromatic
(aryl) residue, Ahp. Other toxin peptide analogs such as [Ala23]ShK
and [Arg30]ShK also conferred Kv1.3 selectivity, albeit with
significantly less potency of inhibition at Kv1.3.
[0407] In the preparation of the toxin peptide analog molecules a
+16 Da side product was observed during the synthesis, folding, and
lyophilization processes. Given that the ShK peptide contains a
single methionine residue at position 21 (see SEQ ID NO:1) and this
sulfur containing residue is well-documented to oxidize to its
Met[O] form (+16 Da) it was desirable to replace the residue at
position 21 relative to SEQ ID NO:4 with another non-oxidizable
residue. A well known isosteric substitute for Met is norleucine
(Nle), where the isoteric side chain group consists of the same
number of atoms but differs by the substitution of the sulfur atom
(present in the Met residue) with a methylene (--CH.sub.2--) unit
in the Nle residue. The incorporation of Nle was reasonably well
tolerated at position 21, although Kv1.3 inhibition potency was
slightly reduced (Table 17). Position scans at residue 21 showed
that this position affects Kv1.3 potency and selectivity. Aside
from Nle substitution, Met[O], Asn, Gln, Tyr, Val, Leu, Abu, Chg,
Phe and Nva were reasonably well tolerated as determined by IWQ
assay. Of these changes Asn, Tyr, Val, Leu, Abu, Chg, Phe and Nva
still resulted in good selectivity for Kv1.3 over Kv1.1. In the
whole blood assay, [Lys16, Nle21]ShK-Ala (SEQ ID NO:236), [Lys16,
Met(0).sub.2-1]ShK-amide (SEQ ID NO:257), and [Lys16,
Nva21]ShK-amide (SEQ ID NO:260) showed more potency with IL-2
response. (Table 15 and Table 17).
[0408] To explore more ShK structure-activity and also avoid a
C-terminal cysteinyl carboxylate, we sought to identify more
appropriate C-terminal functionality. C-terminal cysteinyl
carboxylate may increase chemical synthetic difficulties related to
their preparation. We examined C-terminal amidation which involves
the changing of the C-terminus from a free carboxylate to a
carboxamide (Table 5 and Table 14). Changing [Lys16]ShK (SEQ ID
NO:13) to [Lys16]ShK-amide (SEQ ID NO:14) resulted in a molecule
with roughly equivalent activity in blocking Kv1.3 (IWQ IC50=0.2
nM) and WB IL-2 secretion (IC50=0.1 nM). However, combination of
the Lys16, Nle 21 double substitutions with C-terminal amidation
resulted in a molecule (SEQ ID NO:15) with improved Kv1.3
selectivity (over Kv1.1), that despite being equipotent in blocking
Kv1.3 (IWQ IC50=0.15 nM) had about 7-fold less activity in the WB
IL-2 assay of inflammation (IC50=0.82 nM) relative to [Lys16]ShK
(SEQ ID NO:13; IC50=0.11) (See Table 5 and Table 14).
Interestingly, [His16; Nle21]ShK-amide (SEQ ID NO:157; Table 11),
which substitutes Lys16 with the basic and larger histidine residue
at residue 16, is potent at Kv1.3 but does not show selectivity for
Kv1.3 (IWQ IC50=139 pM) over Kv1.1 (IWQ IC50=15 pM). Of the toxin
peptide analogs containing a C-terminal amide (Table 14), the
following analogs showed improved Kv1.3 selectivity and retained
high potency (IC50<270 pM) in blocking Kv1.3 and WB IL-2
secretion:
[Ala15, Lys16]ShK-amide (SEQ ID NO:186);
[Glu4, Lys16]ShK-amide (SEQ ID NO:188);
[Lys16, Glu30]ShK-amide (SEQ ID NO:190);
[Lys16, Glu18]ShK-amide (SEQ ID NO:195);
[Glu9, Lys16, Nle21]ShK-amide (SEQ ID NO:222);
[Lys16,29;Ala30]ShK-amide (SEQ ID NO:210);
[Lys16,29;Trp27]ShK-amide (SEQ ID NO:202);
[Glu4; Lys16, 29;Arg30]ShK-amide (SEQ ID NO:201);
[Glu4;Lys16,29;Arg18,30]ShK-amide (SEQ ID NO:212);
[Glu4,9;Lys16,29;Arg18,30]ShK-amide (SEQ ID NO:198);
[Glu4,10; Lys16,29; Arg18,30]ShK-amide (SEQ ID NO:199); and
[Glu4, 9,10; Lys16,29; Arg18,30]ShK-amide (SEQ ID NO:200).
[0409] In order to solve potential issues with C-terminal cysteinyl
carboxylate peptides, we explored the extension of the C-terminus
with additional residues. Unexpectedly, addition of a single
alanine residue following the C-terminal Cys35 residue of
[Lys16]ShK (SEQ ID NO:13), caused a significant further improvement
in Kv1.3 specificity over Kv1.1. As shown in Table 15 and Table 16,
extension of the C-terminus of ShK by addition of a non-Cys residue
allows for the reintroduction of the native C-terminal negative
charge and potentially increase potency. Of particular interest,
the Lys16 analog of ShK with an Ala residue added after the
C-terminal Cys35 residue (SEQ ID NO:235, Table 4H and Table 15),
retained Kv1.3 activity and showed a dramatic 262 fold improved
selectivity for Kv1.3 over neuronal Kv1.1 by PatchXpress (PX)
electrophysiology (Table 4H) and 158 fold improved selectivity by
IonWorks (IWQ) electrophysiology (Table 15). This [Lys16]ShK-Ala
(SEQ ID NO:235) toxin peptide analog provided potent blockade of
IL-2 secretion from human whole blood (IC50=138 pM, Table 4H) and
is one of the most Kv1.3 selective (over Kv1.1)
two-amino-acid-change analogs of ShK that we have identified. Other
amino acid analogs of ShK with a C-terminal extension that were
potent in the whole blood assay (IC50<500 pM in blocking IL-2
secretion) and had over 200 fold Kv1.3 versus Kv1.1 selectivity,
included (see Table 15):
[Lys16,Nle21]ShK-Ala (SEQ ID NO:236);
[Glu4,Lys16, Nva21]ShK-Ala (SEQ ID NO:242),
[Glu4,Lys16, Nle21]ShK-Ala (SEQ ID NO:243);
[Lys16, Nle21]ShK-Glu (SEQ ID NO:244); and
[Lys16, Nle21]ShK-Tyr (SEQ ID NO:245).
Example 4
Preparation of Peptides and PEGylated Peptides, including
N.alpha.-(20 kDa PEG)-ShK (SEQ ID NO:8), N.alpha.-(20 kDa
PEG)-[Lys16]ShK (SEQ ID NO: 16), N.alpha.-(20 kDa
PEG)-[Lys16]ShK-Ala (SEQ ID NO:316), and N.alpha.-brPEG-[Lys16]ShK
(SEQ ID NO:315)
[0410] Peptide Synthesis. N.sup..alpha.-Fmoc, side-chain protected
amino acids and H-Cys(Trt)-2Cl-Trt resin were purchased from
Novabiochem, Bachem, or Sigma Aldrich. The following side-chain
protection strategy was employed: Asp(OtBu), Arg(Pbf), Cys(Trt),
Glu(OtBu), His(Trt), Lys(N.sup..epsilon.-Boc), Ser(OtBu), Thr(OtBu)
and Tyr(OtBu). ShK (SEQ ID NO:1),
RSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC (SEQ ID NO:13), or other toxin
peptide analog amino acid sequences, were synthesized in a stepwise
manner on an CS Bio peptide synthesizer by SPPS using DIC/HOBt
coupling chemistry at 0.2 mmol equivalent scale using
H-Cys(Trt)-2Cl-Trt resin (0.2 mmol, 0.32 mmol/g loading). For each
coupling cycle, 1 mmol N.sup..alpha.-Fmoc-amino acid was dissolved
in 2.5 mL of 0.4 M 1-hydroxybenzotriazole (HOBt) in
N,N-dimethylformamide (DMF). To the solution was added 1.0 mL of
1.0 M N,N'-diisopropylcarbodiimide (DIC) in DMF. The solution was
agitated with nitrogen bubbling for 15 min to accomplish
pre-activation and then added to the resin. The mixture was shaken
for 2 h. The resin was filtered and washed three times with DMF,
twice with dichloromethane (DCM), and three times with DMF. Fmoc
deprotections were carried out by treatment with 20% piperidine in
DMF (5 mL, 2.times.15 min). The first 23 residues were single
coupled through repetition of the Fmoc-amino acid coupling and Fmoc
removal steps described above. The remaining residues were double
coupled by performing the coupling step twice before proceeding
with Fmoc-removal.
[0411] Following synthesis, the resin was then drained, and washed
sequentially with DCM, DMF, DCM, and then dried in vacuo. The
peptide-resin was transferred to a 250-mL plastic round bottom
flask. The peptide was deprotected and released from the resin by
treatment with triisopropylsilane (1.5 mL),
3,6-dioxa-1,8-octane-dithiol (DODT, 1.5 mL), water (1.5 mL),
trifluoroacetic acid (TFA, 20 mL), and a stir bar, and the mixture
was stirred for 3 h. The mixture was filtered through a 150-mL
sintered glass funnel into a 250-mL plastic round bottom flask. The
mixture was filtered through a 150-mL sintered glass funnel into a
250-mL plastic round bottom flask, and the filtrate was
concentrated in vacuo. The crude peptide was precipitated with the
addition of cold diethyl ether, collected by centrifugation, and
dried under vacuum.
[0412] Peptide Folding. The dry crude linear peptide (about 600
mg), for example [Lys16]ShK peptide (SEQ ID NO:13) or
[Lys16]ShK-Ala (also known as [Lys16, Ala36]-ShK; SEQ ID NO:235)
peptide, was dissolved in 16 mL acetic acid, 64 mL water, and 40 mL
acetonitrile. The mixture was stirred rapidly for 15 min to
complete dissolution. The peptide solution was added to a 2-L
plastic bottle that contained 1700 mL of water and a large stir
bar. To the thus diluted solution was added 20 mL of concentrated
ammonium hydroxide to raise the pH of the solution to 9.5. The pH
was adjusted with small amounts of acetic acid or NH.sub.4OH as
necessary. The solution was stirred at 80 rpm overnight and
monitored by LC-MS. Folding was usually judged to be complete in 24
to 48 h, and the solution was quenched by the addition of acetic
acid and TFA (pH=2.5). The aqueous solution was filtered (0.45
.mu.m cellulose membrane).
[0413] Reversed-Phase HPLC Purification. Reversed-phase
high-performance liquid chromatography was performed on an
analytical (C18, 5 .mu.m, 0.46 cm.times.25 cm) or a preparative
(C18, 10 .mu.m, 2.2 cm.times.25 cm) column. Chromatographic
separations were achieved using linear gradients of buffer B in A
(A=0.1% aqueous TFA; B=90% aq. ACN containing 0.09% TFA) typically
5-95% over 35 min at a flow rate of 1 mL/min for analytical
analysis and 5-65% over 90 min at 20 mL/min for preparative
separations. Analytical and preparative HPLC fractions were
characterized by ESMS and photodiode array (PDA) HPLC, combined and
lyophilized.
[0414] Mass Spectrometry. Mass spectra were acquired on a single
quadrupole mass spectrometer equipped with an Ionspray atmospheric
pressure ionization source. Samples (25 .mu.L) were injected into a
moving solvent (10 .mu.L/min; 30:50:20 ACN/MeOH containing 0.05%
TFA) coupled directly to the ionization source via a fused silica
capillary interface (50 .mu.m i.d.). Sample droplets were ionized
at a positive potential of 5 kV and entered the analyzer through an
interface plate and subsequently through an orifice (100-120 .mu.m
diameter) at a potential of 60 V. Full scan mass spectra were
acquired over the mass range 400-2200 Da with a scan step size of
0.1 Da. Molecular masses were derived from the observed m/z
values.
[0415] PEGylation, Purification and Analysis. Peptide, e.g.,
[Lys16]ShK (SEQ ID NO:13) or [Lys16]ShK-Ala (SEQ ID NO:235), was
selectively PEGylated by reductive alkylation at its N-terminus,
using activated linear or branched PEG. Conjugation was performed
at 2 mg/ml in 50 mM NaH.sub.2PO.sub.4, pH 4.5 reaction buffer
containing 20 mM sodium cyanoborohydride and a 2 molar excess of 20
kDa monomethoxy-PEG-aldehyde (NOF, Japan). Conjugation reactions
were stirred for approximately 5 hrs at room temperature, and their
progress was monitored by RP-HPLC. Completed reactions were
quenched by 4-fold dilution with 20 mM NaOAc, pH 4 and chilled to
4.degree. C. The PEG-peptides were then purified
chromatographically at 40 C; using SP Sepharose HP columns (GE
Healthcare, Piscataway, N.J.) eluted with linear 0-1M NaCl
gradients in 20 mM NaOAc, pH 4.0. Eluted peak fractions were
analyzed by SDS-PAGE and RP-HPLC and pooling determined by purity
>97%. Principle contaminants observed were di-PEGylated toxin
peptide analog. Selected pools were concentrated to 2-5 mg/ml by
centrifugal filtration against 3 kDa MWCO membranes and dialyzed
into 10 mM NaOAc, pH 4 with 5% sorbitol. Dialyzed pools were then
sterile filtered through 0.2 micron filters and purity determined
to be >97% by SDS-PAGE (FIG. 5A, FIG. 5C, FIG. 5E) and RP-HPLC
(FIG. 5B, FIG. 5D, and FIG. 5F). Reverse-phase HPLC was performed
on an Agilent 1100 model HPLC running a Zorbax.RTM. 5 .mu.m
300SB-C8 4.6.times.50 mm column (Agilent) in 0.1% TFA/H.sub.2O at 1
ml/min and column temperature maintained at 40.degree. C. Samples
of PEG-peptide (20 .mu.g) were injected and eluted in a linear
6-60% gradient while monitoring wavelength 215 nm.
Example 5
N.alpha.-20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16) and other PEGylated
Toxin Peptide Analogs; Pharmacokinetics in Mammalian Species;
Safety Pharmacology Assay; and Isolated Rabbit Heart Assay
[0416] Conjugation of 20 kDa PEG greatly increased the exposure of
a toxin peptide in vivo, as demonstrated by a comparison of the
pharmacokinetics (PK) of ShK-L5 (SEQ ID NO:17) versus N.alpha.-(20
kDa PEG)-ShK (SEQ ID NO:8). (see FIG. 4). ShK-L5 is a potent
peptide inhibitor of Kv1.3 described in earlier publications.
(Beeton et al., Targeting effector memory T cells with a selective
peptide inhibitor of Kv1.3 channels for therapy of autoimmune
diseases, Molec. Pharmacol. 67(4):1369-81 (2005); Chandy et al.,
Analogs of ShK toxin and their uses in selective inhibition of
Kv1.3 potassium channels, WO 2006/042151 A2; Pennington et al.,
Engineering a stable and selective peptide blocker of the Kv1.3
channel in T lymphocytes, Molecular Pharmacology Fast Forward,
published Jan. 2, 2009 as doi:10.1124/mol.108.052704 (2009)).
ShK-L5 (SEQ ID NO:17) contains an N-terminal phophotyrosine moiety
attached to ShK via an 2-(2-(2-aminoethoxy)ethoxy)acetic acid
(AEEA) linker. Despite reports that this molecule has improved
selectivity for mouse Kv1.3 versus Kv1.1, in our hands we found the
ShK-L5 molecule blocked human Kv1.3 and Kv1.1 with about
equipotency (Table 4H, Table 5 and FIG. 6C-D). Whether the
differences in our findings versus those published, results from
differences in the channel species (mouse versus human) used or
differences in the cell lines, is unclear. More recently, M. W.
Pennington et al. described ShK-192 (SEQ ID NO:438) as a stable and
selective peptide blocker of the Kv1.3 channel; ShK-192 (SEQ ID
NO:438) was identified by Pennington et al. as a more stable analog
than ShK-L5 which showed pH-related hydrolysis and oxidation
byproducts that were exacerbated by increasing temperatures.
(Pennington et al., Engineering a stable and selective peptide
blocker of the Kv1.3 channel in T lymphocytes, Molec. Pharmacol.
75(4):762-73 (2009)). Based on this report, we synthesized and
tested ShK-192 for activity on human Kv1.3 and human Kv1.1. Whereas
our studies on the ShK-L5 molecule did not show increased
selectivity at Kv1.3 over Kv1.1 (FIGS. 6C and 6D), the newer
ShK-192 analog showed improved Kv1.3 selectivity (FIG. 29A)
relative to Kv1.1 (FIG. 29B). ShK-192 inhibited human Kv1.3 with an
IC50 of 0.039.+-.0.005 nM, which was about 87 times more potent
than its IC50 on human Kv1.1 (3.39.+-.1.61 nM). Whether the
improved Kv1.3 selectivity of ShK-192 relative to ShK-L5 is a
result of its improved stability, or is due to other reasons, is
unknown.
[0417] Regardless, we also found that ShK conjugated with a
N-terminal 20 kDa PEG moiety (SEQ ID NO:8) was Kv1.3 non-selective
and had roughly equivalent potency on neuronal Kv1.1 (Table 5),
despite being highly potent and having an extended half-life in
vivo. In contrast, [Lys16]ShK toxin peptide analog containing a
N-terminal 20 kDa PEG moiety (SEQ ID NO:16) had greatly improved
Kv1.3 selectivity (Table 5 and FIG. 6A-B), showing 1060 fold
greater activity against Kv1.3 versus Kv1.1 (Table 4H). In
addition, the 20 kDa-PEG-[Lys16]ShK conjugate (SEQ ID NO:16)
retained high potency (sub-nM IC50) in the human whole blood assay
of T-cell inflammation. FIG. 30A-B shows representative
dose-response curves from three lots of 20 kDa-PEG-[Lys16]ShK (SEQ
ID NO:16) for inhibition of IL-2 (FIG. 30A) and IFN.gamma. (FIG.
30B) secretion. The average IC50 of 20 kDa-PEG-[Lys16]ShK (all lots
tested) for inhibition of IL-2 and IFN.gamma. secretion from
thapsigargin stimulated T cells in human whole blood obtained from
multiple donors, was 0.109.+-.0.081 nM (n=34) and 0.240.+-.0.163 nM
(n=34), respectively. Cyclosporin A was about 2000-3000 less
active, inhibiting IL-2 and IFN.gamma. production with IC50 values
of 334.8.+-.172.2 nM (n=64) and 495.2.+-.307.8 nM (n=64),
respectively. Other than its potent activity in blocking T cell
responses in human whole blood, 20 kDa-PEG-[Lys16]ShK also potently
inhibited T cell IL-17 secretion from cynomolgus monkey whole blood
stimulated with thapsigargin (FIG. 31, Example 8) exhibiting an
average IC50 of 0.09 nM in experiments using blood collected from
seven different monkeys. Myelin-specific proliferation of the rat T
effector memory cell line, PAS, was also potently (IC50=0.17 nM)
blocked by the kDa-PEG-[Lys16]ShK molecule (FIG. 32, Example 9).
Therefore, 20 kDa-PEG-[Lys16]ShK was equally effective in
inhibiting T cell responses of three distinct species: human,
monkey and rat. Whereas, the [Lys16]ShK toxin peptide analog alone
(SEQ ID NO:13) showed modest Kv1.3 selectivity (Kv1.1/Kv1.3
IC.sub.50 ratio=15-fold), N-terminal conjugation of this peptide
with a 20 kDa PEG moiety further enhanced its selectivity, with the
conjugate having about 1000-fold selectivity for Kv1.3 over
neuronal Kv1.1. Further ion channel counterscreens revealed that
the novel 20 kDa-PEG-[Lys16]ShK conjugate was 680 fold selective
over Kv1.2, about 500-fold selective over Kv1.6 and >10000 fold
selective over Kv1.4, Kv1.5 and Kv1.7 (Table 4A). Importantly, the
conjugate did not impact ion channels that are known to serve a
role in human cardiac action potential, exhibiting >10000 fold
selectivity over Nav1.5, Cav1.2, Kv4.3, KvLQT1/minK and hERG (Table
4A, see Example 6). The conjugated toxin peptide analog 20
kDa-PEG-[Lys16]ShK (SEQ ID NO:16) also showed no impact on the
calcium-activated K.sup.+ channels, KCa3.1 (IKCa1) and BKCa.
[0418] To further characterize the pharmacology of the conjugate,
it was tested for ex vivo stability in plasma and pharmacokinetics
in vivo. The 20 kDa-PEG-[Lys16]ShK conjugate was stable in rat,
cyno and human plasma for 48 hours at 37.degree. C. (FIG. 10A-D;
see, Example 7). Pharmacokinetic studies in mouse, rat, dog and
cyno (FIG. 11A-B, see, Example 8) indicated the conjugate was
stable in vivo and had a prolonged half-life. As shown in FIG. 11C,
20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16) given as a single 0.5 mg/kg
subcutaneous dose to cynomolgus monkeys showed a prolonged
half-life, achieving a Cmax at 8 hrs of 254 nM (1031 ng/ml) and day
7 serum levels of 28.4 nM (115 ng/ml). The day 7 serum
concentration after this single dose was about 28 times greater
than the conjugate's IC.sub.95 (1.0 nM) in blocking inflammation in
human whole blood and about 315 times its IC.sub.50 (0.09 nM) in
this assay (performed as described in Example 2). Therefore, the
pharmacokinetics of PEG-[Lys16]ShK in cynos suggests weekly dosing
in humans is possible and that very small doses would be needed to
block inflammation. Further details on the pharmacokinetics of 20
kDa-PEG-[Lys16]ShK(SEQ ID NO:16) administered subcutaneously are
provided below and in Table 4B, Table 4C, Table 4D, and Table
4D(a), below.
[0419] The efficacy of 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16)
molecule in an animal model of multiple sclerosis was determined
using the adoptive-transfer (AT)-EAE model, as described in Example
9. PEG-[Lys16]ShK delayed disease onset and caused dose-dependent
reduction in disease severity (FIG. 7 and FIG. 8A-D). The molecule
was highly potent in this model and 4.4 .mu.g/kg/day of
PEG-[Lys16]ShK (SEQ ID NO:16) was estimated as the effective dose
causing 50% reduction in disease severity (ED50), based on EAE
score at day 7.
[0420] To further assess the pharmacology and safety of the 20
kDa-PEG-[Lys16]ShK molecule (SEQ ID NO:16) in vivo, a 12-week
pharmacology study was performed in cynomolgus monkeys (Example
10). The 12-week study involved three pre-dose baseline measure
over two weeks, weekly 0.5 mg/kg SC dosing for one month and six
weeks of follow-up analysis. Further details of the study are
provided in Example 10 and Table 4F, below. Based on earlier
pharmacokinetic studies on the 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16)
molecule weekly dosing at 0.5 mg/kg would provide excess target
coverage ranging from 28 times IC.sub.95 at Cmin to 249 times
IC.sub.95 at Cmax. The PEG-[Lys16]ShK conjugate (SEQ ID NO:16) was
well tolerated. The animals gained weight throughout the study
(FIG. 9D), and there were no changes in CBCs and blood chemistry
relative to pre-dose baseline measures (Table 4G, Example 10).
Using the cyno whole blood pharmacodynamic assay described in
Example 8, T cell inflammation was suppressed for the entire one
month dosing period and no changes were detected over time (FIG.
9A-B). Upon repeat dosing, there was good correlation between
predicted and observed serum drug trough levels (FIG. 9C) over the
one month dosing period. Foreign peptides or proteins can sometimes
be immunogenic and produce clearing or neutralizing antibodies that
can alter serum drug levels and/or pharmacodynamic coverage. Since
no such alterations were observed in cynomolgus monkeys after
repeat dosing with 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16), these data
imply that this PEG-conjugate did not produce neutralizing or
clearing antibodies in monkeys.
[0421] There are typically three reasons cited for peptides having
a short half-life in vivo: target-mediated clearance, rapid
proteolytic degradation or rapid renal clearance. For a given class
of peptides the reason for short half-life is often not obvious.
Although there are established methods for reducing renal clearance
(e.g., PEGylation, Fc fusion), these methods would be futile if the
peptide in question is either proteolytically susceptible (rapidly
hydrolysed) in vivo or cleared rapidly as a result of
target-mediated clearance or non-specific (charge) binding. In
addition, since only large PEG or Fc fusions reduce renal
clearance, it needs to be determined empirically whether a given
class of peptides retains potency following conjugation. Conjugates
that exhibit a significant loss in potency, erase any benefit
gained by improved in vivo half-life since the level of target
coverage in animals is directly dependent on both the molecules
potency and stability in vivo. For a new class of peptides, it is
not obvious that one can successfully form conjugates to a large
PEG or Fc moiety and retain activity. The reasons for this are that
distinct classes of peptides have distinct primary and tertiary
structures, whose biophysical stability can be significantly
altered upon conjugation. Any significant reduction in stability
(in vitro or in vivo) would reduce the molecule's activity and
eliminate its utility as a therapeutic.
[0422] The native ShK peptide (SEQ ID NO:1) is a potent Kv1.3/Kv1.1
inhibitor that has a short half-life in vivo (C. Beeton et al.,
2001, Proc. Natl. Acad. Sci. 98, 13942-13947). The major cause of
ShK's poor stability in vivo is unknown, with Norton et al.
(Current Medicinal Chemistry 11, 3041-3052, 2004) indicating more
detailed analyses of its absorption, distribution, metabolism and
excretion were necessary to identify the cause. To assess whether
proteolysis was the major cause, Beeton et al. (J. Biol. Chem. 283:
988-997, 2008) described an extensive effort to generate a
D-diastereomer analog of ShK containing D-amino acids in order to
limit proteolysis and improve circulating half-life. The D-allo-ShK
molecule was about 2800 times less active than native ShK in
blocking the Kv1.3 target. The authors reported that the D-allo
analog of ShK did not improve its half-life in vivo, suggesting
proteolysis did not explain the short half-life of ShK. To improve
the therapeutic potential of ShK, we generated novel PEG and Fc
conjugates of ShK and demonstrated they have prolonged half-life in
vivo (Example 8), and importantly remain highly Kv1.3 potent. Since
our molecules are highly potent against Kv1.3 and have prolonged
half-life, our data imply that Kv1.3 target-mediated clearance does
not explain the short half-life of the ShK peptide. In addition,
since it is unlikely our conjugates would protect against
proteolysis, our findings imply that proteolysis may not play a
role in ShK's short half-life in vivo. A reduction in renal
clearance may be the key reason our ShK conjugates have prolonged
half-life in vivo, but to determine this definitively, further
studies would be necessary to assess absorption, distribution,
metabolism and excretion of the conjugate.
[0423] In our efforts to identify Kv1.3 inhibitors with improved
therapeutic potential, we performed systematic multi-pass analoging
of ShK to identify analogs with improved Kv1.3 versus Kv1.1
selectivity, formed PEG or Fc-conjugates of the analogs and tested
each for potency, selectivity and stability in vivo. The
afore-mentioned [Lys16]ShK analog (single amino acid change of ShK
Gln16 to Lys16; SEQ ID NO:13) was highly potent and showed improved
selectivity, that was unexpectedly further enhanced upon
conjugation to PEG. Of particular importance, this conjugate
retained high potency and blocked Kv1.3 and T cell inflammation
with an IC50 in the picomolar range. Despite the success here,
several ShK analogs identified with improved Kv1.3 selectivity,
after conjugation with a 20 kDa PEG moiety showed too large a
reduction in potency to be therapeutically useful. Some examples
include 1-Nal16 (SEQ ID NO:11) and 1-Nal27 (SEQ ID NO:95) analogs
of ShK, that as free peptides showed sub nM potency in blocking
Kv1.3 and had improved Kv1.3/Kv1.1 selectivity (Table 8), but as
PEG conjugates showed about 57 times less activity (SEQ ID NO:159
and SEQ ID NO:160, respectively; Table 12). On the other hand, the
[Lys16]ShK-Ala toxin peptide analog (SEQ ID NO:235; see, Example 3)
was N-terminally conjugated with a 20 kDa PEG moiety. The
N.alpha.-20 kDa-PEG-[Lys16]ShK-Ala conjugate (SEQ ID NO:316)
retained high potency, blocked Kv1.3 and T cell inflammation with
sub-nM IC50 and exhibited >3500 fold selectivity for Kv1.3 over
neuronal Kv1.1 (Table 4H). This conjugate, with an N-terminal 20
kDa-PEG moiety and a C-terminal alanine addition, represents one of
the most potent and selective conjugates yet identified.
[0424] Conjugation of a peptide or protein with PEG molecules of
differing size (molecular weight) and form can significantly affect
the bioactivity, pharmacokinetics, distribution and metabolism of
the molecule. To explore the impact of alternative PEG forms other
than N.alpha.-20 kDa-PEG, the Kv1.3-selective [Lys16]ShK (SEQ ID
NO:13) peptide inhibitor of Kv1.3 was N-terminally conjugated with
either a 30 kDa PEG moiety (SEQ ID NO:158) or a branched PEG moiety
comprised of two 10 kDa PEG units (N.alpha.-brPEG-[Lys16]ShK; SEQ
ID NO:315). The purified brPEG-[Lys16]ShK molecule (SEQ ID NO:315)
was a potent blocker of T cell cytokine secretion from whole blood
(IC50=198 pM for blocking IL-2) and exhibited about 750-fold
selectivity for lymphocyte Kv1.3 over neuronal Kv1.1 (Table 4H).
The N.alpha.-30 kDa-PEG-[Lys16]ShK molecule (SEQ ID NO:158) was
also highly active with an IC50 of 282 pM in blocking
thapsigargin-stimulated IL-2 secretion from human whole blood
(Table 12). Given the N.alpha.-20 kDa PEG-[Lys16]ShK (SEQ ID NO:16)
peptide conjugate had an IC50 of about 100 pM in this same assay,
these data imply that the Kv1.3 selective [Lys16]ShK toxin peptide
analog (SEQ ID NO:13) tolerates and retains activity upon
N-terminal conjugation with PEG moieties of a variety of different
sizes and shapes.
[0425] Comparison of PK characteristics across mammalian species.
PEG-[Lys16]ShK (SEQ ID NO:16) preclinical pharmacokinetics
performed in mouse, rat, cynomolgus monkey and dog were generally
supportive of a weekly dosing regimen in humans. Details on the
pharmacokinetics of 20 kDa-PEG-[Lys16]ShK(SEQ ID NO:16)
administered subcutaneously are provided in Table 4B, Table 4C, and
Table 4D, below. The preclinical pharmacokinetics were generally
less favorable in rodents than non-rodent species when considering
parameters such as exposure, half life and bioavailability. Rat
pharmacokinetics were the least favorable of the four species that
were tested and should present an on-going challenge to using this
species effectively for experiments requiring consistent or
long-term exposure of PEG-[Lys16]ShK (SEQ ID NO:16) (e.g.,
pharmacology or toxicology). There is also a growing body of
evidence that rats exhibit a species specific sensitivity to mast
cell degranulation caused by dosing of PEG-[Lys16]ShK (SEQ ID
NO:16) (See, Example 10). The mast cell degranulation is
accompanied by the series of physical changes which may have an
effect on rat pharmacokinetics including drug adsorption,
distribution and clearance. (Example 10).
[0426] Preclinical pharmacokinetic parameters for PEG-[Lys16]ShK
(SEQ ID NO:16) administered intravenously are shown in Table 4D(a),
below. The doses for the intravenous (IV) experiments were 0.2
mg/kg for all species, while subcutaneous administration (SC) was
performed at either 0.5 or 2 mg/kg. In general, the pharmacokinetic
properties of PEG-[Lys16]ShK (SEQ ID NO:16) were more favorable in
dog and cynomolgus monkey when compared to the rodent species.
Volume of distribution at steady state (Vss) was small in the case
of mouse, cynomolgus monkey and dog. Rat was an outlier when
considering its Vss, which was approximately 3-4 times larger per
unit weight than the other three species. Both IV and SC PK
experiments indicated that exposure in rat was mediated by a large
initial distribution phase which was less pronounced in other
species. Terminal elimination half-lives measured after IV dosing
were 12.0 hr, 16.1 hr, 21.0 hr and 28.3 hr for rat, mouse,
cynomolgus monkey and dog, respectively, indicating delineation
between rodent and non-rodent species, concerning terminal
half-life. PEG-[Lys16]ShK (SEQ ID NO:16) was observed to exhibit
variable absorption across species upon subcutaneous dosing. Mouse
had a more rapid subcutaneous absorption than other species;
T.sub.max was obtained after 4 hrs, compared to mean T.sub.max
values from 8 hr to 40 hr for other species. Relative ranking of
the subcutaneous absorption rate constants (K.sub.a) following
subcutaneous injection were Mouse (0.244 h.sup.-1)>Cynomolgus
monkey (0.197 h.sup.-1)>Dog (0.191 h.sup.-1)>Rat (0.065
h.sup.-1).
[0427] In more detail, clearance values for PEG-[Lys16]ShK (SEQ ID
NO:16) measured after IV dosing (0.2 mg/kg) were 43.9, 8.2, 5.94
and 2.68 mL/hr/kg, for rat, mouse, cynomolgus cynomolgus monkey and
dog, respectively. These clearance values resulted in terminal
elimination half-lives of 12.0, 16.1, 21.0 and 28.3 hours,
respectively for PEG-[Lys16]ShK (SEQ ID NO:16). Clearance was
roughly proportional to glomerular filtration in these species and
provided further support to the expectation that renal filtration
is a main route of elimination for PEG-[Lys16]ShK (SEQ ID NO:16).
Volumes of distribution at steady state (Vss) measured after IV
dosing (0.2 mg/kg) were 87.3, 289, 68.4, and 77.0 mL/kg for mouse,
rat, cynomolgus cynomolgus monkey and dog, respectively (Table
4D(a)). Rat had a 3 times larger volume of distribution than the
next species making it an outlier in this regard.
[0428] Bioavailabilities ranged from 15% to 100% in the various
subcutaneous PK experiments that were performed. Bioavailability
was observed to increase with increasing dose in the rat. Exposure
upon subcutaneous dosing was species dependent, with some
delineation observed between rodent and non-rodents. As an example
of this, exposure in rat was less than 4% of that observed in
cynomolgus monkey and dog after giving a 0.5 mg/kg subcutaneous
dose of PEG-[Lys16]ShK (SEQ ID NO:16) (AUC.sub.0-inf of
3,220,137,000, and 103,000 nghr/mL for rat, cynomolgus monkey and
dog, respectively). The difference in exposure is attributed to a
combination of lower bioavailability, higher volume of
distribution, and higher elimination in rat as compared to
non-rodent species. In general, subcutaneous exposure in
Sprague-Dawley rats specifically could be characterized as either
low or inconsistent across the various experiments that were
performed where PK was measured, although it was usually consistent
within individual rat experiments.
[0429] PEG-[Lys16]ShK (SEQ ID NO:16) was tested for dose
proportionality in male Sprague-Dawley rats using subcutaneous
doses of 0.1, 0.5, 2.0 and 5.0 mg/kg. Exposures were twice that
predicted from previous subcutaneous experiments in rats and
increased at a greater than dose proportional rate, probably due to
an increase in bioavailability with increasing dose. PEG-[Lys16]ShK
(SEQ ID NO:16) was also tested for dose proportionality in female
C57BL/6 mice using subcutaneous doses of 0.1, 0.3, 0.6 and 2.5
mg/kg per animal. There was a similar tendency in this species for
greater than dose proportional increases with dose, which added an
additional 20-35% greater AUC than would have been expected from
the next lower dose. This study confirmed results of high exposure
observed previously in toxicology studies using female C57BL/6 mice
and also confirmed that there was a 6 to 8-fold increase in
exposure in female C57BL/6 versus male CD-1 mice when given a
similar dose.
[0430] Safety Pharmacology Assay. Safety pharmacology studies
investigate the potential undesirable pharmacodynamic effects of
chemicals or pharmaceutical compounds on vital organs or systems
which are essential for sustaining life. The International
Conference on Harmonisation (ICH) Safety Expert Working Group has
developed a hierarchy of organ systems with respect to their life
supporting functions. The most important functions are those of the
cardiovascular, respiratory or central nervous systems. Drug
induced effects on these systems should be investigated prior to
the first administration of substances to humans. Other organ
systems (e.g. the renal or gastrointestinal system), the functions
of which can transiently be disrupted by adverse pharmacodynamic
effects without causing irreversible harm, are of less immediate
investigative concern (S. Whitebread et al., DDT, 10:1421-1433
(2005); R. Porsolt et al., Drug. Dev. Res., 64:83-89 (2005)).
PEG-[Lys16]ShK (SEQ ID NO:16) was tested in the Cerep safety
pharmacology assay at 10 .mu.M to evaluate binding capacity to
various human targets such as receptors, enzymes and ion channels.
Of 151 targets, there were 9 hits (>50% inhibition; Table
4D(b)), 5 of which were ion channels. IC50 values were determined
for each hit and a functional assay was performed for neuropeptide
Y1. Compared to the IC50 value for the K Channel (expected target),
the IC50's for the other hits are >25 fold higher. In addition,
there was no impact on Neuropeptide Y1 in a functional assay. These
data imply that these off-target binding events are unlikely to
lead to unexpected toxicities.
[0431] Isolated rabbit heart assay. Potential cardiovascular effect
of 1 .mu.M PEG-[Lys16]ShK (SEQ ID NO:16) was evaluated in the
isolated rabbit heart assay. In this preparation, the test article
was perfused through the coronary circulation and cardiac
electrical (ECG), mechanical (left ventricular contractility), and
hemodynamics (coronary blood flow) were assessed after 20 min of
perfusion. There was no effect of PEG-[Lys16]ShK (SEQ ID NO:16) on
ECG, contractility or hemodynamic parameters in the isolated heart
model, which indicates that it was devoid of any effect cardiac ion
channel function.
TABLE-US-00008 TABLE 4A Potency and selectivity of N.alpha.-(20 kDa
PEG)-[Lys16]ShK (SEQ ID NO: 16). Assay IC50 nM (n) Human whole
blood 0.092 (14) (TG induced IL-2) Kv1.1 997 (3) Kv1.2 639 (3)
Kv1.3 0.94 (5) Kv1.4 >10000 (3) Kv1.5 >30000 (3) Kv1.6 466
(7) Kv1.7 >10000 (3) IKCa1 >10000 (4) BKCa >10000 (4) hERG
(IKr) >10000 (3) Nav1.5 (INa) >30000 (3) Cav1.2 (ICa)
>30000 (3) Kv4.3 (Ito) >30000 (3) KvLQT1/minK (IKs) >30000
(4)
TABLE-US-00009 TABLE 4B Pharmacokinetic data showing PEG-ShK (SEQ
ID NO: 8) has a prolonged half-life in Sprague-Dawley rats compared
to the small peptide ShK-L5 (SEQ ID NO: 17). Administration was by
subcutaneous injection. CMPD Dose Tmax Cmax AUC0-t AUC0-inf DNAUC
CL/F MRT (n) (mg/kg) (h) (ng/ml) (ng hr mL.sup.-1) (ng hr
mL.sup.-1) (0-inf) (mL hr.sup.-1 kg.sup.-1) (h) PEG- 2 24 .+-. 0
1138 .+-. 404 42347 .+-. 10,228 42374 .+-. 10,237 21187 49 .+-. 14
37 .+-. 3 ShK (3) PEG- 0.3 24 165 6011 6,020 20065 52 27 ShK (2)
ShK- 0.2 0.5 50 93 97 487 2052 2 L5 (6).sup.a .sup.aBeeton et al.,
Molec. Pharmacol. 67(4): 1369-81 (2005).
TABLE-US-00010 TABLE 4C Pharmacokinetic data for PEG-[Lys16]ShK
(SEQ ID NO: 16) in mice (dose = 2.0 mg/kg), rats (dose = 2.0 mg/kg)
and cynomolgus monkeys (dose = 0.5 mg/kg) administered by
subcutaneous injection. HL Tmax Cmax AUC0-t AUC0-inf Vz/F CL/F MRT
Species (h) (h) (ng/ml) (ng hr mL.sup.-1) (ng hr mL.sup.-1) (ml/kg)
(mL hr.sup.-1 kg.sup.-1) (h) Cyno 64.5 .+-. 14.9 8.0 1010 .+-. 105
71500 .+-. 607 74900 .+-. 3260 621 .+-. 143 6.68 .+-. 0.29 87 .+-.
16 Mouse* 14.9 4.0 1860 37000 37000 1170 54.1 16.6 Rat N/A 40 .+-.
14 531 .+-. 90 21900 .+-. 2770 21900 .+-. 2760 N/A 92 .+-. 13 36
.+-. 2 *Sparse sampling PK experiment. No standard deviations were
calculated for PK parameters.
TABLE-US-00011 TABLE 4D Pharmacokinetic data for PEG-[Lys16]ShK
(SEQ ID NO: 16) administered by subcutaneous injection (dose = 0.5
mg/kg) to dog (beagle) or monkey (cyno). HL Tmax Cmax AUC0-t
AUC0-inf Vz/F CL/F MRT Species (h) (h) (ng/ml) (ng hr mL.sup.-1)
(ng hr mL.sup.-1) (ml/kg) (mL hr.sup.-1 kg.sup.-1) (h) Cyno(a) 64.5
.+-. 14.9 8.0 1010 .+-. 105 71500 .+-. 607 74900 .+-. 3260 621 .+-.
143 6.68 .+-. 0.29 87 .+-. 16 Cyno(b) 37.4 .+-. 13.2 18.7 .+-. 9.2
1480 .+-. 258 125000 .+-. 31600 137000 .+-. 45500 197 .+-. 9.3 3.90
.+-. 1.10 70.5 .+-. 20.sup. Beagle 42.6 .+-. 4.21 18.7 .+-. 9.24
1270 .+-. 347 95200 .+-. 31300 103000 .+-. 37300 322 .+-. 98 5.37
.+-. 2.14 66.1 .+-. 13.5
TABLE-US-00012 TABLE 4D(a) Preclinical pharmacokinetics of of
PEG-[Lys16]ShK (SEQ ID NO: 16) in mouse, rat, cynomolgus monkey and
dog. Mouse Rat Monkey Dog Parameter (Male CD-1) (Male SD) (Male
Cynomolgus) (Male Beagle) IV t.sub.1/2 (h) 0.2 mg/kg (1) 16.1 (1)
12.0 .+-. 3.7 (1) 21.0 .+-. 13.7 (1) 28.3 .+-. 4.2 (1) 2.0 mg/kg
(2) ND (2) 16.8 (2) ND (2) ND (2) IV MRT (h) 0.2 mg/kg (1) 11.0 (1)
6.6 .+-. 0.7 (1) 13.3 .+-. 7.5 (1) 27.2 .+-. 8.6 (1) 2.0 mg/kg (2)
ND (2) 8.5 .+-. 0.5 (2) ND (2) ND (2) IV CI (mL/h/kg) 0.2 mg/kg (1)
8.2 (1) 43.9 .+-. 1.6 (1) 5.94 .+-. 2.30 (1) 2.68 .+-. 0.54 (1) 2.0
mg/kg (2) ND (2) 60 .+-. 17 (2) ND (2) ND (2) IV Vss (ml/kg) 0.2
mg/kg (1) 87.3 (1) 289.3 .+-. 19.3 (1) 68.4 .+-. 24.7 (1) 77.0 .+-.
8.4 (1) 2.0 mg/kg (2) ND (2) 511 .+-. 170 (2) ND (2) ND (2) SC
AUC.sub.0.fwdarw..infin.(ng h/mL) 0.5 mg/kg (1) 24300 (1) 4570 .+-.
160 (1) 36700 .+-. 11700 (1) 76500 .+-. 14000 (1) 2.0 mg/kg (2) ND
(2) 35500 .+-. 9900 (2) ND (2) ND (2) SC T.sub.max (h) 0.5 mg/kg
(1) ND (1) 24.0 .+-. 0.0 (1) 8 .+-. 0.0 (1a) 18.7 .+-. 9.2 (1) 2.0
mg/kg (2) 4.0 (2) 40 .+-. 14 (2) 18.7 .+-. 9.2 (1b) ND (2) ND (2)
SC C.sub.max (ng/mL) 0.5 mg/kg (1) ND (1) 94.5 .+-. 30 (1) 1031
.+-. 105 (1a) 1270 .+-. 347 (1) 2.0 mg/kg (2) 1860 (2) 531 .+-. 90
(2) 1480 .+-. 258 (1b) ND (2) ND (2) SC AUC.sub.0.fwdarw..infin.(ng
h/mL) 0.5 mg/kg (1) ND (1) 3220 .+-. 781 (1) 75000 .+-. 3,260 (1a)
103000 .+-. 37300 (1) 2.0 mg/kg (2) 37000 (2) 21900 .+-. 2,760 (2)
137000 .+-. 45500 (1b) ND (2) ND (2) SC Bioavailability(%)* 15.2
(2) 28 (1), 62 (2) 82 (1a), >100 (1b) 54 (1) *= Bioavailability
calculated using 0.2 mg/kg IV dose
TABLE-US-00013 TABLE 4D(b) PEG-[Lys16]ShK (SEQ ID NO: 16) was
tested in the Cerep safety pharmacology assay. IC50 Functional
Target % Inhibition (.mu.M) Assay NMDA 71 10 ND Glycine (Strychnine
101 1.7 ND insensitive) Lysophosphotodic A1 62 5.5 ND Neuropeptide
Y1 51 22 No agonistic or antagonistic effects Ca Channel (N) 58 3.0
ND K Channel (KATP) 54 2.7 ND K Channel (Kv) 90 0.023 ND Nitric
Oxide 90 0.59 ND (endothelial) Phosphodiestrase 1B 68 27 ND Hits
are shown. ND = Not done
Example 6
Ion Channel Counterscreens
[0432] Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.6 and Kv1.7,
PatchXpress.RTM., planar patch-clamp electrophysiology. Ion channel
currents were recorded at room temperature using PatchXpress.RTM.
7000A electrophysiology system from MDC using methods and cells
described in Example 1 above. The voltage protocols for each
channel are shown in Table 4E, below.
TABLE-US-00014 TABLE 4E Voltage protocols and recording conditions.
Ion Pulse Time between Holding Benchmark Channel Voltage Step
Duration pulses Potential Inhibitor hKv1.1 From -80 mV 200 ms 10 s
-80 mV 10 nM DTX-k to +30 mV hKv1.2 From -80 mV 400 ms 10 s -80 mV
1 nM MgTx to +60 mV hKv1.3 From -80 mV 200 ms 30 s -80 mV 1 nM ShK
to +30 mV hKv1.4 From -80 mV 200 ms 30 s -80 mV 1 mM 4-AP to +30 mV
hKv1.6 From -80 mV 500 ms 15 s -80 mV 1 mM 4-AP to +60 mV hKv1.7
From -80 mV 1000 ms 15 s -80 mV 1 mM 4-AP to +30 mV
[0433] Cardiac ion channel Counterscreens (hERG, hKvLQt1/hminK,
hNav1.5, hKv1.5, hCav1.2, hKv4.3).
[0434] Cell lines. HEK293 cells stably transfected with
hKvLQT1/hminK and hERG were from Amgen or Cytomyx, Inc. HEK293
cells stable transfected with human hNav1.5 were purchased from
Cytomyx, Inc. HEK293 cells stably expressing hKv4.3 and CHO cells
stably expressing hKv1.5 were from ChanTest. CHO cells stably
expressing the human L-type calcium channel Cav1.2 were from
ChanTest and contained the human CACNA1C gene encoding hCav1.2 and
coexpressed the beta 2 subunit encoded by human CACNB2 and
alpha2delta1 encoded by the CACNA2D1 gene.
[0435] FASTPatch.RTM. studies were performed at ChanTest to examine
the impact of peptides and conjugates on the cloned human L-type
calcium channel hCav1.2, cloned hKv4.3 and cloned hKv1.5 involved
PatchXpress (Model 7000A, Molecular Devices, Union City, Calif.)
electrophysiology at room temperature. The extracellular recording
solution (HB-PS) contained 137 mM NaCl, 4 mM KCl, 1.8 mM
CaCl.sub.2, 1 mM MgCl.sub.2, 10 mM HEPES and 10 mM Glucose adjusted
to pH 7.40 with NaOH. The intracellular recording solution for
hKv4.3 and hKv1.5 contained 130 mM potassium aspartate, 5 mM
MgCl.sub.2, 5 mM EGTA, 4 mM ATP and 10 mM HEPES adjusted to pH 7.2
with KOH. The intracellular solution for hCav1.2 contained 130 mM
cesium aspartate, 5 mM MgCl.sub.2, 5 mM EGTA, 4 mM ATP, 2 mM EDTA,
1 mM CaCl.sub.2, 0.1 mM GTP and 10 mM HEPES adjusted to pH 7.2 with
N-methyl-D-glucamine. In preparation for recording, intracellular
solution is loaded into the intracellular compartments of the
Sea/chip.sub.16 planar electrode. Cell suspensions are pipetted
into the extracellular compartments of the Sea/chip.sub.16 planar
electrode. After establishing a whole-cell configuration, membrane
currents are recorded using dual-channel patch clamp amplifiers in
the PatchXpress.RTM. system. Before digitization, the currents were
low-pass filtered at one-fifth of the sampling frequency. Three
concentrations of peptide conjugates (test article) diluted into
HB-PS with 1% BSA are applied at five minute intervals to naive
cells. Solution exchange were performed in quadruplicate and the
duration of exposure to each test article concentration was five
minutes. Vehicle controls were also applied to naive cells and
after a solution exchange positive controls are applied to verify
sensitivity to ion channel blockade. All positive controls were
diluted into HB-PS with 0.3% DMSO. Positive controls for blockade
of channels included: nifedipine (0.01 .mu.M) which produced about
75% hCav1.2 current block, flecamide (0.1 mM) which produced about
75% inhibition of the hKv4.3 current and 4-aminopyridine (2 mM)
which blocked about 80% of the hKv1.5 current. Valid whole-cell
recordings must meet the following criteria: (1) membrane
resistance (Rm).gtoreq.200 M.OMEGA., (2) leak current.ltoreq.25%
channel current. The test procedures for hCav1.2, hKv4.3 and hKv1.5
were as follows:
[0436] a.) hCav1.2 test procedure. Onset and steady state block of
hCav1.2/.beta.2/.alpha.2.delta. channels were measured using a
stimulus voltage pattern consisting of a depolarizing test pulse
(duration, 200 ms; amplitude, 10 mV) at 10-s intervals from a -40
mV holding potential. Test article concentrations may be applied
cumulatively in ascending order without washout between
applications. Peak current was measured during the step to 10 mV.
Saturating concentration of nifedipine (10 .mu.M) is added at the
end of each experiment to block hCav1.2 current. Leak current was
digitally subtracted from the total membrane current record.
[0437] b.) hKv4.3 test procedure. Onset and steady state block of
hKv4.3 current were measured using a pulse pattern with fixed
amplitudes (depolarization: 0 mV for 300 ms) repeated at 10-s
intervals from a holding potential of -80 mV. Peak and sustained
test pulse current amplitudes were measured during the step to zero
mV.
[0438] c.) hKv1.5 test procedure. Onset and steady state block of
hKv1.5 current were measured using a pulse pattern with fixed
amplitudes (depolarization: +20 mV amplitude, 300 ms duration)
repeated at 10-s intervals from a holding potential of -80 mV.
Current amplitude was measured at the end of the step to +20
mV.
[0439] Counterscreens against the cloned human Nav1.5 sodium
channel using the PatchXpress.RTM. system. The extracellular
(HB-PS2) recording solution contained 70 mM NaCl, 67 mM
N-methyl-D-glucamine, 4 mM KCl, 1.8 mM CaCl.sub.2, 1 mM MgCl.sub.2,
10 mM HEPES, 10 mM Glucose adjusted to pH 7.4 with HCl. The
internal recording solution contained 130 mM CsF, 10 mM NaCl, 10 mM
EGTA, 2 mM MgCl.sub.2, 10 mM HEPES adjusted to pH 7.20 with CsOH.
Stock solutions of reference standard or test articles were diluted
into HB-PS2 prior to application. Test articles included either
peptides or peptide conjugates described herein. Lidocaine (1-30
.mu.M) was the reference standard. A standardized step protocol is
used to elicit ionic current through the hNav1.5 sodium channel.
Cells are held at -80 mV. Onset and steady state block of hNav1.5
sodium current due to Test Article was measured using a pulse
pattern with fixed amplitudes (conditioning prepulse: -120 mV for
50 ms; depolarizing test step to -30 mV for 20 ms) repeated at 10-s
intervals. Currents are filtered at 3 kHz and acquired at 10 kHz,
in episodic mode. When a good recording was established, cells were
washed for 2 minutes, following by applying control vehicle for 5
minutes. Then control and each concentration of test article was
applied for 5 minutes. There were 3 additions for each
concentration with 1-minute interval. Dispense speed was 40 .mu.L/s
with suction on. To determine IC.sub.50, Test Article at 1 .mu.M, 3
.mu.M, 10 .mu.M and 30 .mu.M was applied to cells (n=3 cells)
cumulatively (without washout between test article concentrations)
in ascending order, to each cell (n=3 where n=number of cells).
Each concentration of test article was applied for 5 minutes. There
were 3 additions for each concentration with a 1-minute interval.
Electrophysiological data acquisition was performed using
PatchXpress Commander v1.4 (Axon Instruments, Union City, Calif.)
and analyses was performed using DataXpress v1.4 (Axon Instruments,
Union City, Calif.). The 5 peak currents before and after test
article application were used to calculate the percentage of
current inhibition at each concentration. Acceptance criteria for a
good recording include: (1) seal resistance>200 M.OMEGA., (2)
access resistance<10 M.OMEGA., (3) peak tail current>200 pA,
(4) leakage current<25% of the peak tail current, (5)
rundown<2.5%/minute in control vehicle.
[0440] Counterscreens against the human IKs (hKvLQT1+hminK)
potassium channel using the PatchXpress.RTM. system. The
extracellular recording solution was HB-PS. The internal recording
solution contained 20 mM KF, 90 mM KCl, 10 mM NaCl, 10 mM EGTA, 5
mM K2ATP, 1 mM MgCl.sub.2, 10 mM HEPES adjusted to pH 7.20 with
KOH. Stock solutions of reference standard or test articles were
diluted into HB-PS prior to application. Test articles included
either peptides or peptide conjugates described herein. Chromanol
293B (0.3-10 .mu.M) was the reference standard. A standardized step
protocol was used to elicit ionic current through the IKs potassium
channel. Cells were held at -80 mV. Onset and steady state block of
IKs potassium current due to Test Article was measured using a
pulse pattern with fixed amplitudes (depolarizing test step to +50
mV for 5s) repeated at 10-s intervals. Currents is filtered at 3
kHz and acquired at 10 kHz, in episodic mode. When a good recording
was established, cells were washed for 2 minutes, following by
applying control vehicle for 5 minutes. Then control and each
concentration of test article were applied for 5 minutes. There
were 3 additions for each concentration with 1 minute interval.
Dispense speed was 40 .mu.L/s with suction on. Test article at 1
.mu.M, 3 .mu.M, 10 .mu.M and 30 .mu.M were applied to cells (n=3
cells) cumulatively (without washout between test article
concentrations) in ascending order, to each cell (n=3 where
n=number of cells). Each concentration of test article was applied
for 5 minutes. There were 3 additions for each concentration with a
1 minute interval. Electrophysiological data acquisition was
performed using PatchXpress Commander v1.4 (Axon Instruments, Union
City, Calif.) and analyses is performed using DataXpress v1.4 (Axon
Instruments, Union City, Calif.). The 5 peak currents before and
after test article application were used to calculate the
percentage of current inhibition at each concentration. Acceptance
criteria for a good recording includes: (1) seal resistance>200
M.OMEGA., (2) access resistance<10 M.OMEGA., (3) peak tail
current>200 pA, (4) leakage current<25% of the peak current,
(5) rundown<2.5%/minute in control vehicle.
[0441] Counterscreens against the human IKr (hERG or hKv11.1)
potassium channel by conventional whole cell patch clamp
electrophysiology. One to 2 drops of the cell suspension is added
to a 35 mm poly-d-lysine coated cover slip for overnight incubation
before electrophysiology experiments. Whole-cell currents were
recorded from single cells by using tight GS2 seal configuration of
the patch-clamp technique. A 35 mm cover slip was transferred to
the recording stage after rinsing and replacing the culture medium
with extracellular recording buffer containing 135 mM NaCl, 5 mM
KCl, 1.8 mM CaCl.sub.2, 10 mM HEPES, and 5 mM Glucose (pH was
adjusted to 7.40 with NaOH and osmolarity was set at 300 mOsm).
Cells were continuously perfused with the extracellular recording
buffer via one of the glass capillaries arranged in parallel and
attached to a motorized rod, which places the glass capillary
directly on top of the cell being recorded. For hERG profiling, the
recording pipette solution contained 130 mM KF, 2 mM MgCl.sub.2, 10
mM EGTA, and 10 mM HEPES adjusted to pH 7.40 with KOH and
osmolarity set at 280 mOsm. Experiments were performed at room
temperature and recorded using Multiclamp 700A amplifier (Molecular
Devices Inc.). Pipette resistances were typically 2-3 M.OMEGA..
Cells were held at a potential of -80 mV. To achieve a baseline or
reference point for the peak outward tail current, a step to -50 mV
for 500 ms was used. This was followed by a depolarizing step to
+20 mV for 2 s to drive the channels to the inactivated state. A
step back to -50 mV for 2s allowed the inactivation to be relieved
and peak hERG current to be measured. Pulses were repeated once
every 10 s. Total hERG current was measured as the difference
between the peak current at the repolarizing -50 mV step and the
baseline current at -50 mV. Test articles (up to 10 .mu.M), which
included the peptides and peptide conjugates described herein, were
mixed into the extracellular recording buffer containing 0.1%
bovine serum albumin (BSA) and subsequently transferred to glass
perfusion reservoirs. Electronic pinch valves controlled the flow
of the test articles from the reservoirs onto the cell being
recorded. IC50 values and curve fits were estimated using the four
parameter logistic fit of XLfit software. The hERG channel
inhibitor, cisapride, was used to validate the assay.
[0442] Counterscreens against calcium-activated potassium channels
human IKCa1 and BKCa by conventional whole cell patch clamp
electrophysiology. CHO IKCa and BKCa cell lines were obtained from
BioFocus DPI (A Galapagos Company). One to 2 drops of the hIKCa1 or
BKCa cell suspension is added to a 35 mm poly-d-lysine coated cover
slip for overnight incubation before electrophysiology experiments.
Whole-cell currents were recorded from single cells by using tight
G.OMEGA. seal configuration of the patch-clamp technique. A 35 mm
cover slip was transferred to the recording stage after rinsing and
replacing the culture medium with the extracellular recording
buffer containing 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl.sub.2, 10 mM
HEPES, and 5 mM Glucose (pH was adjusted to 7.40 with NaOH and
osmolarity was set at 300 mOsm). Cells were continuously perfused
with the extracellular recording buffer via one of the glass
capillaries arranged in parallel and attached to a motorized rod,
which places the glass capillary directly on top of the cell being
recorded. The recording pipette solution contained 130 mM potassium
aspartate, 1 mM MgCl.sub.2, 1.26 mM CaCl.sub.2, 2 mM EGTA, 2 mM
Mg-ATP and 10 mM HEPES adjusted to pH 7.40 with KOH and osmolarity
set at 280 mOsm. Experiments were performed at room temperature and
recorded using Multiclamp 700A amplifier (Molecular Devices Inc.).
Cells were held at potential of -80 mV. Both BK and IK currents
were activated as calcium ion diffused into the cell from recording
pipette solution. Activation of the calcium dependent outward
potassium current by calcium diffusion generally takes 3 to 5 min
for full activation. Outward currents were continuously monitored
at holding potential of +50 mV before and during drug exposure.
Alternatively, 400 ms voltage ramps from -120 to +60 mV were
repeated once every 10 s to characterize the current voltage
relation for both channels before and during drug exposure. Test
articles (up to 10 .mu.M), which included the peptides and peptide
conjugates described herein, were mixed into the extracellular
recording buffer containing 0.1% bovine serum albumin (BSA) and
subsequently transferred to glass perfusion reservoirs. Electronic
pinch valves controlled the flow of the test articles from the
reservoirs onto the cell being recorded. Pipette resistances were
typically 2-3 M.OMEGA.. IC50 values and curve fits were estimated
using the four parameter logistic fit of XLfit software. A IKCa and
BK peptide inhibitor, charybdotoxin (100 nM), was applied at the
conclusion of the assay procedures for pharmacological validation
of the assay.
Example 7
Plasma Stability Studies
[0443] The stability of 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16) in
rat, cynomolgus monkey and human plasma was tested by spiking the
peptide conjugate into 100% plasma to a final concentration of 200
ng/mL and incubating for various periods of time at 37.degree. C.
At the end of each incubation period, samples were frozen until
analysis. As a control, an aliquot of conjugate stock solution was
diluted into plasma and immediately frozen to generate a time zero,
untreated control. Analysis of stability included an ELISA analysis
of drug levels (see Example 8, "Protocol 1") to confirm retention
of the ShK immunoreactivity and whole blood assessment of potency
to confirm retention of its bioactivity in blocking T cell
responses. To assess bioactivity, serial dilutions of each
stability sample were tested in the human whole blood bioassay
described in Example 2 which measures T cell IL-2 and IFN-g
production after thapsigargin stimulation. In brief, stability
samples or standard were pre-incubated with whole blood for 30-60
minutes, prior to addition of thapsigargin to induce cytokine
secretion. Supernatants were removed 48 hours later to measure the
level of cytokine secretion. Since Kv1.3 inhibitors suppress
cytokine secretion in this assay, the level of cytokine suppression
is a direct measure of the peptide conjugates bioactivity. As a
control to define full T cell activation and cytokine secretion in
the absence of inhibitor, each plate contained whole blood samples
that were treated with thapsigargin alone or unstimulated.
Dilutions from plasma stability study samples that provide 30%-70%
inhibition of the thapsigargin-induced cytokine response were used
in analysis of stability. FIG. 10A-D, indicates 20
kDa-PEG-[Lys16]ShK (SEQ ID NO:16) is stable when incubated in rat,
cyno and human plasma for up to 48 hrs at 37deg C. and retains
bioactivity and immunoreactivity. FIG. 10A shows [Lys16]ShK (SEQ ID
NO:13) structural moiety of SEQ ID NO:16 is stable in plasma over
time as indicated by retention of the immunological epitope
quantified by ELISA analysis as described in Example 8, below.
Recovery % refers to the ELISA measured level of 20
kDa-PEG-[Lys16]ShK (SEQ ID NO:16) in the plasma stability sample,
compared to the expected or initial 200 ng/ml concentration. As an
example, a plasma stability sample measured to have 160 ng/ml,
would be reported with a recovery % of 80. FIGS. 10B-D show the
bioactivity of various dilutions of the plasma stability sample
added to the thapsigargin whole blood assay to measure the extent
of cytokine inhibition as a measure of the bioactivity of the 20
kDa-PEG-[Lys16]ShK (SEQ ID NO:16) molecule. The final dilution
factor of these samples is listed to the right of FIG. 10B and FIG.
10D. Roughly half of the IL-2 cytokine response (measured in
relative light units, RLU) was suppressed by 0.93-2.78% of the
plasma stability samples and there was no significant change in the
level of response (RLU units) over time at 37.degree. C. which
indicates the 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16) molecule retains
bioactivity and was stable in plasma.
Example 8
Pharmacokinetic & Pharmacodynamic Studies
[0444] Antibodies to ShK. Rabbit polyclonal and mouse monoclonal
antibodies to ShK were generated by immunization of animals with
the Fc-ShK peptibody conjugate. Anti-ShK specific polyclonal
antibodies were affinity purified from antisera to isolate only
those antibodies specific for the ShK portion of the conjugate.
Following fusion and screening, hybridomas specific for ShK were
selected and isolated. Mouse anti-ShK specific monoclonal
antibodies were purified from the conditioned media of the clones.
By ELISA analysis, purified anti-ShK polyclonal and monoclonal
antibodies reacted only to the ShK peptide alone and did not
cross-react with Fc.
[0445] Pharmacokinetic (PK) studies on 20 kDa-PEG-ShK (SEQ ID NO:8)
and 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16) peptide conjugates in
mice, rats, beagles and monkeys. Single subcutaneous doses were
delivered to animals and serum was collected at various time points
after injection. Studies in rats, dogs (beagles), and cynomolgus
monkeys involved two to three animals per dose group, with blood
and serum collection occurring at various time points over the
course of the study. Male Sprague-Dawley (SD) rats (about 0.3 kg),
male beagles (about 10 kg) and male cynomolgus monkeys (about 4 kg)
were used in the studies described herein (n=3 animals per dose
group). Approximately 5 male CD-1 mice were used per dose and time
point in our mouse pharmacokinetic studies. Serum samples were
stored frozen at -80.degree. C., until analysis in an enzyme-linked
immunosorbent assay (ELISA).
[0446] A brief description of the ELISA protocol for detecting
serum levels of PEG-ShK and PEG-[Lys16]ShK is provided below:
[0447] (1) Protocol 1, (a)-(g) below, detects PEG-ShK and
PEG-[Lys16]ShK, as well as the ShK and [Lys16]ShK peptides
alone:
[0448] (a) Streptavidin microtiter plates were coated with 250
ng/ml biotinylated-anti-ShK mouse monoclonal antibody (mAb2.10,
Amgen) in I block buffer [per liter: 1000 mL 1.times.PBS without
CaCl.sub.2, MgCl.sub.2, 5 ml Tween 20 (Thermo Scientific), 2 g I
block reagent (Tropix)] at 4.degree. C., incubated overnight
without shaking
[0449] (b) Plates were washed three times with KPL wash buffer
(Kirkegaard & Perry Laboratories).
[0450] (c) Standards (STD), quality controls (QC) and sample
dilutions were prepared with 100% pooled sera, then diluted 1/5
(pretreatment) in I block buffer. Pretreated STDs, QCs and samples
were added to the washed plate and incubated at room temperature
for 2 hours. (Serial dilutions of STDs, QCs were prepared in 100%
pooled sera. Samples needing dilution were also prepared with 100%
pooled sera. The pretreatment was done to both stds, QCs and
samples to minimize the matrix effect.)
[0451] (d) Plates were washed three times with KPL wash buffer.
[0452] (e) A HRP-labeled rabbit anti-ShK polyclonal Ab at 250 ng/ml
in I block buffer was added and plates were incubated at room
temperature for 1 hour with shaking
[0453] (f) Plates were again washed three times with KPL wash
buffer and the Femto [Thermo Scientific] substrate was added.
[0454] (g) The plate was read with a Lmax II 384 (Molecular
Devices) luminometer.
[0455] Pharmacokinetic (PK) studies on Fc-, Ig-, or Ab conjugates
of ShK and [Lys16]ShK were performed in male SD rats. Single
subcutaneous doses were delivered to animals and serum was
collected at various time points after injection. Three animals
were used per dose group, with blood and serum collection occurring
at various time points over the course of the study. Serum samples
were stored frozen at -80.degree. C., until analysis in an
enzyme-linked immunosorbent assay (ELISA). A brief description of
the ELISA protocol for detecting serum levels of Fc-, Ig-, or
Ab-conjugates of ShK and [Lys16]ShK is provided below. Protocol 2,
below detects both the human Ig, Fc or Ab portion of the molecule,
as well as the ShK peptide portion. Protocol 3, below is an early
assay that detects the human Fc region alone and was used for early
assessment of serum levels of Fc-ShK peptibodies in rodent
pharmacokinetic studies. A brief description of these ELISA
protocols is provided:
[0456] (2) Protocol 2, (a)-(g) below, detects both the human Ig, Fc
or Ab portion of the molecule, as well as the ShK peptide
portion:
[0457] (a) Streptavidin microtiter plates were coated with 250
ng/ml biotinylated-anti-ShK mouse monoclonal antibody (mab 2.10,
Amgen) in I block buffer [per liter: 1000 ml 1.times.PBS without
CaCl.sub.2, MgCl.sub.2, 5 ml Tween 20 (Thermo Scientific), 2 g I
block reagent (Tropix)] at 4.degree. C., overnight without
shaking;
[0458] (b) Plates were washed three times with KPL wash buffer
(Kirkegaard & Perry Laboratories)
[0459] (c) Standards (STD), quality controls (QC) and sample
dilutions with 100% pooled sera were prepared, then were diluted
1/5 (pretreatment) in I block buffer. Pretreated STDs, QCs and
samples were added to the washed plate. Incubation was at room
temperature for 2 hours. (Serial dilutions of STDs, QCs were
prepared in 100% pooled sera. Samples needing dilution were also
prepared with 100% pooled sera. The pretreatment was done to both
stds, QCs and samples to minimize the matrix effect.);
[0460] (d) Plates were washed three times with KPL wash buffer;
[0461] (e) A HRP-labeled Ab35 (against human IgG Fc) at 150 ng/ml
in I block buffer was added and plates were incubated at room
temperature for 1 hour with shaking
[0462] (f) Plates were washed three times with KPL wash buffer and
the Femto [Thermo Scientific] substrate was added;
[0463] (g) The plate was read with a Lmax II 384 [Molecular
Devices] luminometer.
[0464] (3) Protocol 3, (a)-(h) below, is an early assay that
detects the human Fc region alone and was used for early assessment
of serum levels of Fc-ShK peptibodies in rodent pharmacokinetic
studies:
[0465] (a) Costar 3590 96-well EIA/RIA plates were coated with 0.1
mL/well of 2 .mu.g/mL Goat anti-HuFc, F(ab').sub.2, (Sigma 1-3391)
diluted in 1.times. Coating Buffer (10.times. Coating Buffer: 1.59
g Na.sub.2CO.sub.3, 2.93 g NaHCO.sub.3 in 100 ml H.sub.2O). Plates
were sealed and incubated at 4.degree. C. overnight;
[0466] (b) Plates were washed three times with PBST (PBS+0.1%
Tween-20) and blocked by addition of 0.3 ml of blotto (PBS, 0.1%
Tween-20, 5% non-fat dry milk) to each well and incubated for 1 h
at room temperature (RT) with shaking;
[0467] (c) Plates were washed with a KP Wash Solution (Cat
#50-63-00, KPL, Gaithersburg, Md.);
[0468] (d) Diluted serum samples and controls/standards in Dilution
Buffer (PBS, 0.1% BSA, 0.1% Tween-20) plus rat serum, if needed,
were brought to 10% rat serum final and 0.1 ml sample was added per
well. Plates were incubated at room temperature with shaking for 1
hour;
[0469] (e) Plates were washed with a KP Wash Solution (Cat
#50-63-00, KPL, Gaithersburg, Md.);
[0470] (f) A HRP labeled secondary antibody (Pierce #31416-HRP Goat
.alpha.-Hu IgG Fc) was diluted 1:5000 in PBST and then 100
.mu.l/well is added and incubated at RT with shaking for 1
hour;
[0471] (g) Plates were washed with a KP Wash Solution (Cat
#50-63-00, KPL, Gaithersburg, Md.) and 100 .mu.l/well of ABTS
substrate (ABTS Microwell Substrate 1-Component, Cat#50-66-018,
KPL) was added;
[0472] (h) At appropriate times after substrate addition and
shaking, the plate was read with a SpectraMax340 [Molecular
Devices] plate reader.
[0473] Comparison of the rat pharmacokinetics of our novel toxin
conjugates to the published pharmacokinetic profile of the small
ShK-L5 (SEQ ID NO:17) peptide. The pharmacokinetics of a single 0.2
mg/kg subcutaneous dose of ShK-L5 (SEQ ID NO:17) in rats was
reported by Beeton et al. (Mol. Pharmacol. 67, 1369-1381, 2005).
ShK-L5 was reported to have a circulating half-life estimated to be
about 50 min. Beeton et al. (ibid) provide two figures showing
serum ShK-L5 concentrations (nM) verus time after a single 0.2
mg/kg subcutaneous injection. Based on the data from those figures,
a single 0.2 mg/kg dose of ShK-L5 was estimated to provide the
following ng/ml serum concentration per unit time in minutes after
subcutaneous injection: 10(5), 23(10), 28(20), 50(30), 34(60),
19(120), 10(180), 2(420), where values in parenthesis are time in
minutes and the ShK-L5 molecular weight (MW) was set to about 4050
Da. FIG. 4 illustrates that 20 kDa-PEG-ShK (SEQ ID NO:8) molecule
has an extended half-life and provides much greater exposure in
rats than the ShK-L5 (SEQ ID NO:17) molecule. Because only the
peptide portion of 20 kDa-PEG-ShK (SEQ ID NO:8) was used in
calculating mg/mL stock concentrations, and this ShK peptide
portion (4055 Da) is of similar MW to ShK-L5 (SEQ ID NO:17),
equivalent mg/kg doses of these two molecules generate
approximately equivalent nmol/kg doses. The calculated
pharmacokinetic values from this analysis of ShK-L5 (SEQ ID NO:17)
and the 20 kDa-PEG-ShK (SEQ ID NO:8) molecule, is provided in Table
4B. The clearance (CL/F) of PEG-ShK (SEQ ID NO:8) in rats is about
40 times slower than ShK-L5 (SEQ ID NO:17) and the mean residence
time (MRT) of PEG-ShK is 13-18 times longer. Example 12 and Tables
41-K, provide results from rat pharmacokinetic studies on exemplary
Fc, Ig and Ab-ShK toxin peptide analog conjugate embodiments that
demonstrate they have extended half-life in vivo and improved
therapeutic potential. The anti-KLH-Ab-[Lys16]ShK Ab molecules, in
both bivalent and monovalent forms (represented schematically in
FIG. 12G and FIG. 12F, respectively), exhibit an extended half-life
in vivo, which is approximately 32-60 times as long as that
reported by Beeton et al. (2005, ibid) for ShK-L5. (See, Example
12, FIG. 21) Since the anti-KLH-Ab-[Lys16] ShK Ab molecules have a
MW of about 150 kDa, a 6 mg/kg dose of these molecules on a nmol/kg
basis would be equivalent to a 0.16 mg/kg of the smaller (4 kDa)
ShK (SEQ ID NO:1) or ShK-L5 (SEQ ID NO:17) peptide. Therefore, the
0.2 mg/kg PK study on ShK-L5 provides a nmol/kg dose that is very
similar (within 25%) to that achieved with a 6 mg/kg dose of the
larger anti-KLH-Ab-[Lys16]ShK Ab molecules. Our monovalent
anti-KLH-Ab-[Lys16]ShK molecule exhibited very slow clearance in
rats (CL/F=10.9 mLhr.sup.-1kg.sup.-1), a rate that was about 188
times slower than ShK-L5 (CL/F=2052 mLhr.sup.-1kg.sup.-1). The
monovalent KLH-Ab-[Lys16]ShK molecule provided about 6100 times
greater drug exposure in rats (AUC0-inf=594000 nghrmL.sup.-1)
compared to the small ShK-L5 peptide (AUC0-inf=97 nghrmL.sup.-1)
(see Table 4B and Table 4J). If one normalizes for MW differences
between these two molecules (150 kDa//4 kDa), the monovalent
KLH-Ab-[Lys16]ShK molecule provided about 163 times greater
exposure (based on AUC0-inf) than the small ShK-L5 (SEQ ID NO:17)
molecule. The monovalent aKLH-AbLoop-[Lys16]ShK Ab molecule,
described in detail in Example 11 and Example 12, exhibited the
slowest clearance in rats of all the novel toxin-conjugates that we
have examined (FIG. 22 and Table 4L). This molecule's clearance
(CL/F=3.11 mLhr.sup.-1kg.sup.-1) was about 660 times slower than
ShK-L5.
[0474] Ex vivo cynomolgus monkey whole blood assay to measure the
potency of 20 kDa-PEG-[Lys16]ShK and its level of pharmacodynamic
coverage in vivo. The potency and level of coverage of cynomolgus
monkey T cell responses was determined with an ex vivo whole blood
assay measuring thapsigargin-induced IL-4, IL-5 and IL-17. To
determine potency of peptides and peptide conjugates, cynomolgus
whole blood was obtained from healthy, naive, male monkeys in a
heparin vacutainer. DMEM complete media was Iscoves DMEM (with
L-glutamine and 25 mM Hepes buffer) containing 0.1% human albumin
(Gemini Bioproducts, #800-120), 55 .mu.M 2-mercaptoethanol (Gibco),
and lx Pen-Strep-Gln (PSG, Gibco, Cat#10378-016). Thapsigargin was
obtained from Alomone Labs (Jerusalem, Israel). A 10 mM stock
solution of thapsigargin in 100% DMSO was diluted with DMEM
complete media to a 40 .mu.M, 4.times. solution to provide the
4.times. thapsigargin stimulus for calcium mobilization. The Kv1.3
inhibitor peptide ShK (Stichodacytla helianthus toxin, Cat# H2358)
and the BKCa1 inhibitor peptide IbTx (Iberiotoxin, Cat# H9940) were
purchased from Bachem Biosciences, whereas the Kv1.1 inhibitor
peptide DTX-k (Dendrotoxin-K) was from Alomone Labs (Israel). The
calcineurin inhibitor cyclosporin A (CsA) is available commercially
from a variety of vendors. Whereas the BKCa inhibitor IbTx and the
Kv1.1 inhibitor DTX-k do not inhibit the cytokine response, the
Kv1.3 inhibitor ShK and the calcineurin inhibitor CsA inhibit the
cytokine response and are used routinely as standards or positive
controls. Ten 3-fold serial dilutions of standards, ShK analogs or
ShK-conjugates were prepared in DMEM complete media at 4.times.
final concentration and 50 .mu.l of each were added to wells of a
96-well Falcon 3075 flat-bottom microtiter plate. Whereas columns
1-5 and 7-11 of the microtiter plate contained inhibitors (each row
with a separate inhibitor dilution series), 50 .mu.l of DMEM
complete media alone was added to the 8 wells in column 6 and 100
.mu.l of DMEM complete media alone was added to the 8 wells in
column 12. To initiate the experiment, 100 .mu.l of whole blood was
added to each well of the microtiter plate. The plate was then
incubated at 37.degree. C., 5% CO.sub.2 for one hour. After one
hour, the plate was removed and 50 .mu.L/well of the 4.times.
thapsigargin stimulus (40 .mu.M) was added to all wells of the
plate, except the 8 wells in column 12. The plates were placed back
at 37.degree. C., 5% CO.sub.2 for 48 hours. To determine the amount
of IL-4, IL-5 and IL-17 secreted in whole blood, 100 .mu.L of the
supernatant (conditioned media) from each well of the 96-well plate
was transferred to a storage plate. For Meso Scale Discovery (MSD)
electrochemiluminesence analysis of cytokine production, the
supernatants (conditioned media) were tested using MSD Multi-Spot
Custom Coated plates (Meso Scale Discovery, Gaithersburg, Md.). The
working electrodes on these plates were coated with seven Capture
Antibodies (hIL-2 hIL-4, hIL-5, hIL-10, hTNFa, hIFNg and hIL-17) in
advance. After blocking plates with MSD Human Serum Cytokine Assay
Diluent, and then washing with PBS containing 0.05% of BSA, 25
.mu.L/well of conditioned medium was added to wells of the MSD
plate. The plates were covered and placed on a shaking platform for
1 hr. Next, 25 .mu.L of a cocktail of Detection Antibodies in MSD
Antibody Diluent were added to each well. The cocktail contained
seven Detection Antibodies (hIL-2, hIL-4, hIL-5, hIL-10, hTNFa,
hIFNg and hIL-17) at 1 .mu.g/mL each. The plates were covered and
placed on a shaking platform overnight (in the dark). The next
morning the plates were washed three times with PBS buffer. 150
.mu.l of 2.times.MSD Read Buffer T was added to wells of the plate
before reading on the MSD Sector Imager. Since the 8 wells in
column 6 of each plate received only the thapsigargin stimulus and
no inhibitor, the average MSD response here was used to calculate
the "High" value for a plate. The calculate "Low" value for the
plate was derived from the average MSD response from the 8 wells in
column 12 which contained no thapsigargin stimulus and no
inhibitor. Percent of control (POC) is a measure of the response
relative to the unstimulated versus stimulated controls, where 100
POC is equivalent to the average response of thapsigargin stimulus
alone or the "High" value. Therefore, 100 POC represents 0%
inhibition of the response. In contrast, 0 POC represents 100%
inhibition of the response and would be equivalent to the response
where no stimulus is given or the "Low" value. To calculate percent
of control (POC), the following formula is used: [(MSD response of
well)-("Low")]/[("High")-("Low")].times.100. The potency of the
molecules in whole blood was calculated after curve fitting from
the inhibition curve (IC) and IC50 was derived using standard curve
fitting software. Although we describe here measurement of cytokine
production using a high throughput MSD electrochemillumenescence
assay, one of skill in the art can readily envision lower
throughput ELISA assays are equally applicable for measuring
cytokine production.
[0475] Ex vivo cynomolgus monkey pharmacodynamic (PD) assay to
measure the level of T cell Kv1.3 coverage in vivo following dosing
of animals. To determine the level of target coverage in monkeys
prior to or following dosing with the Kv1.3 inhibitor 20
kDa-PEG-[Lys16]ShK (SEQ ID NO:16), whole blood was removed and
tested for level of T cell cytokine secretion following
thapsigargin stimulation. Example 10 provides the results using
this assay of levels of coverage in a 12-week study in cynomolgus
monkeys, involving one month of weekly dosing with 20
kDa-PEG-[Lys16]ShK (SEQ ID NO:16). To determine level of
suppression in animals, prior to thapsigargin addition, each whole
blood sample was split into two aliquots, one aliquot being
untreated to measure level of drug in the animal and the second
aliquot spiked with an excess of 20 kDa-PEG-[Lys16]ShK (SEQ ID
NO:16; 100 nM) as a control of full suppression. After
preincubation at 37.degree. C., 5% CO.sub.2 for one hour,
thapsigargin is added to blood to stimulate cytokine secretion and
conditioned media is collected 48 hours later. A detailed
description of the procedure follows. Cynomolgus whole blood was
collected from untreated or 20 kDa-PEG-[Lys16]ShK-treated healthy,
naive, male cynomolgus monkeys by arm-pull into a heparin
vacutainer. Monkeys voluntarily presented arms for a grape
incentive, allowing for injections and blood draws in the absence
of any sedatives or stress. DMEM complete media was Iscoves DMEM
(with L-glutamine and 25 mM Hepes buffer) containing 0.1% human
albumin (Gemini Bioproducts, #800-120), 55 .mu.M 2-mercaptoethanol
(Gibco), and 1.times. Pen-Strep-Gln (PSG, Gibco, Cat#10378-016).
Thapsigargin was obtained from Alomone Labs (Jerusalem, Israel). A
10 mM stock solution of thapsigargin in 100% DMSO was diluted with
DMEM complete media to a 40 .mu.M, 4.times. solution to provide the
4.times. thapsigargin stimulus for calcium mobilization. PEG
peptide inhibitor of Kv1.3, 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16)
was prepared in DMEM complete media to a 400 nM, 4.times. final
concentration. The assay was set up in a 96-well Falcon 3075
flat-bottom microtiter plate. 100 .mu.l/well of DMEM complete media
was added to columns 1 and 2 as a negative control. Columns 3-7
received 50 .mu.l/well of DMEM complete media, while columns 8-12
received 50 .mu.l/well of the 4.times.20 kDa-PEG-[Lys16]ShK
inhibitor (400 nM) in DMEM complete media. To initiate the
experiment, 100 .mu.L of whole blood from one monkey was added to
each well of one row of the microtiter plate. The plate was then
incubated at 37.degree. C., 5% CO.sub.2 for one hour. After one
hour, the plate was removed and 50 .mu.l of the 4.times.
thapsigargin stimulus (40 .mu.M) was added to wells in columns
3-12. The plates were placed back at 37.degree. C., 5% CO.sub.2 for
48 hours. To determine the amount of IL-4, IL-5 and IL-17 secreted
in whole blood, 100 .mu.L of the supernatant (conditioned media)
from each well of the 96-well plate was transferred to a storage
plate. For MSD electrochemiluminesence analysis of cytokine
production, supernatants (conditioned media) were tested on MSD
Multi-Spot Custom Coated plates (Meso Scale Discovery,
Gaithersburg, Md.). The working electrodes on these plates were
coated with seven Capture Antibodies (hIL-2 hIL-4, hIL-5, hIL-10,
hTNFa, hIFNg and hIL-17) in advance. After blocking plates with MSD
Human Serum Cytokine Assay Diluent, and then washing with PBS
containing 0.05% of BSA, 25 .mu.l of conditioned media was added to
the MSD plate. The plates were covered and placed on a shaking
platform for 1 hr. Next, 25 .mu.L/well of a cocktail of Detection
Antibodies in MSD Antibody Diluent were added to each well. The
cocktail contained seven Detection Antibodies (hIL-2, hIL-4, hIL-5,
hIL-10, hTNFa, hIFNg and hIL-17) at 1 .mu.g/mL each. The plates
were covered and placed on a shaking platform overnight (in the
dark).The next morning the plates were washed three times with PBS
buffer. 150 .mu.l of 2.times.MSD Read Buffer T was added to the
plate before reading on the MSD Sector Imager. Since the wells in
columns 3-7 of each plate received only the thapsigargin stimulus
and had no spike with excess inhibitor, the average MSD response
here was used to calculate the "Test" value in a row for each
animal to detect the in vivo level of drug present in each animal.
The calculated "Negative Control" value for each animal, was
calculated from the average MSD response in columns 1-2 of each
row, which received neither thapsigargin nor inhibitor spike. The
"Positive Control" value for the animal was derived from the
average MSD response from the 5 wells in columns 8-12 which
contained thapsigargin stimulus and a spike of exogenous inhibitor.
Percent of Inhibition (POI) is a measure of the relative
thapsigargan-induced cytokine response of the same blood samples
either spiked with exogenous inhibitor or untreated, where 100 POI
is equivalent to the average response of thapsigargin stimulus plus
exogenous inhibitor spike. Therefore, 100 POI represents 100%
inhibition of the response. In contrast, 0 POI represents 0%
inhibition of the response. To calculate percent of inhibition
(POI), the following formula is used: [("Positive Control" ECL
counts)-("Negative Control" ECL counts)]/[("Test" ECL
counts)-("Negative Control" ECL counts)].times.100. Percent of
control (POC) is calculated using the formula, POC=(100-POI).
Although we describe here measurement of cytokine production using
a high throughput MSD electrochemilumenescence assay, one of skill
in the art can readily envision lower throughput ELISA assays are
equally applicable for measuring cytokine production.
Example 9
Adoptive-Transfer EAE Model of Efficacy
[0476] Using an adoptive transfer experimental autoimmune
encephalomyelitis (AT-EAE) model of multiple sclerosis in rats
described earlier [C. Beeton et al. (2001) J. Immunol. 166, 936],
we examined the activity in vivo of our Kv1.3 selective 20
kDa-PEG-[Lys16]ShK (SEQ ID NO:16) and compared its efficacy to the
less selective 20 kDa-PEG-ShK molecule (SEQ ID NO:8). The 20
kDa-PEG-[Lys16]ShK molecule delivered subcutaneously (SC) daily at
10, 100 and 1000 .mu.g/kg (day-1 to day 7) significantly reduced
disease severity and increased survival, whereas rats treated with
vehicle developed severe disease and died (FIG. 7 and FIG. 8A-D).
Rats treated daily with 0.04 .mu.g/kg 20 kDa-PEG-ShK also showed
significant disease amelioration.
[0477] The encephalomyelogenic CD4+ rat T cell line, PAS, specific
for myelin-basic protein (MBP) was kindly provided by Dr. Evelyne
Beraud. The maintenance of these cells in vitro and their use in
the AT-EAE model has been described earlier [C. Beeton et al.
(2001) PNAS 98, 13942]. PAS T cells were maintained in vitro by
alternating rounds of antigen stimulation or activation with MBP
and irradiated thymocytes (2 days), and propagation with T cell
growth factors (5 days). Activation of PAS T cells
(3.times.10.sup.5/ml) involved incubating the cells for 2 days with
10 .mu.g/ml MBP and 15.times.10.sup.6/ml syngeneic irradiated (3500
rad) thymocytes. On day 2 after in vitro activation,
10-15.times.10.sup.6 viable PAS T cells were injected into 6-12
week old female Lewis rats (Charles River Laboratories) by tail IV.
Daily subcutaneous injections of vehicle (2% Lewis rat serum in
PBS), 20 kDa-PEG-[Lys16]ShK or 20 kDa-PEG-ShK were given from day-1
to day 7 (FIG. 7 and FIG. 8A-D), where day-1 represents 1 day prior
to injection of PAS T cells (day 0 in FIG. 7). Serum was collected
by retro-orbital bleeding at day 4 and by cardiac puncture at day 8
(end of the study) for analysis of levels of inhibitor. Rats were
weighed on days-1 and days 4-8. Animals were scored blinded once a
day from the day of cell transfer (day 0) to day 3, and twice a day
from day 4 to day 8. Clinical signs were evaluated as the total
score of the degree of paresis of each limb and tail. Clinical
scoring ("EAE Score" in FIG. 7 and FIG. 8A-D): 0=No signs,
0.5=distal limp tail, 1.0=limp tail, 2.0=mild paraparesis, ataxia,
3.0=moderate paraparesis, 3.5=one hind leg paralysis, 4.0=complete
hind leg paralysis, 5.0=complete hind leg paralysis and
incontinence, 5.5=tetraplegia, 6.0=moribund state or death. Rats
reaching a score of 5.5 were euthanized. The in vivo activity of
other conjugates in this model, are described within the Examples
disclosed herein. Under some circumstances prior to testing peptide
conjugates for efficacy in vivo, conjugates were tested in vitro
for their activity in inhibiting antigen (myelin)-mediated
proliferation (3H-thymidine incorporation) of the rat T effector
memory cell line, PAS. The methods employed here were similar to
those described in C. Beeton et al., PNAS 98, 13942 (2001), and are
well known to those skilled in the art.
[0478] Treatment of rats with the Kv1.3 blocker 20
kDa-PEG-[Lys16]ShK (SEQ ID NO:16) prior to the onset of EAE caused
a lag in the onset of disease, inhibited the progression of
disease, and prevented death in a dose-dependent manner (FIG. 7).
Onset of disease in rats treated with vehicle alone occurred on day
4.0, whereas onset was delayed until day 4.5 to day 5.0 in animals
treated with 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16). In addition to
delaying onset, treatment with 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16)
significantly reduced disease severity in a dose-dependent manner.
Whereas vehicle treated rats on day 6 had severe disease (EAE score
of 6) and were sacrificed, animals treated with the two highest
doses of 20 kDa-PEG-[Lys16] ShK (SEQ ID NO:16) had only mild
disease and an EAE score of about 1. The 20 kDa-PEG-[Lys16]ShK (SEQ
ID NO:16) molecule provided potent blockade of experimental
autoimmune encephalomyelitis and had a calculated ED50 of about 4.4
.mu.g/kg, based on a dose response assessment of the data of day 7,
pm (FIG. 8C).
Example 10
Pharmacology Studies in Cynomolgus Monkeys; Determination of
PEG-[Lys16]ShK (SEQ ID NO:16) in Cerebral Spinal Fluid (CSF);
Toxicologic Exploration & Mast Cell Degranulation Studies
[0479] A repeat-dose pharmacology study was designed and
implemented in order to investigate the long-term effects of the 20
kDa-PEG-[Lys16]ShK molecule (SEQ ID NO:16) in nonhuman primates.
Prior to initiating the study, 6 to 10 male cynomolgus monkeys were
profiled for a period of 3-10 weeks to allow for assessment of the
end-points stability over time and selection of 6 cynos for the
study. End-points measured, included complete blood counts (CBCs),
blood chemistry, FACS analysis of lymphocyte subsets and the ex
vivo whole blood PD assay measuring cytokine response and target
coverage, as described in Example 8. Subsets analyzed by FACS
included: lymphocytes, CD4.sup.+, CD4+ naive, CD4.sup.+ T.sub.CM,
CD4.sup.+ T.sub.EM, CD4.sup.+CD28.sup.-CD95.sup.-, CD8.sup.+,
CD8.sup.+ naive, CD8.sup.+ T.sub.CM, CD8.sup.+ T.sub.EM,
CD8.sup.+CD28.sup.-CD95.sup.-, B cells, NK cells, and NKT cells.
Monkeys with the highest level of CD4+ effector memory T cells were
chosen. Using the cyno whole blood PD assay, 20 kDa-PEG-[Lys16]ShK
had an IC.sub.50 of 0.09.+-.0.08 nM for blockade of the IL-17
response and an IC.sub.50 of 0.17.+-.0.13 nM for blockade of the
IL-4 response (n=7 monkeys). A 0.5 mg/kg weekly 20
kDa-PEG-[Lys16]ShK dose was selected which would provide excess
target coverage for a period of one month. This dose, based on
early pharmacokinetic studies (see, Example 4) was estimated to
provide about 28 fold coverage (28 nM) of the 20 kDa-PEG-[Lys16]ShK
IC.sub.95 (1.0 nM) in human whole blood. Table 4F illustrates the
design of the 12-week cynomolgus pharmacology study.
[0480] Male Chinese cynomolgus monkeys were used in this study that
were naive (no earlier exposure to drugs). Care was taken to avoid
undue stress. All injections and blood draws were done by arm-pull,
with the monkeys voluntarily presenting their arm for a grape
incentive. The study involved baseline measures for two weeks (3
predose samples), one month of Kv1.3 block (qw dosing of 20
kDa-PEG-[Lys16]ShK [SEQ ID NO:16]) and 6 weeks follow-up
analysis.
[0481] Excess coverage of the Kv1.3 target for one month by 20
kDa-PEG-[Lys16]ShK was well tolerated. Clinical pathology measures
of hematology and blood chemistry showed no changes relative to
baseline measures (Table 4G) over the entire one month period.
Animal weight increased progressively over the study and
PEG-[Lys16]ShK had no impact on weight gain (FIG. 9D). The PD assay
of coverage, as measured by IL-4 and IL-17 serum concentration
(FIG. 9A-B), indicated the target was fully suppressed over the
entire one month dosing period (from week 3 though week 6) and
there was no evidence of loss of coverage over time. Based on this
finding, there was no evidence of a clearing antibody response to
the [Lys16]ShK conjugate or compensatory ion channels, since both
of these responses would be expected to result in release of the PD
suppression over time. In addition, since predicted and observed
serum drug levels of 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16) matched
closely (FIG. 9C), these data also indicate that the PEG-[Lys16]ShK
(SEQ ID NO:16) conjugate did not generate an immunogenic response
resulting in clearing antibodies over the one month dosing period.
Others have suggested that PEGylation of peptides and proteins can
reduce immunogenicity and have a beneficial effect in shielding or
protecting peptides and proteins from an immunological response (F.
M. Veronese and A. Mero, Biodrugs 22: 315 (2008)). Since ShK (SEQ
ID NO:1) and [Lys16]ShK (SEQ ID NO:13) are peptides foreign to
cynos, PEGylated toxin peptide (or toxin peptide analog)-conjugates
may have enhanced protection over the unconjugated, free peptides
alone. Alternatively, it was thought that ShK (SEQ ID NO:1) and
[Lys16]ShK (SEQ ID NO:13) may be intrinsically of low
immunogenicity since these peptides have a very small compact
structure. After dosing cessation, there was no evidence of a
"rebound" effect based on cytokine response, blood chemistry and
cellular profiling. During dosing and follow-up analysis there were
no changes in CD4 or CD8 T cell subsets, NK cells, NKT cells and B
cells relative to pre-dose, baseline measures. In conclusion, 20
kDa-PEG-[Lys16]ShK (SEQ ID NO:16) provided potent blockade of T
cell responses in cynomolgus monkeys and was well tolerated at
doses providing excess coverage of the target for an extended
period of time.
[0482] Although the 12-week pharmacology study on PEG-[Lys16]ShK
(SEQ ID NO:16) indicated weekly dosing for one month provided
sustained coverage and expected drug levels, further analysis of
serum samples from this study indicated 4 of the 6 monkeys had
anti-drug antibodies which emerged during the third week of dosing
(week 5, Table 4F) and peaked at week 9. To examine further the
immunogenicity of PEG-[Lys16]ShK (SEQ ID NO:16), 5 of the 6
cynomolgus monkeys dosed more than a year earlier with
PEG-[Lys16]ShK (SEQ ID NO:16) were available and re-challenged with
drug. The study involved baseline measures for two weeks, 0.5 mg/kg
PEG-[Lys16]ShK (SEQ ID NO:16) weekly dosing for seven weeks and
four weeks of follow up analysis. Pharmacokinetics were measured
after the first and sixth week of dosing, and serum and blood were
collected weekly throughout the study to measure drug exposure,
CBCs and blood chemistry, levels of anti-drug antibody and the
pharmacodynamic level of target coverage. PEG-[Lys16]ShK (SEQ ID
NO:16) was well tolerated upon re-challenge. All monkeys gained
weight over the course of the study and there were no changes in
CBCs or blood chemistry. Upon re-challenge, an antibody response in
3 of the 5 animals with binding specificity against the [Lys16]ShK
peptide was detected, and antibody concentrations to the drug
exceeding 10 .mu.g/ml were observed in two monkeys. These 2 animals
also tested positive for neutralizing antibodies in a bioassay. In
the re-challenge study, after the sixth dose of PEG-[Lys16]ShK (SEQ
ID NO:16) (0.5 mg/kg, weekly), exposure as measured by area under
the curve (AUC.sub.0-96h) was observed to increase by 1.8, 2.0,
3.0, 8.8 and 9.2 fold in the five monkeys, using an LC-MS/MS method
detecting total drug levels. The changes in AUC for PEG-[Lys16]ShK
(SEQ ID NO:16) upon multiple dosing were also accompanied by
increases in Cmax and apparent half-life. While higher than
expected accumulation occurred in all animals, the highest AUC
increases (8.8 and 9.2 fold) were observed in animals having the
highest antibody concentration. The same two monkeys exhibited
extended whole blood pharmacodynamic (PD) coverage after dosing
cessation, which was consistent with the higher exposure to
PEG-[Lys16]ShK (SEQ ID NO:16). It can be concluded that the
development of anti-drug antibodies in these two animals may have
been associated with the excessive accumulation of drug and
extended PD impact in these animals. Because more, not less,
pharmacodynamic coverage was observed in the two animals showing
anti-drug antibodies, it is possible that prolonged suppression of
the Kv1.3-dependent whole blood PD response is caused by both
elevated drug levels and a PEG-ShK[Lys16] (SEQ ID NO:16) affinity
for T-cell Kv1.3 that is higher than the PEG-ShK[Lys16] (SEQ ID
NO:16) affinity for the antibody. Further studies are necessary to
clarify this.
[0483] Despite the occurrence of anti-drug antibodies in monkeys,
it is unclear whether a similar response would occur in humans
since preclinical immunogenicity is not predictive of human
immunogenicity (Ponce et al. Regulatory Toxicology and Pharmacology
54, 164-182 (2009)). Exenatide represents an example of an approved
drug for treatment of type 2 diabetes mellitus that shows anti-drug
antibodies in humans, but remains safe and effective (Faludi et al.
Peptides 30, 1771-1774 (2009); Malone et al. Expert Opin. Investig.
Drugs 18, 359-367 (2009); Schnabel et al. Peptides 27, 1902-1910
(2006)).
[0484] Determination of PEG-[Lys16]ShK (SEQ ID NO:16) in Cerebral
Spinal Fluid (CSF). To address concerns that ShK peptide-based
inhibitors have some propensity for non specific targeting of the
Kv1.1 (a potassium channel that has been shown to be important in
the regulation of neuronal activity in the brain), we evaluated the
ability of PEG-[Lys16]ShK (SEQ ID NO:16) to enter cerebral spinal
fluid (CSF). PEG-[Lys16]ShK (SEQ ID NO:16) concentrations were
evaluated in the CSF of cynomolgus monkeys and Sprague-Dawley rats.
In one study, PEG-[Lys16]ShK (SEQ ID NO:16) was measured in the CSF
of monkeys 24 and 48 hours after subcutaneous administration. These
data are shown in Table 4G(a) below. CSF concentrations were small
in comparison to serum concentrations for both monkeys and rats. As
shown in Table 4G(a), the highest concentration of PEG-[Lys16]ShK
(SEQ ID NO:16) measured in the CSF of monkeys was 7.62 ng/mL. This
sample was observed visually to be contaminated with blood. The
highest concentration measured in an uncontaminated sample was 3.83
ng/mL, which represents a serum (ng/mL) to CSF (ng/mL) ratio of
292. Table 4G(b) shows the CSF concentrations of PEG-[Lys16]ShK
(SEQ ID NO:16) measured in rats. The highest concentrations were 43
ng/mL and 12.7 ng/mL, representing serum (ng/mL) to CSF (ng/mL)
ratios of 46.5 and 470, respectively. Both these samples were taken
from rats sacrificed early in the study (6 hrs). Of the rats that
survived to the end of the study (48 hr), only one had an
PEG-[Lys16]ShK (SEQ ID NO:16) concentration that could be detected
above the assay limit of quantitation (1 ng/mL). This CSF sample
had a concentration of 5.70 ng/mL, which was nearly identical to
the measured serum concentration, making it an outlier compared to
all other samples and supporting the conclusion that it was also
contaminated with serum during sampling.
[0485] Mast cell degranulation studies. Because the native peptide
ShK impacts Kv1.3 and neuronal Kv1.1, concerns have been expressed
thata small fraction of drug crossing the blood-brain barrier could
cause off-target impacts on neuronal Kv1.1 and paralysis (Kalman et
al., J. Biol. Chem. 273, 32697-32707 (1998)). In pharmacokinetic
studies of ShK, PEG-ShK and PEG-[Lys16]ShK (SEQ ID NO:16) in
Sprague-Dawley rats, we noted severe adverse events immediately
following injection of large IV bolus doses. Shortly after 2.0 and
5.0 mg/kg bolus IV injections of PEG-[Lys16]ShK (SEQ ID NO:16)
which generated serum C.sub.0/C.sub.max levels greater than 10,000
ng/ml (>2500 nM) which were more than 25,000 times the molecules
IC.sub.50 in blocking T cell cytokine production in whole blood
(0.1 nM), rats were moribund and at the bottom of the cage. Two of
the three animals died at the 5 mg/kg high dose group, whereas all
animals receiving the 2 mg/kg dose survived. Other observations
included discolored skin (blue paws, ears) and thick blood.
Initially these transient effects were attributed to an impact on
neuronal Kv1.1, however, treated rats exhibited no seizures,
expressed other symptoms inconsistent with a CNS impact (e.g. thick
blood) and the Kv1.3 selective PEG-[Lys16]ShK conjugate (SEQ ID
NO:16) caused similar effects despite being 1000 fold less potent
on Kv1.1. In single-dose subcutaneous pharmacokinetic studies in
rats, whereas animals receiving low doses (0.1 and 0.5 mg/kg)
appeared normal and gained weight, 2 of 3 animals treated with 5.0
mg/kg were lethargic, lost weight and exhibited mild swelling of
the forelimb and hindlimb 24 hours post-dose. The occurrence of
symptoms 24 hours post-dose correlated with Cmax and the animals
recovered over time and gained weight. Interestingly, these effects
were not observed in mouse, cyno or dog pharmacokinetic studies
despite equal or higher exposure. In an attempt to understand the
unique findings in rats, literature was reviewed for studies that
described molecules that produced related symptoms and exhibited
species selectivity. Basic secretagogues, such as mast cell
degranulating peptide (MCDP), were reported to show a differential
impact on mast cells from different species, with rat showing
increased sensitivity to some cationic molecules (Barrett &
Pearce Int. Archs. Allergy appl Immun. 72, 234-238 (1983); M.
Mousli et al. Immunopharmacology 27, 1-11 (1994); Keller Int. Arch.
Allergy 34, 139-144 (1968); Leung & Pearce Br. J. Pharmac. 81,
693-701 (1984)). Since ShK is a basic peptide like MCDP and mast
cell degranulation could cause swelling, discolored skin and
vascular collapse, we embarked on studies to test the hypothesis
that the adverse events in rats following large bolus doses of
PEG-[Lys16]ShK are due to mast cell degranulation and an
anaphylactoid response. Sera from rats receiving 2 and 5 mg/kg
bolus IV or subcutaneous injections of PEG-[Lys16]ShK were tested
for levels of histamine. As shown in FIG. 37, histamine levels were
profoundly elevated 5 and 15 minutes following bolus IV injection
of PEG-[Lys16]ShK achieving levels that were greater than 2,000
ng/ml which quickly declined over time. This level of histamine
would be expected to cause anaphylactoid shock and vascular
collapse. Subcutaneous administration of PEG-[Lys16]ShK at doses of
0.1, 0.5, 2.0 and 5.0 mg/kg produced 24 hour post-dose C.sub.max
drug levels of 52 ng/ml (13 nM), 233 ng/ml (58 nM), 988 ng/ml (247
nM) and 2570 ng/ml (642 nM) corresponding to drug levels that were
130-6420 times greater than the molecules IC.sub.50 (0.1 nM) in the
whole blood pharmacodynamic assay of target coverage. As shown in
FIG. 38, at the 2.0 and 5.0 mg/kg dose groups, a slight elevation
in serum histamine was observed 24-48 hours after dosing. In the 5
mg/kg highest dose group, the two animals (rat #10 and #12) with
mild swelling of the limbs exhibited increased serum histamine,
whereas the animal (rat #11) without these symptoms did not show
significant histamine elevation.
[0486] To examine further the impacts of PEG-[Lys16]ShK in rats,
further toxicology studies were undertaken. In addition, toxicology
was assessed in a second species, cynomolgus monkeys. In vitro
studies were also performed to directly assess the effect of
PEG-[Lys16]ShK on mast cells. Further details on these studies are
provided in the sections below.
[0487] In one toxicology study, PEG-[Lys16]ShK (SEQ ID NO:16) was
administered by intravenous infusion (over 30 minutes) to male
Sprague Dawley rats at single doses of 0.04, 0.12, 0.2, 0.4, 1.36,
and 4.0 mg/kg (n=1-3/dose group). At 4.0 mg/kg, 1 animal was found
dead and the other was euthanized due to clinical signs of
hypoactivity/lethargy, lateral recumbence, discolored skin (blue
paws, tail, and/or ears), and coldness to touch. Hypoactivity and
lateral recumbence were also observed at 0.4 mg/kg. The 1 animal
given 1.36 mg/kg was euthanized due to clinical signs of swelling
and discolored skin. Swelling was observed in all test
article-treated animals, except for the 1 animal dosed at 0.12
mg/kg. The severity of swelling was not dose dependent, and all
clinical observations noted in all groups that survived to their
scheduled euthanasia resolved by approximately 24 hours postdose.
At 4.0 and 1.36 mg/kg, test article-related effects included
degeneration/necrosis of renal tubular epithelium,
necrosis/depletion of thymic lymphocytes, inflammation (increased
neutrophils and monocytes), and altered fluid homeostasis
(dehydration, decreased sodium and chloride, and decreased albumin
and globulin). In animals given 0.4 and 4.0 mg/kg, there were
vacuoles in the renal tubular epithelium, similar to those
described following administration of pegylated biomolecules
(Bendele et al. Short Communication Renal tubular vacuolation in
animals treated with polyethylene-glycol-conjugated proteins, Tox
Sci. 42:152-157 (1998)). Other changes noted at lower doses were
limited to decreased lymphocytes and triglycerides at 0.4
mg/kg.
[0488] In another toxicology study, PEG-[Lys16]ShK (SEQ ID NO:16)
was administered by subcutaneous injection once weekly to male
Sprague Dawley rats (n=5/dose group) at doses of 0.1, 0.5, or 2.0
mg/kg/dose for 14 days (2 doses). At the 0.5- and 2.0-mg/kg dose
levels, all animals showed clinical signs of discolored skin (red),
which was more severe in the high dose group. In addition,
chromorhinorrhea was noted in animals dosed at 2.0 mg/kg. All
clinical signs were resolved by day 11. A decrease in body weight
was noted in the 2-mg/kg dose group 48 hours after both doses and a
decrease in body weight gain (days 1-14) was noted in the 2.0 mg/kg
dose group compared with controls. Hematologic effects in animals
given .gtoreq.0.5 mg/kg/day were consistent with increased red cell
turnover, and correlated with increased spleen weights, and
increased splenic hemopoiesis. Increased neutrophils and
eosinophils were noted in all test article-treated animals.
Increased bone marrow eosinophil hematopoeisis and splenic
hemopoiesis was noted histologically at all doses of PEG-[Lys16]ShK
(SEQ ID NO:16).
[0489] In another toxicology study, PEG-[Lys16]ShK (SEQ ID NO:16)
was administered by subcutaneous injection to male Sprague Dawley
rats (n=5/dose group) at doses of 0.1, 0.3, 0.7, 2.5, or 5.0
mg/kg/dose once every 72 hours for 12 days (4 doses). Exposures
increased with increasing dose up to 2.5 mg/kg, but exposures were
similar in rats given 2.5 and 5.0 mg/kg. All animals survived the
12-day treatment period. Redness in the ears and paws was observed
in all animals 24 hours after each dose in the 0.3, 0.7, 2.5 and
5.0 mg/kg/dose groups. In the 0.1 mg/kg/dose group, one of five
animals showed redness, but only after the second dose. There was a
decrease in body weight and body weight gain noted in the 2.5 and
5.0 mg/kg/dose groups. Hematologic effects were noted at
doses.gtoreq.2.5 mg/kg/day and were consistent with increased red
cell turnover. An increase in platelet production was noted at
doses.gtoreq.0.7 mg/kg/dose. In the kidney, vacuoles were noted in
the proximal convoluted tubules of rats given 5 mg/kg/day. Urine
chemistry values were increased in a few animals/group at 0.5 or
2.0 mg/kg/day, but these changes were inconsistent among and within
animals, and considered unlikely to be related to treatment. There
were no test article-related effects on biomarkers of renal injury
(beta-2 microglobulin, calbindin, clusterin, cystatin C,
glutathione S-transferase alpha, kidney injury molecule-1,
neutrophil gelatinase-associated lipocalin, osteopontin, tissue
inhibitor of metalloproteinase-1, and vascular endothelial growth
factor). There was a dose-dependent increase in incidence/severity
of hematopoiesis in the bone marrow, spleen, liver, and mesenteric
lymph node of treated rats. This change was more prominent in the
spleen and bone marrow compared with the liver and mesenteric lymph
node. In the spleen, the increased hematopoiesis was composed of
erythroid, myeloid, and platelet precursors; whereas in the bone
marrow, it was primarily composed of myeloid precursors. The
majority of the myeloid precursors in the spleen and bone marrow
were associated with eosinophil precursors. In the liver and
mesenteric lymph node, hematopoiesis was composed of scattered
groups of erythroid and eosinophil precursors. In the tibia and/or
femur, there was osteoblastic new bone formation in the medullary
canal, which was primarily composed of woven bone. In addition,
there was increased porosity of cortical bone accompanied by a
subtle increase in woven bone. In the adrenal gland of most treated
rats, there was minimal hypertrophy of the cortex. In 1 rat given
5.0 mg/kg/dose, there was erosion of the glandular epithelium. In
the kidney, vacuoles were noted in the proximal convoluted tubules
of rats given 5 mg/kg/dose. These vacuoles are consistent with
PEG-associated renal vacuoles and were not associated with any
other renal tubular change (Bendele et al. Short Communication:
Renal tubular vacuolation in animals treated with
polyethylene-glycol-conjugated proteins, Tox Sci. 42:152-157
(1998)).
[0490] In another toxicology study, a single subcutaneous dose of
PEG-[Lys16]ShK (SEQ ID NO:16) (1 mg/kg in A4S and DPBS vehicle),
relatively Kv1.3-inactive PEG-[1-Nal 16]ShK (SEQ ID NO:159) (1
mg/kg), anti-KLH Ab-[Lys16]ShK (heterotetramer of SEQ ID NOS:338,
339, 338, 342) (37 mg/kg), Fc/Fc-[Lys16]ShK (heterodimer of SEQ ID
NOS:337, 348) (14 mg/kg), or PEG (5 mg/kg) was administered to male
Sprague Dawley rats (n=3/dose group). There was no mortality nor
effects on body weights in test article-treated rats. The dose
given to each group was intended to provide an equimolar equivalent
of the ShK peptide or an equivalent load of PEG. On a molar basis,
measured AUC exposure for groups given PEG-[Lys16]ShK (SEQ ID
NO:16) (in A4S and DPBS) and anti-KLH Ab-[Lys16]ShK were roughly
equal. Compared with PEG-[Lys16]ShK (SEQ ID NO:16) (in A4S and
DPBS) and anti-KLH Ab-[Lys16]ShK (heterotetramer of SEQ ID NOS:338,
339, 338, 342), molar AUC exposures for the groups given PEG-[1-Nal
16]ShK (SEQ ID NO:159) and Fc/Fc-[Lys16]ShK (heterodimer of SEQ ID
NOS:337, 348) were .about.6-8.times. and .about.0.5.times.,
respectively. Rats given PEG-[Lys16]ShK (SEQ ID NO:16) in both A4S
and DPBS had red ears/paws, while rats given PEG alone did not.
Rats in all treatment groups given a ShK construct had similar
pathology findings. In the bone marrow, there were increased
numbers of cells of the myeloid series (increased myeloid:erythroid
ratio), many of which were eosinophils. In the spleen, there was
increased hemopoiesis with equal erythroid and myeloid cell
populations. Increased levels in serum histamine were noted in all
groups given a ShK construct. This study demonstratesd that
different constructs (PEG, Fc/Fc, anti-KLH) using the active or
inactive ShK moiety can cause similar changes in rats. Further,
these changes were independent of the vehicle used and are not
caused by PEG alone.
[0491] In a separate study, PEG-[Lys16]ShK (SEQ ID NO:16) at 5
mg/kg was administered as a single subcutaneous injection to male
Sprague Dawley, Lewis, Wistar, or Fischer rats (n=3/dose
group/strain) to determine if the changes in previous studies were
rat strain specific. There were no mortalities or test
article-related effects on body weight. All 4 rat strains exhibited
test article-related clinical observations of chromorhinorrhea,
chromodacryorrhea, and/or discolored skin (red), and had
statistically significant increases in plasma histamine levels
compared with their respective vehicle control group. Although
there were some minor differences in mean exposures (AUC.sub.0-48)
and C.sub.max between strains, the PEG-[Lys16]ShK (SEQ ID NO:16)
serum concentration profiles were generally similar for all 4 rat
strains. Plasma fibrinogen was consistently increased to a similar
degree in all strains, and was usually accompanied by other
indicators of inflammation, including increased neutrophils and
globulins and decreased albumin. One Fischer rat had adrenal
necrosis and focal degeneration/necrosis of the papillary muscle in
the heart. A similar but less severe change in the adrenal gland of
2 Wistar rats was minimal, multifocal necrosis of individual
cortical cells. In the Sprague Dawley, Lewis and Wistar strains,
there was a cellular infiltrate in the adrenal cortex and/or
medulla. This infiltrate was either an inflammatory response to
adrenal cortical necrosis, extramedullary hematopoiesis, or a
combination of inflammation and hematopoiesis. Adrenal cortex
hypertrophy was noted in the Sprague Dawley and Wistar strains,
which may be due to physiologic stress and/or the effects of mast
cell degranulation and histamine release on ACTH and corticosterone
secretion (Bugajski et al., Influence of cyclooxygenase inhibitors
on the central histaminergic stimulation of
hypothalamic-pituitary-adrenal axis, J Phys and Pharm.
54(4):643-652 (2003)). There was lymphoid depletion in the cortex
of the thymus, the periarteriolar lymphoid sheaths of the spleen,
and the paracortex of the mesenteric lymph node. Lymphoid depletion
was most pronounced in the thymus of the Fischer rat, whereas
lymphoid depletion in the spleen and lymph node occurred in the
Sprague Dawley, Lewis, and Wistar strains. Additional changes in
the spleen included increased hemopoiesis, which involved both
erythroid and myeloid series and generally correlated with
increased spleen weight in the Sprague Dawley, Lewis and Wistar
strains, and sinus neutrophilia, which correlates with the
increased neutrophils in the CBC. There was increased cellularity
in the bone marrow, which was primarily due to an increase in early
myeloid progenitors. Kupffer cells in the livers of Sprague Dawley
and Wistar strains were prominent, which is likely due to increased
activity (e.g. increased phagocytosis, release of cytokines). In
the ear pinna of all treated rats, small veins in the superficial
dermis were congested and the endothelium was lined by marginated
neutrophils; this change is related to the clinical observation of
red skin. In the deep dermis/subcutis of the injection site of
treated rats, there was an inflammatory infiltrate composed
primarily of macrophages, with fewer lymphocytes and neutrophils.
The nature of the inflammatory infiltrate is consistent with the
injection of foreign material into the subcutis. A less severe
inflammatory infiltrate was noted in controls and was primarily
composed of lymphocytes. In the kidney, vacuoles were noted in the
proximal convoluted tubules of all treated rats at this 5 mg/kg
dose of PEG-[Lys16]ShK (SEQ ID NO:16). These vacuoles are
consistent with PEG-associated renal vacuoles, and were not
associated with any other renal tubular change (Bendele,
ibid.).
[0492] In an assessment of positive wheal and flare reactions,
PEG-[Lys16]ShK (SEQ ID NO:16) was administered by intradermal
injection to male Sprague Dawley rats at various dose levels
ranging between 0.4 to 40,000 pmol in a 50 .mu.l injection volume.
To determine the incidence of positive wheal reactions at 30
minutes post-dose, a total of 8 rats on two separate days were
given PBS negative control and a series of doses ranging from 0.4
to 4,000 pmol. To determine whether intradermal injection could
induce a systemic response, groups of 3 rats were given a single
dose of 40, 400, 4,000 or 40,000 pmol. Mean exposures
(AUC.sub.0-24) were roughly dose proportional from 400 to 40,000
pmol for PEG-[Lys16]ShK (SEQ ID NO:16), but there was no systemic
exposure measured at 40 pmol. There were no mortalities during the
study. Thirty minutes after intradermal injection, there was a
noticeable wheal reaction (edema), the size of which was dose
dependent. There was no flare reaction (redness due to vascular
congestion) noted after 30 minutes, but redness at the injection
site was noted at 8 and 24 hours postdose and correlated with
inflammation noted histologically. Positive wheal reactions were
noted in individual animals at doses.gtoreq.0.4 pmol. The incidence
of positive wheal reactions was dose responsive, and it plateaued
(100% incidence) at 400 pmol. The increase in wheal area and wheal
thickness was also dose responsive. Dispersion of mast cell
granules and margination/congestion of blood vessels were noted
histologically at dose levels of 40, 400, and 4,000 pmol 30 minutes
postdose. At 24 hours postdose coagulative necrosis of the dermis
and epidermis was noted in 2 of 3 animals given 40,000 pmol. In
addition, doses of 400, 4,000, and 40,000 were associated with test
article-related inflammation, and mast cells were no longer
apparent in toluidine blue stained sections. Compared with the
inflammation noted at 24 hours, there was a significant reduction
in inflammation at 72 hours and no inflammation at 168 hours.
Increased fibrinogen and histamine levels in rats dosed with 40,000
pmol suggest that systemic exposure in this group induced a
systemic response. The NOEL for the systemic reaction was 4,000
pmol. This study demonstrates that there is a large margin between
a wheal reaction and a systemic response, suggesting that relevant
intradermal doses could be administered to humans with low risk of
an undesirable systemic reaction.
[0493] In a further rat study, PEG-[Lys16]ShK (SEQ ID NO:16) was
administered by subcutaneous injection to telemeterized male
Sprague Dawley rats (n=3/dose group) at doses of 0.7 and 2.5 mg/kg
and mean arterial pressure and heart rate were monitored
continuously for 24 hours. At the 24 hour timepoint after dosing,
there was a dose-dependent decrease in mean arterial pressure and
an increase in heart rate (reflex-mediated) relative to
vehicle-treated rats (Table 4G(c)). In addition, PEG-[Lys16]ShK
(SEQ ID NO:16) exhibited clinical observations of redness, swelling
and pinna irritation, which were considered consistent with
histamine release.
[0494] In another study, a dose of 2.5 mg/kg PEG-[Lys16]ShK (SEQ ID
NO:16) was administered by subcutaneous injection to telemeterized
female Sprague Dawley rats (n=5/dose group) following pretreatment
with Compound 48/80, which is known to cause mast cell
degranulation and depletion of granule contents (Blom et al., A
method for determining whether hypotension caused by novel
compounds in preclinical development results from histamine
release. J Pharm and Toxicol Methods, 49:31-37 (2004)).
Pretreatment with Compound 48/80 caused complete inhibition of
PEG-[Lys16]ShK (SEQ ID NO:16)-induced hypotension. The cause of the
decreased mean arterial pressure and was attributed to mast cell
degranulation, and the increased heart rate was attributed to a
reflex response.
[0495] In summary, the adverse observations at higher doses in the
in vivo rat toxicology studies were consistent with the biologic
consequences of mast cell activation/degranulation and the release
of a variety of preformed and/or synthesized inflammatory
mediators. Histamine level is a reliable marker of mast cell
degranulation, and was elevated in some animals treated with
test-article. Histamine is a potent vasoactive mediator that
produces increases in blood flow and vasodilation, which explains
many of the clinical observation, as well as the results of the
telemetry studies. Histamine can also produce gastric ulcers in
rats (Hase et al., Prostaglandin E2 aggravates gastric mucosal
injury induced by histamine in rats through EP1 receptors, Life
Sci. 74:629-641 (2003)), and increase ACTH and corticosterone
secretion, which may have caused adrenal hypertrophy (Bugajski et
al., Influence of cyclooxygenase inhibitors on the central
histaminergic stimulation of hypothalamic-pituitary-adrenal axis, J
Phys and Pharm. 54(4):643-652 (2003)). Histamine may play a role in
bone metabolism and has been shown to regulate osteoclastogenesis,
which may have caused the bone changes following repeat dosing
(Biosse-Duplan et al., Histamine promotes osteoclastogenesis
through the differential expression of histamine receptors on
osteoclasts and osteoblasts. Am J Pathol. 174:1426-1434 (2009)).
Other mast cell-derived cytokines, such as IL-3, IL-5 and GM-CSF,
are important in eosinophil maturation and chemotaxis, and can
promote eosinophil hematopoiesis in tissues, which was noted in
several studies (Shakoory et al., The role of human mast
cell-derived cytokines in eosinophil biology, J Interferon and
Cytokine Res. 24:271-281 (2004)).
[0496] Interestingly, the putative mast cell related effects, which
were observed in Sprague-Dawley,Lewis, Wistar, and Fischer rat
strains, were not observed in mouse, cynomolgus monkey, or dog
studies despite equal or higher exposure than in the rats.
[0497] For example, in a study in mice, PEG-[Lys16]ShK (SEQ ID
NO:16) was administered daily by subcutaneous injection to female
C57BL/6 mice (n=3/dose group) at doses of 0.6, 2.5, or 5 mg/kg/day
for 14 days. PEG-[Lys16]ShK (SEQ ID NO:16) exposure at the end of
day 14 was 833.+-.185 ng/ml (208.+-.46 nM), 10066.+-.2219 ng/ml
(2516.+-.555 nM), and 31334.+-.5800 ng/ml (7834.+-.1450 nM) for
mice receiving daily doses of 0.6, 2.5 and 5.0 mg/kg, respectively.
All mice survived the dosing period. No test article-related
clinical observations or body weight changes were noted in any
animal. There was dose-dependent vacuole formation in the renal
tubular epithelium, and no vacuole formation in hepatic sinusoidal
macrophages or resident macrophages in mesenteric lymph node and
spleen.
[0498] In another study in cynomolgus monkey, PEG-[Lys16]ShK (SEQ
ID NO:16) was administered by subcutaneous injection to male
cynomolgus monkeys (n=3/dose group) at doses of 0.7 mg/kg every
third day or 0.1, 0.5, or 2.0 mg/kg weekly for 2 weeks. No adverse
effects were seen in cynomolgus monkeys administered PEG-[Lys16]ShK
(SEQ ID NO: 16). There were no treatment-related effects on body
weight. There were no test article-related organ weight changes, or
macroscopic or microscopic findings. Further, no qualitative or
quantitative electrocardiogram (ECG) abnormalities were
attributable to administration of PEG-[Lys16]ShK (SEQ ID NO:16) at
doses of 0.1, 0.5, or 2 mg/kg/dose. The only findings considered
possibly test-article related were minimal increases in serum urea
nitrogen and serum creatinine for animals given 0.7 mg/kg every
third day. No histopathologic correlates to the clinical pathology
changes were observed. Renal vacuoles were not observed, and there
were no changes in urinary biomarkers of renal injury (alpha-1
microglobulin, beta-2 microglobulin, calbindin, clusterin,
connective tissue growth factor, creatinine, cystatin C,
glutathione S-transferase alpha, kidney injury molecule-1,
microalbumin, neutrophil gelatinase-associated lipocalin,
osteopontin, Tamm-Horsfall protein, tissue inhibitor of
metalloproteinase-1, trefoil factor 3, and vascular endothelial
growth factor). Thus, the No-Observable-Adverse-Effect-Level in
cynomolgus monkey is >2 mg/kg. In this study, the 2 mg/kg dose
group had a mean AUC value of 584,000 ng.times.hr/mL. Binding
antibodies against PEG-[Lys16]ShK (SEQ ID NO:16) were detected in
17% (2/12) of the PEG-[Lys16]ShK (SEQ ID NO:16) treated animals.
Both of the antibody-positive animals were in the 0.5 mg/kg dose
group. The detected antibodies were capable of binding to both
PEG-[Lys16]ShK (SEQ ID NO:16) and the ShK peptide (SEQ ID NO:1).
Antibodies were not detectable in the low or high dose groups.
[0499] Renal tubule vacuolization is a well recognized consequence
of dosing with PEGylated compounds. It is noteworthy that
PEG-[Lys16]ShK (SEQ ID NO:16) caused renal vacuoles in mice and
rats (NOEL=.about.20.times.), but not in cynomolgus monkeys
(NOEL>.about.150.times.). Moreover, there was no evidence of
overt renal toxicity, including the evaluation of sensitive
biomarkers of renal toxicity, in any species.
[0500] In an additional study of wheal and flare reactions,
cynomolgus monkeys (n=3) received intradermal injections of 0.01,
0.1, 1.0 and 10 .mu.g Compound 48/80 (positive control for mast
cell degranulation) and 0.4, 4, 40, 400, 4000, 40000 pmol
PEG-[Lys16]ShK (SEQ ID NO:16) per dose site. The two compounds were
given at least 7 days apart and each animal received the complete
range of doses on a single day. Positive wheal reactions were noted
at 30 minutes post-dose in all 3 animals at the dose sites
receiving 10 .mu.g of Compound 48/80, and 40,000 pmol of
PEG-[Lys16]ShK (SEQ ID NO:16). The 40,000 pmol of PEG-[Lys16]ShK
(SEQ ID NO:16) dose sites were also positive at 4 hours post-dose.
One of three dose sites receiving 4,000 pmol of PEG-[Lys16]ShK (SEQ
ID NO:16) had a positive reaction 30 minutes post-dose only. Wheal
reactions were not observed at dose sites receiving 0.4 to 400 pmol
PEG-[Lys16]ShK (SEQ ID NO:16). Compound 48/80-induced wheal
reactions were larger, but persisted for a shorter duration than
the PEG-[Lys16]ShK (SEQ ID NO:16)-induced wheal reactions.
Histologically, there were no PEG-[Lys16]ShK (SEQ ID NO:16)-related
findings. In contrast, Compound 48/80 caused slight inflammation in
sites given 10 .mu.g, and there were decreased toluidine blue
positive mast cells at sites given .gtoreq.0.1 .mu.g. Compared to
the positive wheal reactions in the rat, the positive wheal
reactions in the cynomolgus monkey were smaller and not associated
with histologic evidence of a biologic effect. Based on the data
from this study, it is uncertain if the PEG-[Lys16]ShK (SEQ ID
NO:16)--induced wheal reactions in the cynomolgus monkey are due to
mast cell degranulation followed by edema, or to the injection of
high concentration of PEGylated peptide.
[0501] Telemeterized male cynomolgus monkeys were used to evaluate
the impact of PEG-[Lys16]ShK (SEQ ID NO:16) on mean arterial
pressure, heart rate and cardiac intervals. Several weeks prior to
the administration of test article, each animal was treated with
vehicle (5 ml/kg) or Compound 48/80 (0.3 or 3 mg/kg SC) to validate
the animal model. Subcutaneous injection of 3 mg/kg Compound 48/80
caused a reduction in systolic and mean arterial pressure in the
cynomolgus monkey, whereas administration of vehicle did not have
any impact on these parameters. After validation of the animal
model, PEG-[Lys16]ShK (SEQ ID NO:16) was administered at doses of
0.5 or 2.0 mg/kg and monitored continuously for 72 hours.
PEG-[Lys16]ShK (SEQ ID NO:16) did not have any effect on
hemodynamics or cardiac intervals compared to vehicle control
treatment.
[0502] Mast cell degranulation leading to a systemic response
occurred at low exposures of PEG-[Lys16]ShK (SEQ ID NO:16) in the
rat. In contrast, very high exposures in the cynomolgus monkey
showed no adverse effects and no concrete evidence of the
biological consequences of mast cell degranulation. It is unknown
whether humans will be sensitive to ShK-induced mast cell
degranulation; however, the intradermal studies demonstrate an
opportunity to explore this with low risk of a systemic response in
dosed humans.
[0503] To examine further the impacts of PEG-[Lys16]ShK (SEQ ID
NO:16) on mast cells, isolated peritoneal mast cells from rat and
mice, as well as, human CD34-derived mast cells were tested to
assess mast cell degranulation and histamine release in vitro.
PEG-[Lys16]ShK (SEQ ID NO:16), monovalent aKLH HC-ShK(1-35, Q16K)
Ab and monovalent Fc-L10-ShK(1-35, Q16K) caused degranulation of
rat peritoneal mast cells (FIG. 39), similar to positive control
basic secretagogues MCDP, compound 48/80 and substance P.
Importantly, PEG-[Lys16]ShK (SEQ ID NO:16) and monovalent
Fc-L10-ShK(1-35, Q16K) caused little to no degranulation of human
mast cells (FIG. 40), whereas the basic secretagogues MCDP,
compound 48/80 and substance P were highly active. To further
assess the species specificity of PEG-[Lys16]ShK-induced mast cell
degranulation, it was also tested for activity against mouse
peritoneal mast cells and mast cells from a second strain of rat.
PEG-[Lys16]ShK (SEQ ID NO:16) caused degranulation of peritoneal
mast cells from both strains of rats, but was inactive against
mouse peritoneal mast cells and human mast cells (FIG. 41). To
confirm that PEG-[Lys16]ShK degranulation of rat peritoneal mast
cells was a result of an off-target impact and not due to Kv1.3 or
another ShK-sensitive potassium channel, electrophysiology studies
were performed on rat peritoneal mast cells. As shown in FIG.
54A-B, rat peritoneal mast cells did not show an outward current
resembling Kv1.3, and the observed current was insensitive to high
concentrations of the potent Kv1.3 inhibitors PEG-[Lys16]ShK and
charybdotoxin (ChTx). The ShK analog, ShK-192 (SEQ ID NO:438),
which is a selective Kv1.3 inhibitor (Pennington et al. Molecular
Pharmacology 75, 762-773 (2009)) also caused degranulation of rat
mast cells through this off-target pathway, but did not degranulate
mouse or human mast cells. ShK-192 (SEQ ID NO:438) exhibited
greater potency in inducing rat mast cell degranulation (EC.sub.50
about 0.25 uM) than the larger 20 kDa-PEG-[Lys16]ShK (SEQ ID NO:16)
molecule (EC.sub.50 about 1.63 uM), however, neither molecule
showed any significant degranulation of human or mouse mast cells.
The micromolar EC.sub.50 of PEG-[Lys16]ShK (SEQ ID NO:16) for
degranulation of rat peritoneal mast cells implies that high
concentrations of drug would be necessary to cause 50% or more of
the mast cells to degranulate in vivo. Based on pharmacokinetic
studies in rats, this level of drug would occur only after large
bolus doses. Indeed, severe overt anaphylactoid shock and vascular
collapse were only observed when high serum drug concentrations
were achieved. Due to the fact that human mast cells appear orders
of magnitude less sensitive than rat, even higher drug levels might
be necessary in human, making this unlikely to occur. Irrespective,
the 0.1 nM IC.sub.50 of PEG-[Lys16]ShK (SEQ ID NO:16) in blocking
Kv1.3-dependent human T cell responses is at least 10,000 times
lower than its EC.sub.50 in activating rat mast cells (1000-2000
nM), making it conceivable that there would be a good safety margin
against mast cells from a less sensitive species, such as
human.
[0504] The Mas-related gene receptors (Mrg), were originally
identified as a family of orphan GPCRs expressed in a specific
subset of nociceptive sensory neurons that may serve a role in
modulating sensation and pain (Dong et al., Cell 106, 619-632
(2001)). Recently, rat and human mast cells were also reported to
express members of the Mas-related GPCR family which allowed for
non-IgE mediated signaling by basic secretagogues via the
peptidonergic pathway (Tatemoto et al Biochem. Biophys. Res.
Commun. 349, 1322-1328 (2006)). Whereas rat mast cells expressed
the family member MrgB3 to elicit mast cell degranulation, human
mast cells expressed the family member MrgX2. The basic
secretagogues MCDP and substance P were shown, not only, to induce
human and rat mast cell degranulation, but also to activate cells
transfected with human MrgX2 and rat MrgB3. Consistent with earlier
observations that basic secretagogues signal through a pertussis
toxin (PTX) sensitive pathway in mast cells, substance P activation
of cell lines expressing human MrgX2 was also shown to be
PTX-sensitive. We have similarly found that PEG-[Lys16]ShK (SEQ ID
NO:16) mediated degranulation of rat peritoneal mast cells is
PTX-sensitive. Thus, the family of Mas-related GPCRs (Mrg) may
represent the off-target responsible for the species specific
effect of ShK and PEG-[Lys16]ShK on rat mast cells. Indeed, members
of the Mas-related GPCR family members exhibit poor conservation
across species. Humans express just four members (MrgX1, MrgX2,
MrgX3, MrgX4), whereas rats express one each of the MrgA, MrgC, and
MrgD genes and ten MrgB genes (Tatemoto et al. (ibid); Dong (ibid);
Zylka et al., PNAS100, 10043-10048 (2003)). Despite MrgB3
originally being described as a pseudo-gene (Zylka, ibid), Tatemoto
et al. (ibid) find this gene is expressed. The impact of
PEG-[Lys16]ShK (SEQ ID NO:16) on rat mast cells and the apparent
lack of effects on human mast cells, may reflect the unique Mrg
expression pattern in rat mast cells and the fact that the human
genome is without a homolog to rat MrgB3. Monkey, however,
expresses the same four MrgX family members as human (Burstein et
al. Br. J. Pharmacol. 147, 73-82 (2006); Zhang et al., Molecular
Brain Res. 133, 187-197 (2005)), making it a more relevant species
to humans. Importantly, toxicology studies in cynomolgus monkeys
indicate PEG-[Lys16]ShK (SEQ ID NO:16) is well tolerated at doses
providing a margin of coverage 54-197 times the predicted human
efficacious dose (data not shown).
[0505] Materials and Methods for Mast Cell Degranualtion
Studies
[0506] Reagents. MCDP was obtained from Alomone Labs (Israel).
Compound 48/80, substance P, A23187 were obtained from Sigma (Saint
Louis, Mich.). STEMPRO-34 SFM complete medium was obtained from
Invitrogen (Carlsbad, Calif.). Recombinant human SCF was prepared
in-house. Human IL-6 was obtained from R&D Systems
(Minneapolis, Minn.). Human IL-3 was obtained from Invitrogen
(Camarillo, Calif.). Histamine Elisa kit was obtained from NEOGEN
(Lexington, Ky.). Tyrode's buffer was made in-house (10 mM Hepes,
pH7.4), 130 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.1% BSA, 1 mM
CaCl.sub.2, 0.6 mM MgCl.sub.2). Diff Quick Stain was obtained from
Dade Behring (NEWARK, DE)
[0507] Cells. Human peripheral blood CD34+ derived mast cells were
obtained by long-term culture of peripheral blood progenitor CD34+
cells (Allcells) in vitro in STEMPRO-34 serum-free medium
containing 100 ng/ml of human SCF, 40 ng/ml of human IL-6 and 30
ng/ml of human IL-3 for one week, then terminally differentiated in
the same serum-free medium containing only 100 ng/ml of human SCF
and 40 ng/ml of human IL-6 for about 4 weeks. Terminally
differentiated mast cells were determined by Flow Cytometry
analysis for the surface c-Kit expression (>95% of the cells
expressed c-Kit, data not shown) and IgE/anti-IgE induced histamine
release (about 40% of histamine released of the total histamine
content, data not shown).
[0508] Rat peritoneal fluid was collected in Tyrode's buffer from 8
weeks old of female Sprague Dawley or Lewis. (Gillespie et al.,
Histamine release from rat peritoneal mast cells: inhibition by
colchicine and potentiation, 1968; 154-1). Mouse peritoneal fluid
was collected in Tyrode's buffer from 8 weeks old of female C57B6.
Percentage of rat and mouse peritoneal mast cells were determined
by staining with Diff Quick Stain.
[0509] Measurement of rat serum histamine. Male Sprage Dawley Rats
serum samples from intravenous single-dose or subcutaneous
single-dose were diluted in Tyrode's buffer and seeded on 96-well
plates. The histamine was quantified by ELISA (followed the
manufacturer's instruction). The absorbance was measured at 450 nm
and 650 nm in microplate reader. Molecular Devices, SPECTRA mac 340
pc). (FIG. 37 and FIG. 38).
[0510] Measurement of mast cell degranulation. Rat or mouse
peritoneal fluid collected in Tyrode's buffer or human mast cells
in Tyrode's buffer containing 200 ng/ml of human SCF and 80 ng/ml
of human IL-6 was seeded on 96-well plates (rat or mouse peritoneal
mast cells, 4-5000 cells/well; human mast cells, 20,000 cells/well)
with half log diluted control molecules: MCDP, Compound 48/80,
Substance P and A23187 and test peptides: 20 kDa-PEG-[Lys16]ShK
(SEQ ID NO:16), monovalent aKLH Ab-[Lys16]ShK (tetramer of SEQ ID
NOS:338, 339, 338, 342) and monovalent Fc/Fc-L10-[Lys16]ShK
(heterodimer of SEQ ID NOS:337, 348) in Tyrode's buffer at 37 C for
1 h. Cells were centrifuged at 800 rpm, 4 C for 15 min. Supernatant
was collected, released histamine was quantified by ELISA. The
absorbance was measured at 450 nm and 650 nm in microplate reader.
Total histamine content of the cells (0.1% Triton X-100) and
spontaneous release were also measured to determine the percent of
histamine release (FIGS. 39-41). Percent histamine release was
calculated by the following formulam (III):
% histamine release=(ng/ml histamine release by test article-ng/ml
spontaneous histamine release in Tyrode's buffer)/(ng/ml total
histamine content of cells lysed in 0.1% Triton X-100-ng/ml
spontaneous histamine release in Tyrode's buffer).times.100.
(III)
TABLE-US-00015 TABLE 4F Design of repeat-dose pharmacology study on
PEG-[Lys16]ShK (SEQ ID NO: 16) in cynomolgus monkeys. Week Activity
1 2 3 4 5 6 7 8 9 10 11 12 0.5 mg/kg 20 kDa-PEG- x x x x ShK[Lys16]
Weekly SC Doses (n = 6, male cynos) Whole Blood Sample x x x x x x
x x x x x x (1 mL) for CBC Analysis Whole Blood Sample x x x x x x
x x x x x x (3 mL) for FACS & PD Analysis Serum Sample for
Clinical x x x x x x x x x x x x Chemistry and PK Analysis a. 1.0
mL for clinical pathology b. ~0.5 mL for PK Analysis
TABLE-US-00016 TABLE 4G Average pre-dose baseline CBCs and clinical
pathology measures of monkeys (n = 6 male cynomolgus monkeys; 3
pre-dose samples collected for each cyno over two weeks). Pre-Dose
Pre-Dose (Average) (Stdev) Hematology WBC 10.sup.3/.mu.L 10.944
1.898 RBC 10.sup.6/.mu.L 4.886 0.289 HGB g/dL 12.011 0.610 HCT %
36.961 1.334 MCV fL 75.733 2.828 MCH pg 24.594 0.727 MCHC g/dL
32.500 0.849 RDW % 12.556 0.315 PLT 10.sup.3/.mu.L 381.278 27.296
MPV fL 8.294 0.538 NEUT % 35.611 12.571 LYMPHS % 59.833 11.994 MONO
% 3.611 0.574 EOS % 1.333 0.667 BASO % 0.000 0.000 BANDS % 0.000
0.000 Neut 10.sup.3/.mu.L 4.080 1.925 Lymphs 10.sup.3/.mu.L 6.369
1.447 Mono 10.sup.3/.mu.L 0.403 0.083 Eos 10.sup.3/.mu.L 0.139
0.068 Baso 10.sup.3/.mu.L 0.000 0.000 Bands 10.sup.3/.mu.L 0.000
0.000 Retic % 1.233 0.250 Retic count 10.sup.3/.mu.L 60.026 10.687
Blood Chemistry Alb g/dL 4.417 0.278 Total Pro g/dL 7.072 0.226 ALP
U/L 349.389 145.717 ALT U/L 30.444 3.250 AST U/L 32.389 8.747 CPK
U/L 632.389 322.364 T bili mg/dL 0.161 0.033 Dir Bili mg/dL 0.011
0.017 BUN mg/dL 20.611 2.195 Creatinine mg/dL 0.856 0.054 Calcium
mg/dL 10.206 0.406 Choles mg/dL 122.833 23.122 Glucose mg/dL 69.278
4.041 Phosphor mg/dL 5.344 0.449 GGT U/L 71.278 11.275 CO2 mEq/L
22.944 1.163 Na mEq/L 146.500 1.670 K mEq/L 4.339 0.272 Cl mEq/L
106.778 1.129 Globulin mg/dL 2.656 0.154 A/G Ratio 1.678 0.185 B/C
Ratio 24.167 1.964 Ind Bili mg/dL 0.150 0.028 ANION mEq/L 21.056
1.782
TABLE-US-00017 TABLE 4G(a) PEG-[Lys16]ShK (SEQ ID NO: 16) Cerebral
Spinal Fluid and Serum Concentrations at 24 and 48 hours in Three
Cynomolgus Monkeys After a 0.5 mg/kg Subcutaneous Dose of
PEG-[Lys16]ShK (SEQ ID NO: 16). Cyno - Serum Conc. CSF Conc.
Serum/CSF sample time (ng/mL) (ng/mL Ratio 4-24 h 1410 1.68 839
4-48 h 1360 1.72 791 5-24 h 1270 2.55 498 5-48 h 910 2.14 425 6-24
h 1420 2.30 617 6-48 h* 1080 7.62 142 *Sample visually contaminated
with blood.
TABLE-US-00018 TABLE 4G(b) PEG-[Lys16]ShK (SEQ ID NO: 16) Cerebral
Spinal Fluid and Serum Concentrations at 6 and 48 hours in Male
Sprague-Dawley Rats Receiving Various Intravenous Doses of
PEG-[Lys16]ShK (SEQ ID NO: 16). Dose(mg/kg) - Serum Conc. CSF Conc.
Serum/CSF timepoint (ng/mL) (ng/mL) Ratio 0.04-48 h* 6.46 5.7 1.13
0.04-48 h 8.68 BQL >8.68 0.04-48 h 4.75 BQL >4.75 0.12-48 h
31.2 BQL >31.2 0.20-48 h 40.6 BQL >40.6 0.20-48 h 69.2 BQL
>69.2 0.40-48 h 173 BQL >173 0.40-48 h 151 BQL >151 1.20-6
h** 5970 12.7 470 4.00-6 h** 2000 43 46.5 *Sample visually
contaminated with blood. **Animals sacrificed early due to adverse
events. BQL--Below the assay limit of quantitation of 1 ng/mL.
TABLE-US-00019 TABLE 4G(c) PEG-[Lys16]ShK (SEQ ID NO: 16) was
administered by subcutaneous injection to telemeterized male
Sprague Dawley rats (n = 3/dose group). Mean Arterial Heart Rate
Pressure (bpm) (mm Hg) Vehicle 336 .+-. 6 115 .+-. 10 0.7 mg/kg 413
.+-. 22 90 .+-. 5 PEG-[Lys16]ShK (+23%) (-13%) 2.5 mg/kg 464 .+-.
12 84 .+-. 8 PEG-[Lys16]ShK (+38%) (-26%) Mean arterial pressure
and heart rate were monitored continuously for 24 hours.
Example 11
Expression and Purification of Immunoglobulin- and/or Fc
domain-Toxin Peptide Analog Fusions
[0511] An assortment of bivalent and monovalent structures were
expressed and purified as exemplary embodiments of the invention.
Those included IgG2 Fc/Fc-ShK variants (see FIG. 12A), aKLH
IgG2/Fc-ShK variants (see FIG. 12E), and anti-KLH IgG2-ShK variants
(see FIG. 12F-L). For example, bivalent Fc-L10-ShK[1-35], bivalent
Fc-L10-ShK[2-35], monovalent Fc/Fc-L10-ShK[2-35] fusions were made
by recombinant methods as described in Sullivan et al., WO
2008/088422 A2, and in particular Examples 1, 2, and 56,
incorporated by reference in its entirety, or as modified herein.
Monovalent anti-Keyhole Limpet Hemocyanin (KLH) immunoglobulin
heavy chain-[Lys16]ShK fusion antibody (designated "aKLH
HC-[Lys16]ShK Ab"; see FIG. 12F), and monovalent anti-KLH
immunoglobulin light chain-[lys16]ShK antibody fusions (designated
"aKLH LC-[Lys16]ShK Ab"; see FIG. 12J) were made by similar
recombinant methods and as further modified herein.
[0512] Transient expression system used to generate toxin peptide
analog-Fc fusions ("peptibodies") or other immunoglobulin fusion
embodiments. HEK 293-6E cells were maintained in 3 L Fernbach
Erlenmeyer Flasks between 2e5 and 1.2e6 cells/ml in F17 medium
supplemented with L-Glutamine (6 mM) and Geneticin (25 .mu.g/ml) at
37.degree. C., 5% CO2, and shaken at 65 RPM. At the time of
transfection, cells were diluted to 1.1.times.10.sup.6 cells/mL in
the F17 medium mentioned above at 90% of the final culture volume.
DNA complex was prepared in Freestyle 293 medium at 10% of the
final culture volume. DNA complex includes 500 ug total DNA per
liter of culture and 1.5 ml PEImax per liter of culture. DNA
complex is briefly shaken once ingredients are added and incubated
at room temperature for 10 to 20 minutes before being added to the
cell culture and placed back in the incubator. The day after
transfection, Tryptone N1 (5 g/L) was added to the culture from
liquid 20% stock. Six days after transfection, culture was
centrifuged at 4,000 RPM for 40 minutes to pellet the cells and the
cultured medium was harvested through a 0.45 um filter.
[0513] In preparing the DNA complex, the ratio of plasmids was
proportional to the desired molar ratio of the peptides needed to
generate the intended product. The components of the IgG2 Fc/Fc-ShK
include IgG2 Fc and IgG2 Fc-ShK at a 1:1 ratio. During expression
these assemble into IgG2 Fc homodimers, IgG2 Fc/Fc-ShK
heterodimers, and IgG2 Fc-ShK homodimers. The IgG2 Fc/Fc-ShK
heterodimer (monovalent form) was isolated during purification
using cation exchange chromatography.
[0514] IgG2 Fc-ShK[2-35]; IgG2 Fc Shk[2-35, Q16K];IgG2
Fc-Shk[1-35]; IgG2 Fc-ShK[1-35, Q16K] mammalian expression. DNA
sequences coding for the immunoglobulin Fc domain of human
IgG2:
MEWSWVFLFFLSVTTGVHSERKVECPPCPAPPVAGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//(SEQ ID NO:337),
fused in-frame to a monomer of the Kv1.3 inhibitor peptide
ShK[2-35] or a mutated ShK[2-35, Q16K] were constructed using
standard PCR technology. The ShK[2-35] or ShK[2-35, Q16K] and the
10 amino acid linker portion of the molecule were generated in a
PCR reaction using the original Fc-2.times.L-ShK[2-35] in
pcDNA3.1(+)CMVi as a template (see Sullivan et al., WO 2008/088422
A2, Example 2, FIG. 15A-B). The ShK[1-35] was generated in a PCR
reaction using the original Fc-2.times.L-ShK[1-35] in
pcDNA3.1(+)CMVi as a template (Sullivan et al., WO 2008/088422 A2,
Example 1, FIG. 14A-B).These ShK constructs have the following
modified VH21 Signal peptide amino acid sequence of
MEWSWVFLFFLSVTTGVHS//SEQ ID NO:318 generated from a
pSelexis-Vh21-hIgG2-Fc template with the following oligos:
TABLE-US-00020 (SEQ ID NO: 319) 5'- CAT GAA TTC CCC ACC ATG GAA TGG
AGC TGG -3'; and (SEQ ID NO: 320) 5'- CA CGG TGG GCA CTC GAC TTT
GCG CTC GGA GTG GAC ACC -3'.
[0515] Wild Type ShK[2-35] with N-terminal linker extension (amino
acid sequence
GGGGSGGGGSSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC//SEQ ID NO:322) was
encoded by the DNA sequence below:
GGAGGAGGAGGATCCGGAGGAGGAGGAAGCAGCTGCATCGACACCAT
CCCCAAGAGCCGCTGCACCGCCTTCCAGTGCAAGCACAGCATGAAGTA
CCGCCTGAGCTTCTGCCGCAAGACCTGCGGCACCTGC//(SEQ ID NO:321). A fragment
containing this coding sequence (SEQ ID NO:321) was generated using
the oligos below (SEQ ID NO:323 and SEQ ID NO:324)--and the
original Fc-L10-ShK[2-35] in pcDNA3.1(+)CMVi as a template
(Sullivan et al., WO 2008/088422 A2, Example 2, FIG. 15A-B,
incorporated by reference):
TABLE-US-00021 (SEQ ID NO: 323) 5'-GTC CAC TCC GAG CGC AAA GTC GAG
TGC CCA CCG TGC C-3'; and (SEQ ID NO: 324) 5'-TCC TCC TCC TTT ACC
CGG AGA CAG GGA GAG -3'//.
[0516] Mutant ShK[2-35, Q16K] was generated using site directed
mutagenesis with Stratagene's QuikChange Multi site-Directed
Mutagenesis kit cat#200531 per the manufacturer's instruction.
Oligos used to generate the mutagenesis were:
5'-GCT GCA CCG CCT TCA AGT GCA AGC ACA GC 3' (SEQ ID NO:325);
and
[0517] 5'-GCT GTG CTT GCA CTT GAA GGC GGT GCA GC-3' (SEQ ID
NO:326); and using the original Fc-L10-ShK[2-35] in pcDNA3.1(+)CMVi
as a template (Sullivan et al., WO 2008/088422 A2, Example 2, FIG.
15A-15B) resulting in the DNA coding sequence
GGAGGAGGAGGATCCGGAGGAGGAGGAAGCAGCTGCATCGACACCAT
CCCCAAGAGCCGCTGCACCGCCTTCAAGTGCAAGCACAGCATGAAGTA
CCGCCTGAGCTTCTGCCGCAAGACCTGCGGCACCTGC//(SEQ ID NO:327), which
encodes the amino acid sequence
GGGGSGGGGSSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC//SEQ ID NO:328).
[0518] ShK[1-35]WT fragment was generated using the original
Fc-2.times.L-ShK[1-35] in pcDNA3.1(+)CMVi as a template (Sullivan
et al., WO 2008/088422 A2, Example 1, FIG. 14A-B) and oligos:
TABLE-US-00022 (SEQ ID NO: 323) 5'-GTC CAC TCC GAG CGC AAA GTC GAG
TGC CCA CCG TGC C-3'; and (SEQ ID NO: 324) 5'-TCC TCC TCC TTT ACC
CGG AGA CAG GGA GAG-3'.
[0519] The IgG2Fc region was generated using oligos:
5'-CCG GGT AAA GGA GGA GGA GGA TCC GGA G-3' (SEQ ID NO:329);
and
[0520] 5'-CAT GCG GCC GCT CAT TAG CAG GTG-3' (SEQ ID NO:330), and
the pSelexis Vh21-hIgG2-Fc template resulting in a fragment
containing the following DNA coding sequence:
GCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC
AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTG
GTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCA
GTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCA
GGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAG
GCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGC
CCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGA
CCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCA
GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC
TACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCT
ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC
TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAG
AAGAGCCTCTCCCTGTCTCCGGGTAAA//SEQ ID NO:331, which encodes the amino
acid sequence
APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG
VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLP
APIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK SEQ ID NO:332).
[0521] The PCR fragments were generated and the products were run
out on a gel. After gel purification, the DNA fragments were put
together in a PCR tube and sewn together with outside primers:
TABLE-US-00023 (SEQ ID NO: 319) 5'- CAT GAA TTC CCC ACC ATG GAA TGG
AGC TGG -3'; and (SEQ ID NO: 330) 5'- CAT GCG GCC GCT CAT TAG CAG
GTG -3'.
[0522] The PCR products were digested with EcoRI and NotI (Roche)
restriction enzymes and agarose gel purified by Gel Purification
Kit. At the same time, the pTT14 vector (an Amgen vector containing
a CMV promoter, Poly A tail and a Puromycin resistance gene) was
digested with EcoRI and NotI restriction enzymes and the large
fragment was purified by Gel Purification Kit. Each purified PCR
product was ligated to the large fragment and transformed into
OneShot Top10 bacteria. DNAs from transformed bacterial colonies
were isolated and subjected to EcoRI and NotI restriction enzyme
digestions and resolved on a one percent agarose gel. DNAs
resulting in an expected pattern were submitted for sequencing.
Although, analysis of several sequences of clones yielded a 100%
percent match with the above sequence, only one clone of each
construct was selected for large scaled plasmid purification. The
final pTT14-VH1SP-IgG2-Fc construct encoded IgG2-Fc-L10-ShK(2-35)
fusion polypeptide having the following sequence:
TABLE-US-00024 (SEQ ID NO: 345)
MEWSWVFLFFLSVTTGVHSERKVECPPCPAPPVAGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS
LSPGKGGGGSGGGGSSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGT C//.
[0523] The pTT14-VH21SP-IgG2-Fc-ShK2-35Q16K construct encoded a
IgG2-Fc-L10-ShK(2-35, Q16K) fusion polypeptide sequence:
TABLE-US-00025 SEQ ID NO: 346
MEWSWVFLFFLSVTTGVHSERKVECPPCPAPPVAGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS
LSPGKGGGGSGGGGSSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGT C//;
and pTT14-VH21SP-IgG2-Fc ShK1-35 construct contained a coding
sequence for IgG2 Fc-L10-ShK(1-35) fusion polypeptide having the
following sequence:
TABLE-US-00026 (SEQ ID NO: 334)
MEWSWVFLFFLSVTTGVHSERKVECPPCPAPPVAGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGG
SGGGGSRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC//.
[0524] Generating the VH21 SP-IgG2-Fc-only construct in pYD 16 (an
Amgen vector containing a CMV promoter, Poly A tail and a
Hygromycin resistance gene) occurred as follows: The VH21 signal
peptide was generated using the following oligos:
5'-CAT AAG CTT CCC ACC ATG GAA TGG AGC TGG-3' (SEQ ID NO:335);
and
[0525] 5'-CA CGG TGG GCA CTC GAC TTT GCG CTC GGA GTG GAC ACC-3'
(SEQ ID NO:320), and using the pSelexis template as noted
above.
[0526] The Fc region was generated using the pSelexis template
described above and following oligos:
TABLE-US-00027 (SEQ ID NO: 323) 5'-GTC CAC TCC GAG CGC AAA GTC GAG
TGC CCA CCG TGC C-3'; and (SEQ ID NO: 336) 5'- CAT GGA TCC TCA TTT
ACC CGG AGA CAG GGA G -3'.
[0527] The PCR fragments were gel purified and sewn together in
single PCR reaction using outside primers SEQ ID NO:335 and SEQ ID
NO:336. The resulting PCR fragment was gel purified, and digested
by HindIII and BamHI. Concurrently, pYD16 vector (an Amgen vector
containing a CMV promoter, Poly A tail and a Hygromycin resistance
gene) was also cut by HindIII and BamHI and the large vector
fragment was purified by Qiagen's Gel Purification Kit. The
purified PCR product was ligated to the large fragment and
transformed into OneShot Top10 bacteria. DNA from transformed
bacterial colonies were isolated and subjected to HindIII and BamHI
restriction enzyme digestions and resolved on a one percent agarose
gel. DNAs resulting in an expected pattern were submitted for
sequencing. Although, analysis of several sequences of clones
yielded a 100% percent match with the above sequence, only one
clone was selected for large scaled plasmid purification. The final
pYD16-VH21SP-IgG2-Fc construct encoded human IgG2-Fc (SEQ ID NO:337
above).
[0528] Immunizations. Anti-DNP antibodies were generated by
immunizing XenoMouse.RTM. mice with DNP-KLH, over a period of 4
weeks, and by screening for those antibodies that bind to
DNP-lysine. More particularly, XenoMouse.RTM. XMG2 strain of mice
were generated generally as described previously (Mendez et al.,
Nat. Genet. 15:146-156 (1997); published International Patent
Application Nos. WO 98/24893, and WO 00/76310, the disclosures of
which are hereby incorporated by reference) and immunized with
2,4-Dinitrophenyl-Keyhole Limpet Hemocyanin (DNP-KLH conjugate;
BioSearch Technologies, Novato, Calif.), using a range of 10-30
.mu.g/mouse of immunogen emulsified in TiterMax Gold adjuvant
(Sigma-Aldrich, Oakville, Ontario) for the initial immunization of
the XMG2 strain of XenoMouse.TM. according to the methods disclosed
in International Patent Application Nos. WO 98/24893, and WO
00/76310, the disclosures of all of which are hereby incorporated
by reference. Following the initial immunization, subsequent boost
of immunogen (5-20 .mu.g/mouse) were administered on a schedule and
for the duration necessary to induce a suitable anti-DNP titer in
the mice. Titers were determined by enzyme immunoassay using
immobilized DNP-BSA (BioSearch Technologies, Novato, Calif.), this
conjugate was prepared such that the final DNP:BSA molar ratio was
30:1.
[0529] Immunizations to raise anti-KLH antibodies were conducted,
over a period of 4 weeks, using Imject.RTM. Mariculture Keyhole
Limpet hemocyanin (mcKLH; Pierce Biotechnology, Rockford, Ill.;
cat#77600, lot#B144095B). Immunizations were conducted using 10
.mu.g of KLH per mouse in Aluminium Phosphate Gel Adjuvant (HCl
Biosector, Frederikssund, Denmark; Catalog #1452-250); delivered
via footpad injection. The initial immunization of the XMG1K strain
of XenoMouse.RTM. was according to methods previously disclosed
(Mendez et al., Nat. Genet. 15:146-156 (1997); published
International Patent Application Nos. WO 98/24893, and WO 00/76310,
the disclosures of which are hereby incorporated by reference,
which are all hereby incorporated by reference). Following the
initial immunization, subsequent boosts of immunogen (5-10
.mu.g/mouse) were administered on a schedule and for the duration
necessary to induce a suitable anti-KLH titer in the mice. Titers
were determined by enzyme immunoassay using immobilized KLH (Pierce
Biotechnology, Rockford, Ill.).
[0530] Preparation of monoclonal antibodies. Mice exhibiting
suitable titers were identified, and lymphocytes and splenocytes
were obtained from draining lymph nodes and spleen, then were
pooled for each cohort. B cells were dissociated from the tissue by
grinding in a suitable medium (for example, Dulbecco's Modified
Eagle Medium; DMEM; Invitrogen, Carlsbad, Calif.) to release the
cells from the tissues, and were suspended in DMEM. B cells were
selected and/or expanded using standard methods, and fused with
suitable fusion partner, for example, nonsecretory myeloma
P3X63Ag8.653 cells (American Type Culture Collection CRL 1580;
Kearney et al, J. Immunol. 123:1548-1550 (1979)), using techniques
known in the art.
[0531] B cells were mixed with fusion partner cells at a ratio of
1:4. The cell mixture was gently pelleted by centrifugation at
400.times.g for 4 minutes, the supernatant was decanted, and the
cell mixture was gently mixed by using a 1 ml pipette. Fusion was
induced with PEG/DMSO (polyethylene glycol/dimethyl sulfoxide;
obtained from Sigma-Aldrich, St. Louis Mo.; 1 ml per million of
lymphocytes). PEG/DMSO was slowly added with gentle agitation over
one minute followed, by one minute of mixing. IDMEM (DMEM without
glutamine; 2 ml per million of B cells), was then added over 2
minutes with gentle agitation, followed by additional IDMEM (8 ml
per million B-cells) which was added over 3 minutes.
[0532] The fused cells were gently pelleted (400.times.g 6 minutes)
and resuspended in 20 ml Selection medium (for example, DMEM
containing Azaserine and Hypoxanthine [HA] and other supplemental
materials as necessary) per million B-cells. Cells were incubated
for 20-30 minutes at 37.degree. C. and then were resuspended in 200
ml Selection medium and cultured for three to four days in T175
flasks prior to 96-well plating.
[0533] Cells were distributed into 96-well plates using standard
techniques to maximize clonality of the resulting colonies. After
several days of culture, the hybridoma supernatants were collected
and subjected to screening assays as detailed in the examples
below, including confirmation of binding to KLH or DNP,
respectively. Positive cells were further selected and subjected to
standard cloning and subcloning techniques. Clonal lines were
expanded in vitro, and the secreted human antibodies obtained for
analysis. Several cell lines secreting DNP-specific antibodies were
obtained, and the antibodies were further characterized. The
sequences thereof are presented herein and in the Sequence Listing,
and results of various tests using these antibodies are
provided.
[0534] Transient expression to generate recombinant monoclonal
antibodies. Transient transfections were carried out in HEK 293-6E
cells as follows. The human embryonic kidney 293 cell line stably
expressing Epstein Barr virus Nuclear Antigen-1 (293-6E cells) was
obtained from the National Research Council (Montreal, Canada).
Cells were maintained as serum-free suspension cultures using F17
medium (Invitrogen, Carlsbad, Calif.) supplemented with 6 mM
L-glutamine (Invitrogen, Carlsbad, Calif.), 1.1% F-68 Pluronic
(Invitrogen, Carlsbad, Calif.) and 250 .mu.g/ul Geneticin
(Invitrigen, Carlsbad, Calif.). The suspension cell cultures were
maintained in Erlenmeyer shake flask cultures. The culture flasks
were shaken at 65 rpm at 37.degree. C. in a humidified, 5% CO.sub.2
atmosphere. A stock solution (1 mg/ml) of 25-kDa linear PEI
(Polysciences, Warrington, Pa.) was prepared in water, acidified
with HCl to pH 2.0 until dissolved, then neutralized with NaOH,
sterilized by filtration (0.2 .mu.m), aliquoted, and stored at
-20.degree. C. until used. Tryptone N1 was obtained from
OrganoTechni S.A. (TekniScience, QC, Canada). A stock solution
(20%, w/v) was prepared in Freestyle medium (Invitrogem, Carlsbad,
Calif.), sterilized by filtration through 0.2 lam filters, and
stored at 4.degree. C. until use. Typically, transfections were
performed at the 1 L scale. Cells (293-6E) were grown too a viable
cell density of 1.1.times.106 cells/ml then transfection complexes
were prepared in 1/10th volume of the final culture volume. For a
1-L transfection culture, transfection complexes were prepared in
100 ml F17 basal medium, and 500 .mu.g plasmid DNA (heavy chain and
light chain DNA, 1:1 ratio) was first diluted in 100 ml F17 medium.
After a 5-minute incubation at room temperature, 1.5 ml of PEI
solution was added. The complexes were vortexed mildly, then
incubated for 15 minutes at room temperature. The cells were
transfected by adding the transfection complex mix to the cells in
the shale flask culture. 24 hours post-transfection, Tryptone N1
was added to the transfected culture to a final concentration of
0.5%, and the transfected cultures were maintained on a shaker at
65 rpm at 37.degree. C. in a humidified, 5% CO.sub.2 atmosphere for
another 5 days after which they were harvested. The conditioned
medium was harvested by centrifugation at 4000 rpm, and then
sterile filtered through 0.2 .mu.m filter (Corning Inc.).
[0535] The stably expressed aKLH 120.6 control antibody pool was
created by transfecting CHO d-host cells with expression plasmids
pDC323 anti-KLH 120.6 kappa LC and pDC324 anti-KLH 120.6-IgG2 HC
using a standard electroporation procedure. After transfection, the
cells were grown as a pool in a serum free -GHT selective growth
media to allow for selection and recovery of the plasmid containing
cells. Cell pools grown in -GHT selective media were cultured until
they reached >85% viability. The selected cell pools were
amplified with 150 nm and 300 nM methotrexate (MTX). Upon reaching
>85% viability the 150 nM pools were then further re amplified
in 500 nm MTX. When the viability of the MTX amplified pools
reached >85% viability, the pools were screened using an
abbreviated six day batch production assay with an enriched
production media to assess expression. The expression of the
amplified pools ranged from 120-400 .mu.g/mL. The best pool was
chosen based on the six-day assay and scaled-up using a ten-day fed
batch process. The conditioned media was harvested and purified to
provide protein for analysis.
[0536] The stably expressed aKLH 120.6 antibody pool was created by
transfecting CHO d-host cells with expression plasmids pDC323
anti-KLH 120.6 kappa LC and pDC324 anti-KLH 120.6-IgG2 HC using a
standard electroporation procedure. After transfection, the cells
were grown as a pool in a serum free -GHT selective growth media to
allow for selection and recovery of the plasmid containing cells.
Cell pools grown in -GHT selective media were cultured until they
reached >85% viability. The selected cell pools were amplified
with 150 nm and 300 nM MTX. Upon reaching >85% viability the 150
nM pools were then further re amplified in 500 nm MTX. When the
viability of the MTX amplified pools reached >85% viability, the
pools were screened using an abbreviated six day batch production
assay with an enriched production media to assess expression. The
expression of the amplified pools ranged from 120-400 .mu.g/mL. The
best pool was chosen based on the six day assay and scaled up using
a ten day fed batch process. The conditioned media was harvested
and purified to provide protein for analysis.
[0537] The .alpha.DNP 3A4 antibody stable expression pools were
created by transfecting CHO DHFR(-) host cells with corresponding
heavy chain and light chain expression plasmid sets using a
standard electroporation procedure. Per each antibody molecule, 3-4
different transfections were performed to generate multiple pools.
After transfection the cells were grown as a pool in a serum free
-GHT selective growth media to allow for selection and recovery of
the plasmid containing cells. Cell pools grown in -GHT selective
media were cultured until they reached >85% viability. The
selected cell pools were amplified with 150 nm methotrexate. When
the viability of the methotrexate amplified pools reached >85%
viability, the pools were screened using an abbreviated six day
batch production assay with an enriched production media to assess
expression. The best pool was chosen based on the six day assay
titer and correct mass confirmation.
[0538] Antibody purification and selections. The antibodies were
purified by Mab Select Sure chromatography (GE Life Sciences) using
8 column volumes of Dulbecco's PBS without divalent cations as the
wash buffer and 100 mM acetic acid, pH 3.5, as the elution buffer
at 7.degree. C. The elution peak was pooled based on the
chromatogram and the pH was raised to about 5.0 using 2 M Tris
base. The pool was then diluted with at least 3 volumes of water,
filtered through a 0.22-.mu.m cellulose acetate filter and then
loaded on to an SP-HP sepharose column (GE Life Sciences) and
washed with 10 column volumes of S-Buffer A (20 mM acetic acid, pH
5.0) followed by elution using a 20 column volume gradient to 50%
S-Buffer B (20 mM acetic acid, 1 M NaCl, pH 5.0) at 7.degree. C. A
pool was made based on the chromatogram and SDS-PAGE analysis, then
the material was concentrated about 7-fold and diafiltered against
about 5 volumes of 10 mM acetic acid, 9% sucrose, pH 5.0 using a
VivaFlow TFF cassette with a 30 kDa membrane. The dialyzed material
was then filtered through a 0.22-.mu.m cellulose acetate filter and
the concentration was determined by the absorbance at 280 nm.
[0539] IgG2-Fc ShK[1-35, Q16K] mammalian expression. Using the DNA
pTT5-aKLH120.6-VK1SP-IgG2-HC-L10-ShK[1-35, Q16K] construct, the
fragment containing the DNA coding sequence
GGATCCGGAGGAGGAGGAAGCCGCAGCTGCATCGACACCATCCCCAA
GAGCCGCTGCACCGCCTTCAAGTGCAAGCACAGCATGAAGTACCGCCT
GAGCTTCTGCCGCAAGACCTGCGGCACCTGCTAATGAGCGGCCGCTCG
AGGCCGGCAAGGCCGGATCC//(SEQ ID NO:436)
[0540] was cut out using BamHI/BamHI. This coding sequence (SEQ ID
NO:436) encodes ShK(1-35, Q16K) with an N-terminal linker
sequence:
TABLE-US-00028 (SEQ ID NO: 437)
GSGGGGSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC//.
[0541] At the same time, pTT14-hIgG2-Fc-ShK[1-35]WT construct, was
also digested by BamHI/BamHI, thereby removing the Shk[1-35] coding
region to yield the coding sequence
ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTG
TCCACTCCGAGCGCAAAGTCGAGTGCCCACCGTGCCCAGCACCACCTG
TGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC
TCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGA
GCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGG
AGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGC
ACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTG
AACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGC
CCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAAC
CACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACC
AGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCG
CCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC
ACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGC
TCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT
CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT
CCCTGTCTCCGGGTAAAGGAGGAGGA//(SEQ ID NO:391, encoding the amino acid
sequence MEWSWVFLFFLSVTTGVHSERKVECPPCPAPPVAGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGG//(SEQ ID
NO:392).
[0542] The pTT14-hIgG2-Fc vector with the ShK removed was treated
with Calf Intestine Phosphatase (CIP) to remove the 5' Phosphate
group and Phenol/Chloroform extracted to prevent religation of the
vector upon itself The insert ShK[1-35, Q16K] fragment was gel
purified away from its vector and cleaned up with Qiagen Gel
Purification Kit. The purified insert was ligated to the large
vector fragment and transformed into OneShot Top10 bacteria. DNAs
from transformed bacterial colonies were isolated and subjected to
BamHI restriction enzyme digestion and resolved on a one percent
agarose gel. DNAs resulting in an expected pattern were submitted
for sequencing. Although, analysis of several sequences of clones
yielded a 100% percent match with the above sequence, only one
clone was selected for large scaled plasmid purification. The final
pTT14-IgG2-Fc-ShK[1-35, Q16K] construct encoded the following IgG2
Fc-ShK(1-35, Q16K) fusion protein sequence:
TABLE-US-00029 (SEQ ID NO: 348)
MEWSWVFLFFLSVTTGVHSERKVECPPCPAPPVAGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGG
SGGGGSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC//.
[0543] Mammalian expression of anti-KLH immunoglobulin heavy chain
(HC) and light chain (LC) toxin peptide (and toxin peptide analog)
fusions. The components of the aKLH IgG2/Fc-ShK (schematically
represented in FIG. 12E) included:
[0544] (a) aKLH 120.6 kappa LC:
TABLE-US-00030 (SEQ ID NO: 338)
MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCRASQG
IRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSL
QPEDFATYYCLQHNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC//;
[0545] (b) aKLH 120.6 IgG2 HC:
TABLE-US-00031 SEQ ID NO: 339
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCV
ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN
WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGK//;
[0546] and
[0547] (c) IgG2 Fc-L10-ShK(1-35):
TABLE-US-00032 (SEQ ID NO: 340)
MEWSWVFLFFLSVTTGVHSERKVECPPCPAPPVAGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGG
SGGGGSRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC//.
[0548] The desired aKLH IgG2/Fc-ShK product contained one copy of
each of components (a)-(c), immediately above, configured as in
FIG. 12E. Because of this, the ratio was 1:1:1. This product can be
described as half antibody and half Fc fusion ("hemibody"), coupled
together at the Fc domain. Additional peptide assemblies that had
to be removed from the culture were the aKLH Ab and the Fc-ShK
homodimer.
[0549] The components of the aKLH 120.6 IgG2-ShK fusion antibody
(schematically represented in FIG. 12F) include:
[0550] (a) aKLH 120.6 kappa LC (SEQ ID NO:338, above);
[0551] (b) aKLH 120.6 IgG2 HC (SEQ ID NO:339, above); and
[0552] (c) aKLH 120.6 IgG2 HC-ShK fusion having the following
sequence:
TABLE-US-00033 (SEQ ID NO: 341)
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSRSCIDTIPKSRCTAFQCK
HSMKYRLSFCRKTCGTC//.
[0553] The components of the aKLH 120.6 IgG2 HC-ShK[1-35, Q16K]
fusion Ab (schematically represented in FIG. 12F) include:
[0554] (a) aKLH 120.6 kappa LC (SEQ ID NO:338, above);
[0555] (b) aKLH 120.6 IgG2 HC (SEQ ID NO:339, above); and
[0556] (c) aKLH 120.6 IgG2-ShK[1-35, Q16K] fusion having the
following sequence:
TABLE-US-00034 (SEQ ID NO: 342)
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGOPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVES
CSVMHEALHNHYTOKSLSLSPGGGGGSGGGGSRSCIDTIPKSRCTAFKCK
HSMKYRLSFCRKTCGTC//.
[0557] The components of the monovalent aKLH 120.6 HC-ShK[1-35,
R1A, I4A, Q16K] fusion antibody (schematically represented in FIG.
12F) include the following monomers:
[0558] (a) aKLH 120.6 kappa LC (SEQ ID NO:338);
[0559] (b) aKLH 120.6 IgG2 HC (SEQ ID NO:339); and
[0560] (c) aKLH 120.6 IgG2 HC-ShK[1-35, R1A, I4A, Q16K] fusion
having the following amino acid sequence:
TABLE-US-00035 (SEQ ID NO: 462)
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSASCADTIPKSRCTAFKCK
HSMKYRLSFCRKTCGTC//.
[0561] The desired monovalent aKLH 120.6 IgG2 HC-ShK analogue
product was a full antibody with the ShK peptide fused to the
C-terminus of one heavy chain. With two different heavy chains
sharing one variety of light chain, the ratio of heavy chain:
chain:light chain:heavychain-ShK was 1:2:1. The expected expression
products are aKLH 120.6 IgG2 antibody, monovalent aKLH 120.6 IgG2
HC-ShK peptide analog, and bivalent KLH 120.6 IgG2 HC-ShK peptide
analog. The monovalent aKLH 120.6 IgG2 HC-toxin peptide
fusion-containing antibody was isolated from the mix using cation
exchange chromatography, as described herein.
[0562] The components of the monovalent aKLH 120.6 HC-ShK[1-35,
R1A, Q16K, K30E] fusion antibody (schematically represented in FIG.
12F) included the following monomers:
[0563] (a) aKLH 120.6 kappa LC (SEQ ID NO:338);
[0564] (b) aKLH 120.6 IgG2 HC (SEQ ID NO:339); and
[0565] (c) aKLH 120.6 IgG2-ShK[1-35, R1A, Q16K, K30E] fusion having
the following sequence:
TABLE-US-00036 (SEQ ID NO: 463)
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSASCIDTIPKSRCTAFKCK
HSMKYRLSFCRETCGTC//.
[0566] The desired monovalent aKLH 120.6 IgG2 HC-ShK analogue Ab
product was a full antibody with the ShK peptide fused to the
C-terminus of one heavy chain. With two different heavy chains
sharing one variety of light chain, the ratio of heavy chain:
chain:light chain:heavychain-ShK was 1:2:1. The expected expression
products are aKLH 120.6 IgG2 antibody, monovalent aKLH 120.6 IgG2
HC-ShK peptide analog, and bivalent aKLH 120.6 IgG2 HC-ShK peptide
analog. The monovalent aKLH 120.6 IgG2 HC-toxin peptide
fusion-containing antibody protein was isolated from the mix using
cation exchange chromatography, as described herein.
[0567] The components of the monovalent aKLH 120.6 HC
(IgG2)-ShK[1-35, R1H, I4A, Q16K] fusion Ab (schematically
represented in FIG. 12F) include monomers:
[0568] (a) aKLH 120.6 kappa LC (SEQ ID NO:338);
[0569] (b) aKLH 120.6 IgG2 HC (SEQ ID NO:339); and
[0570] (c) aKLH 120.6 HC IgG2-ShK[1-35, R1H, I4A, Q16K] fusion
having the following amino acid sequence:
TABLE-US-00037 (SEQ ID NO: 464)
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSHSCADTIPKSRCTAFKCK
HSMKYRLSFCRKTCGTC//.
[0571] The desired monovalent aKLH 120.6 IgG2 HC-ShK analogue
product was a full antibody with the ShK peptide fused to the
C-terminus of one heavy chain. With two different heavy chains
sharing one variety of light chain, the ratio of heavy chain:
chain:light chain:heavychain-ShK peptide analog was 1:2:1. The
expected expression products are aKLH 120.6 IgG2 antibody,
monovalent aKLH 120.6 IgG2 HC-ShK peptide analog Ab, and bivalent
KLH 120.6 IgG2 HC-ShK peptide analog Ab. The monovalent aKLH 120.6
IgG2 HC-toxin peptide fusion-containing Ab protein was isolated
from the mix using cation exchange chromatography, as described
herein.
[0572] The components of the monovalent aKLH 120.6 HC-ShK[1-35,
R1H, Q16K, K30E] fusion Ab (schematically represented in FIG. 12F)
included the monomers:
[0573] (a) aKLH 120.6 kappa LC (SEQ ID NO:338);
[0574] (b) aKLH 120.6 IgG2 HC (SEQ ID NO:339); and
[0575] (c) aKLH 120.6 IgG2-ShK[1-35, R1H, Q16K, K30E] fusion having
the following sequence:
TABLE-US-00038 (SEQ ID NO: 465)
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSHSCIDTIPKSRCTAFKCK
HSMKYRLSFCRETCGTC//.
[0576] The desired monovalent aKLH 120.6 IgG2 HC-ShK analogue Ab
product was a full antibody with the ShK peptide fused to the
C-terminus of one heavy chain. With two different heavy chains
sharing one variety of light chain, the ratio of heavy chain:
chain:light chain:heavychain-ShK peptide analog was 1:2:1. The
expected expression products are aKLH 120.6 IgG2 antibody,
monovalent aKLH 120.6 IgG2 HC-ShK peptide analog Ab, and bivalent
KLH 120.6 IgG2 HC-ShK peptide analog Ab. The monovalent aKLH 120.6
IgG2 HC-toxin peptide fusion-containing Ab protein was isolated
from the mix using cation exchange chromatography, as described
herein.
[0577] The components of the monovalent aKLH 120.6 HC-ShK[1-35,
R1K, I4A, Q16K] fusion Ab (schematically represented in FIG. 12F)
included the monomers:
[0578] (a) aKLH 120.6 kappa LC (SEQ ID NO:338);
[0579] (b) aKLH 120.6 IgG2 HC (SEQ ID NO:339); and
[0580] (c) aKLH 120.6 HC (IgG2)-ShK[1-35, R1K, I4A, Q16K] fusion
having the following sequence:
TABLE-US-00039 (SEQ ID NO: 466)
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSKSCADTIPKSRCTAFKCK
HSMKYRLSFCRKTCGTC//.
[0581] The desired monovalent aKLH 120.6 IgG2 HC-ShK analogue Ab
product was a full antibody with the ShK peptide fused to the
C-terminus of one heavy chain. With two different heavy chains
sharing one variety of light chain, the ratio of heavy chain:
chain:light chain:heavychain-ShK peptide analog was 1:2:1. The
expected expression products are aKLH 120.6 IgG2 antibody,
monovalent aKLH 120.6 IgG2 HC-ShK peptide analog Ab, and bivalent
KLH 120.6 IgG2 HC-ShK peptide analog Ab. The monovalent aKLH 120.6
IgG2 HC-toxin peptide fusion-containing Ab protein was isolated
from the mix using cation exchange chromatography, as described
herein.
[0582] The components of the monovalent aKLH 120.6 HC-ShK[1-35,
R1K, Q16K, K30E] fusion Ab (schematically represented in FIG. 12F)
included the monomers:
[0583] (a) aKLH 120.6 kappa LC (SEQ ID NO:338);
[0584] (b) aKLH 120.6 IgG2 HC (SEQ ID NO:339); and
[0585] (c) aKLH 120.6 IgG2-ShK[1-35, R1K, Q16K, K30E] fusion having
the following amino acid sequence:
TABLE-US-00040 (SEQ ID NO: 467)
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSKSCIDTIPKSRCTAFKCK
HSMKYRLSFCRETCGTC//.
[0586] The desired monovalent aKLH 120.6 IgG2 HC-ShK analogue Ab
product was a full antibody with the ShK peptide fused to the
C-terminus of one heavy chain. With two different heavy chains
sharing one variety of light chain, the ratio of heavy chain:
chain:light chain:heavychain-ShK peptide analog was 1:2:1. The
expected expression products are aKLH 120.6 IgG2 antibody,
monovalent aKLH 120.6 IgG2 HC-ShK peptide analog Ab, and bivalent
KLH 120.6 IgG2 HC-ShK peptide analog Ab. The monovalent aKLH 120.6
IgG2 HC-toxin peptide fusion-containing Ab protein was isolated
from the mix using cation exchange chromatography, as described
herein.
[0587] The components of the aKLH 120.6 IgG2-ShK[2-35, Q16K] fusion
Ab (schematically represented in FIG. 12F) include:
[0588] (a) aKLH 120.6 kappa LC (SEQ ID NO:338, above);
[0589] (b) aKLH 120.6 IgG2 HC (SEQ ID NO:339, above); and
[0590] (c) aKLH 120.6 IgG2-ShK[2-35, Q16K] fusion having the
following sequence:
TABLE-US-00041 (SEQ ID NO: 387)
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSSCIDTIPKSRCTAFKCKH
SMKYRLSFCRKTCGTC//.
[0591] The desired aKLH 120.6 IgG2-ShK Ab product was a full
antibody with the ShK peptide fused to the C-terminus of one heavy
chain, configured as in FIG. 12F. With two different heavy chains
sharing one variety of light chain, the ratio of heavy chain:light
chain:heavy chain-ShK was 1:2:1. The expected expression products
are aKLH 120.6 IgG2, monovalent aKLH 120.6 IgG2-ShK Ab, and
bivalent aKLH 120.6 IgG2-ShK Ab. The monovalent aKLH 120.6
IgG2-toxin peptide (or toxin peptide analog) fusion-containing Ab
was isolated from the mix using cation exchange chromatography, as
described herein.
[0592] The aKLH IgG1-loop-ShK Ab also had a single copy of the ShK
peptide sequence inserted into one of the heavy chains, but in this
case it was inserted into an internal conjugation site in the Fc
domain instead of at the C-terminus. (See, e.g., Gegg et al., U.S.
Pat. No. 7,442,778; U.S. Pat. No. 7,655,765; U.S. Pat. No.
7,655,764; U.S. Pat. No. 7,662,931; U.S. Pat. No. 7,645,861;
published U.S. Patent Applications US 2009/0281286; and US
2009/0286964, each of which are incorporated herein by reference in
their entireties). The components of the aKLH IgG1-loop-ShK Ab
included
[0593] (a) aKLH 120.6 kappa LC (SEQ ID NO:338, above);
[0594] (b) aKLH 120.6 IgG1 HC:
TABLE-US-00042 (SEQ ID NO: 343)
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGKH;
[0595] and
[0596] (c) aKLH 120.6 IgG1-loop-ShK:
TABLE-US-00043 (SEQ ID NO: 344)
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELGGRSCIDTIPKSR
CTAFKCKHSMKYRLSFCRKTCGTCGGTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGK//.
[0597] With two different heavy chains sharing one light chain, the
ratio of heavy chain:light chain:heavy chain-ShK is 1:2:1. The
expected expression products are aKLH 120.6 IgG1, monovalent aKLH
120.6 IgG1-loop-ShK Ab, and bivalent aKLH 120.6 IgG1-loop-ShK Ab.
The monovalent aKLH 120.6 IgG1-loop-ShK Ab (represented
schematically by FIG. 12N) was isolated from the mix using cation
exchange chromatography as described herein.
[0598] Monovalent aKLH 120.6 kappa LC-ShK[1-35, Q16K] fusion. The
components of the monovalent aKLH 120.6 kappa LC-ShK[1-35, Q16K]
fusion Ab (schematically represented in FIG. 12J) included the
monomers:
[0599] (a) aKLH 120.6 IgG2 HC (SEQ ID NO:339);
[0600] (b) aKLH 120.6 kappa LC (SEQ ID NO:338); and
[0601] (c) aKLH 120.6 kappa LC-ShK[1-35, Q16K] fusion having the
following sequence:
TABLE-US-00044 (SEQ ID NO: 439)
MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCRASQG
IRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQ
PEDFATYYCLQHNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSRSCIDTI
PKSRCTAFKCKHSMKYRLSFCRKTCGTC//.
This embodiment of monovalent aKLH 120.6 IgG2 LC-ShK[1-35, Q16K] Ab
product was a full antibody with the ShK peptide fused to the
C-terminus of one light chain as shown in FIG. 12J. With two
different light chains sharing one variety of heavy chain, the
ratio of light chain:heavy chain:light chain-ShK[1-35, Q16K] was
1:2:1. The expected expression products are aKLH 120.6 IgG2,
monovalent KLH 120.6 IgG2 LC-ShK[1-35, Q16K] Ab, and bivalent KLH
120.6 IgG2 LC-ShK[1-35, Q16K] Ab. The monovalent KLH 120.6 IgG2
LC-toxin peptide fusion-containing Ab was isolated from the mix
using cation exchange chromatography, as described herein.
[0602] Monovalent aKLH 120.6 kappa LC-ShK[2-35, Q16K] fusion. The
components of the monovalent aKLH 120.6 kappa LC-ShK[2-35, Q16K]
fusion Ab (schematically represented in FIG. 12J) included the
monomers:
[0603] (a) aKLH 120.6 IgG2 HC (SEQ ID NO:339);
[0604] (b) aKLH 120.6 kappa LC (SEQ ID NO:338); and
[0605] (c) aKLH 120.6 kappa LC-ShK[2-35, Q16K] fusion having the
following sequence:
TABLE-US-00045 (SEQ ID NO: 440)
MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDR
VTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGT
EFTLTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGG
GGSSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC//.
[0606] This embodiment of monovalent aKLH 120.6 IgG2 LC-ShK[2-35,
Q16K] Ab product was a full antibody with the ShK peptide fused to
the C-terminus of one light chain as shown in FIG. 12J. With two
different light chains sharing one variety of heavy chain, the
ratio of light chain:heavy chain:light chain-ShK[2-35, Q16K] was
1:2:1. The expected expression products are KLH 120.6 IgG2,
monovalent KLH 120.6 IgG2 LC-ShK[2-35, Q16K] Ab, and bivalent KLH
120.6 IgG2 LC-ShK[2-35, Q16K] Ab. The monovalent KLH 120.6 IgG2
LC-toxin peptide fusion-containing Ab was isolated from the mix
using cation exchange chromatography, as described herein.
[0607] Bivalent aKLH 120.6 kappa LC-ShK[1-35, Q16K] fusion. The
components of the bivalent aKLH 120.6 kappa LC-ShK[1-35, Q16K]
fusion Ab (schematically represented in FIG. 12K) included the
monomers:
[0608] (a) aKLH 120.6 IgG2 HC (SEQ ID NO:339); and
[0609] (b) aKLH 120.6 kappa LC-ShK[1-35, Q16K] fusion (SEQ ID
NO:439), above.
This embodiment of bivalent KLH 120.6 IgG2 LC-ShK[1-35, Q16K] Ab
product was a full antibody with the ShK peptide fused to the
C-terminus of both light chains as shown in FIG. 12K. The ratio of
heavy chain:light chain-ShK[1-35, Q16K] was 1:1. The expected
expression product is bivalent aKLH 120.6 IgG2 LC-ShK[1-35, Q16K]
Ab. The bivalent KLH 120.6 IgG2 LC-ShK[1-35, Q16K] peptide
fusion-containing Ab was isolated from the mix using cation
exchange chromatography, as described herein.
[0610] Bivalent aKLH 120.6 kappa LC-ShK[2-35, Q16K] fusion. The
components of the bivalent aKLH 120.6 kappa LC-ShK[2-35, Q16K]
fusion Ab (schematically represented in FIG. 12K) included the
monomers:
[0611] (a) aKLH 120.6 IgG2 HC (SEQ ID NO:339); and
[0612] (b) aKLH 120.6 kappa LC-ShK[2-35, Q16K] fusion (SEQ ID
NO:440), above.
[0613] This embodiment of bivalent aKLH 120.6 IgG2 LC-ShK[2-35,
Q16K] Ab product was a full antibody with the ShK peptide fused to
the C-terminus of both light chains as shown in FIG. 12K. The ratio
of heavy chain:light chain-ShK[2-35, Q16K] was 1:1. The expected
expression product is bivalent aKLH 120.6 IgG2 LC-ShK[2-35, Q16K]
Ab. The bivalent KLH 120.6 IgG2 LC-ShK[2-35, Q16K] peptide
fusion-containing Ab was isolated from the mix using cation
exchange chromatography, as described herein.
[0614] Trivalent aKLH 120.6 kappa LC-ShK[1-35, Q16K] fusion. The
components of the trivalent aKLH 120.6 kappa LC-ShK[1-35, Q16K]
fusion Ab (schematically represented in FIG. 12L) included the
monomers:
[0615] a) aKLH 120.6 IgG2 HC (SEQ ID NO:339, above);
[0616] (b) aKLH 120.6 IgG2 HC-Shk[1-35, Q16K] fusion having the
amino acid of SEQ ID NO:342, above; and
[0617] (c) aKLH 120.6 kappa LC-ShK[1-35, Q16K] fusion having the
amino acid sequence of SEQ ID NO:439, above.
This embodiment of trivalent aKLH 120.6 IgG2 LC-ShK Ab product was
a full antibody with the ShK[1-35, Q16K] peptide fused to the
C-terminus of both light chains and one heavy chain as shown in
FIG. 12L. With two different heavy chains sharing one variety of
light chain, the ratio of heavy chain:light chain-ShK[1-35,
Q16K]:heavy chain-ShK[1-35, Q16K] was 1:2:1. The expected
expression products were a bivalent aKLH 120.6 IgG2 LC-ShK[1-35,
Q16K] Ab, trivalent aKLH 120.6 IgG2 LC-ShK[1-35, Q16K] Ab, and
tetravalent aKLH 120.6 IgG2 LC-ShK[1-35, Q16K] Ab. The trivalent
KLH 120.6 IgG2 LC-toxin peptide fusion-containing Ab was isolated
from the mix using cation exchange chromatography, as described
herein.
[0618] Trivalent aKLH 120.6 kappa LC-ShK[2-35, Q16K] fusion. The
components of the trivalent KLH 120.6 kappa LC-ShK[2-35, Q16K]
fusion Ab (schematically represented in FIG. 12L) included the
monomers:
[0619] a) aKLH 120.6 IgG2 HC (SEQ ID NO:339);
[0620] (b) aKLH 120.6 IgG2 HC-Shk[2-35, Q16K] fusion (SEQ ID
NO:387), above; and
[0621] (c) aKLH 120.6 kappa LC-ShK[2-35, Q16K] fusion (SEQ ID
NO:440), above.
This embodiment of trivalent aKLH 120.6 IgG2 LC-ShK[2-35, Q16K] Ab
product was a full antibody with the ShK[2-35, Q16K] peptide fused
to the C-terminus of both light chains and one heavy chain as shown
in FIG. 12L. With two different heavy chains sharing one variety of
light chain, the ratio of heavy chain:light chain-ShK[2-35,
Q16K]:heavy chain-ShK[2-35, Q16K] was 1:2:1. The expected
expression products were a bivalent aKLH 120.6 IgG2 LC-ShK[2-35,
Q16K] Ab, trivalent aKLH 120.6 IgG2 LC-ShK[2-35, Q16K] Ab, and
tetravalent aKLH 120.6 IgG2 LC-ShK[2-35, Q16K] Ab. The trivalent
aKLH 120.6 IgG2 LC-toxin peptide fusion-containing Ab protein was
isolated from the mix using cation exchange chromatography, as
described herein.
[0622] Anti-KLH 120.6 Antibody Light Chain mammalian expression.
The XenoMouse.RTM. hybridoma expressing aKLH monoclonal antibody
120.6 was used as a source to isolate total RNA using TRIzol.RTM.
reagent (Invitrogen). First strand cDNA was synthesized using a
random primer with an extension adapter 5'-GGC CGG ATA GGC CTC CAN
NNN NNT-3'(SEQ ID NO:349) and a 5' RACE (rapid amplification of
cDNA ends) was performed using the GeneRacer.TM. Kit (Invitrogen).
For the light chain sequence determination, the forward primer was
5'-GTG GTT GAG AGG TGC CAG ATG TGA CAT TGT GAT GAC TCA GTC TCC-3'
(SEQ ID NO:350) and the reverse primer was 5'-AAC CGT TTA AAC GCG
GCC GCT CAA CAC TCT CCC CTG TTG AA-3' (SEQ ID NO:351). The RACE
product was cloned into pCR4-TOPO (Invitrogen) and the sequences
determined. Consensus sequences were used to determine probable
framework and signal peptide sequence and design primers for
full-length antibody chain PCR amplification.
[0623] The expression clone for the anti-KLH 120.6 kappa light
chain was prepared by PCR. The 5' PCR primer encoded the amino
terminus of the signal sequence, an SalI restriction enzyme site,
and an optimized Kozak sequence 5'-AAG CTC GAG GTC GAC TAG ACC ACC
ATG GAC ATG AGG GTC CCC G-3' (SEQ ID NO:352). The 3' primer encoded
the carboxyl terminus and termination codon, as well as a NotI
restriction site 5'-AAC CGT TTA AAC GCG GCC GCT CAA CAC TCT CCC CTG
TTG AA-3' (SEQ ID NO:351). The resulting product was cloned into
pCR4-TOPO (Invitrogen) and the sequences determined. After the
insert was confirmed, the pCR4-TOPO product was cut with SalI and
NotI, the insert gel isolated and Qiagen purified, and then ligated
into the mammalian expression vector pTT5.
[0624] A PCR was done to change the signal peptide from the native
peptide derived from the hybridoma to the VK1/O12 peptide. The
primers used for the VK1/O12 fragment were 5' AAG CTC GAG GTC GAC
TAG ACC ACC ATG GAC ATG AGG GTG CCC GCT 3' (SEQ ID NO:353) and
5'-TCA TCT GGA TGT CAC ATC TGG CAC C-3' (SEQ ID NO:354). The
primers used for the mature light chain peptide were 5'-GGT GCC AGA
TGT GAC ATC CAG ATG A-3' (SEQ ID NO:355) and (SEQ ID NO:351). The
resulting fragments were joined by overlap PCR using primers SEQ ID
NO:353 and SEQ ID NO:351. The sequence of the resulting clone
encodes the following immunoglobulin kappa LC sequence:
TABLE-US-00046 (SEQ ID NO: 338)
MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCRASQG
IRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQ
PEDFATYYCLQHNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC//.
[0625] Anti-KLH 120.6 Antibody Light Chain-ShK peptide analog
mammalian expression. The Shk[1-35, Q16K] fragment was generated by
PCR using the pTT14-huIgG2-Fc ShK[1-35, Q16K] encoding (SEQ ID
NO:348), described above, as a template and the oligos:
TABLE-US-00047 (SEQ ID NO: 441) 5'- AAC AGG GGA GAG TGT GGA GGA GGA
GGA TCC GGA G -3'; and (SEQ ID NO: 442) 5'- CAT GCG GCC GCT CAT TAG
CAG G -3'.
[0626] The light chain fragment and ShK PCR product were then
amplified by PCR using the outside primers SEQ ID NO: CAT TCT AGA
ACC ACC ATG GAC ATG AGG GTG//(SEQ ID NO:482) and SEQ ID NO:442. The
PCR product was then digested by XbaI and NotI and PCR clean up kit
(Qiagen) purified. At the same time, pYD16 was cut by XbaI and
NotI. The pYD16 vector was run out on a 1% agarose gel and the
larger fragment was cut out and gel purified by Qiagen's Gel
Purification Kit. The purified PCR product was ligated to the large
vector fragment and transformed into OneShot Top10 bacteria. DNAs
from transformed bacterial colonies were isolated and subjected to
XbaI and NotI restriction enzyme digestions and resolved on a one
percent agarose gel. DNAs resulting in an expected pattern were
submitted for sequencing. Although, analysis of several sequences
of clones yielded a 100% percent match with the above sequence,
only one clone was selected for large scaled plasmid purification.
The final pYD16-aKLH120.6-VK1SP-LC-L10-ShK[1-35, Q16K] construct
encoded an aKLH 120.6 LC-L10-ShK[1-35, Q16K] fusion polypeptide
(SEQ ID NO:439).
[0627] The Shk[2-35, Q16K] fragment was generated as described
above using pTT5-aKLH120.6 HC-ShK[2-35, Q16K] as a template and the
oligonucleotide primers SEQ ID NO:441 and SEQ ID NO:442.
[0628] The light chain and ShK PCR products were amplified by PCR
using the outside primers SEQ ID NO:482 and SEQ ID NO:442. The PCR
product was then digested by XbaI and NotI and PCR clean up kit
(Qiagen) purified. At the same time, pYD16 was cut by XbaI and
NotI. The pYD16 vector was run out on a 1% agarose gel and the
larger fragment was cut out and gel purified by Qiagen's Gel
Purification Kit. The purified PCR product was ligated to the large
vector fragment and transformed into OneShot Top10 bacteria. DNAs
from transformed bacterial colonies were isolated and subjected to
XbaI and NotI restriction enzyme digestions and resolved on a one
percent agarose gel. DNAs resulting in an expected pattern were
submitted for sequencing. Although, analysis of several sequences
of clones yielded a 100% percent match with the above sequence,
only one clone was selected for large scaled plasmid purification.
The final pYD16-aKLH120.6-VK1SP-LC-L10-ShK[2-35, Q16K] construct
encoded an aKLH LC-L10-ShK[2-35, Q16K] fusion polypeptide (SEQ ID
NO:440).
[0629] aKLH-IgG2 Heavy Chain-L10-ShK[1-35] and aKLH-IgG2 Heavy
Chain-L10-ShK[1-35, Q16K] mammalian expression.
[0630] Using oligos
5'-CAT TCT AGA CCC ACC ATG GAC ATG AGG GTG-3' (SEQ ID NO:393);
and
5'-GGA TCC TCC TCC TCC ACC CGG AGA CAG GGA GAG G-3' (SEQ ID
NO:358),
[0631] the a-KLH-IgG2-Heavy Chain region was amplified by PCR from
a pTT5-aKLH 120.6-VK1SP-IgG2 Heavy Chain (HC) construct containing
the coding sequence (SEQ ID NO:356; below), encoding aKLH
120.6-VK1SP-IgG2 Heavy Chain (SEQ ID NO:357; below):
ATGGACATGAGGGTGCCCGCTCAGCTCCTGGGGCTCCTGCTGCTGTGG
CTGAGAGGTGCCAGATGTCAGGTGCAGCTGGTGCAGTCTGGGGCTGAG
GTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGA
TACACCTTCACCGGCTACCACATGCACTGGGTGCGACAGGCCCCTGGA
CAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCAC
AAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACA
CGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACG
ACACGGCCGTGTATTACTGTGCGAGAGATCGTGGGAGCTACTACTGGT
TCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCA
CCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCT
CCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCG
AACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGC
ACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTG
CAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTG
AGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGG
CAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA
TGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCC
ACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCAC
GTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAA
CGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCC
CCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCA
CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCA
GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGC
CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA
CACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT
CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC
CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC
CCTGTCTCCGGGT//(SEQ ID NO:356), encoding the amino acid sequence
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCV
ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN
WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPG//(SEQ ID NO:357).
[0632] The ShK[1-35]WT fragment was generated using the original
Fc-L10-ShK[1-35] in pcDNA3.1(+)CMVi as a template (described in
Example 1, FIG. 14A-14B in Sullivan et al., Toxin Peptide
Therapeutic Agents, PCT/US2007/022831, published as WO 2008/088422,
which is incorporated herein by reference in its entirety) and the
oligos:
TABLE-US-00048 (SEQ ID NO: 359) 5'-TCC CTG TCT CCG GGT GGA GGA GGA
GGA TCC GGA G-3'; and (SEQ ID NO: 330) 5'- CAT GCG GCC GCT CAT TAG
CAG GTG -3'
The PCR products were run on a 1% agarose gel. The bands were
punched for an agarose plug and the plugs were placed in a fresh
PCR reaction tube. The agarose plugs were then amplified by PCR
using the outside primers SEQ ID NO:357 and SEQ ID NO:330. The PCR
product was then digested by XbaI and NotI and PCR clean up kit
(Qiagen) purified. At the same time, pTT5 Vector (an Amgen vector
containing a CMV promoter and Poly A tail) was cut by XbaI and
NotI. The pTT5 vector was run out on a 1% agarose gel and the
larger fragment was cut out and gel purified by Qiagen's Gel
Purification Kit. The purified PCR product was ligated to the large
vector fragment and transformed into OneShot Top10 bacteria. DNAs
from transformed bacterial colonies were isolated and subjected to
XbaI and NotI restriction enzyme digestions and resolved on a one
percent agarose gel. DNAs resulting in an expected pattern were
submitted for sequencing. Although, analysis of several sequences
of clones yielded a 100% percent match with the above sequence,
only one clone was selected for large scaled plasmid purification.
The final pTT5-aKLH 120.6-VK1SP-IgG2-HC-L10-ShK[1-35] construct
encoded an IgG2-HC-L10-ShK[1-35] fusion polypeptide with the amino
acid sequence:
TABLE-US-00049 (SEQ ID NO: 394)
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERK
CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYK
CKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSRSCIDTIPKSR
CTAFQCKHSMKYRLSFCRKTCGTC//.
[0633] To generate the ShK[1-35, Q16K] mutant version of this
construct, site-directed mutagenesis was performed using the
Stratagene Quikchange Multi site Directed Mutagenesis Kit
(Cat#200531), per manufacturer's instructions, and oligos:
TABLE-US-00050 (SEQ ID NO: 325) 5'-GCT GCA CCG CCT TCA AGT GCA AGC
ACA GC 3'; and (SEQ ID NO: 326) 5'- GCT GTG CTT GCA CTT GAA GGC GGT
GCA GC -3',.
The final construct pTT5-aKLH120.6-VK1SP-IgG2-HC-L10-ShK[1-35,
Q16K] encoded IgG2-HC-L10-ShK[1-35, Q16K] fusion polypeptide with
the following amino acid sequence:
TABLE-US-00051 (SEQ ID NO: 333)
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCV
ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN
WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSRSCIDTIPKSRCTAFKCK
HSMKYRLSFCRKTCGTC//.
[0634] aKLH-IgG2 Heavy Chain-L10-ShK[2-35, Q16K] mammalian
expression. Using DNA construct pTT5-aKLH 120.6-VK1
SP-IgG2-HC-L10-ShK[1-35] as the vector, the ShK[1-35] was cut out
using BamHI/BamHI. The vector fragment from pTT5-aKLH
120.6-VK1SP-IgG2-HC without ShK[1-35] contained the coding
sequence:
ATGGACATGAGGGTGCCCGCTCAGCTCCTGGGGCTCCTGCTGCTGTGG
CTGAGAGGTGCCAGATGTCAGGTGCAGCTGGTGCAGTCTGGGGCTGAG
GTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGA
TACACCTTCACCGGCTACCACATGCACTGGGTGCGACAGGCCCCTGGA
CAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCAC
AAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACA
CGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACG
ACACGGCCGTGTATTACTGTGCGAGAGATCGTGGGAGCTACTACTGGT
TCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCA
CCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCT
CCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCG
AACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGC
ACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTG
CAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTG
AGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGG
CAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA
TGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCC
ACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCAC
GTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAA
CGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCC
CCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCA
CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCA
GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGC
CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA
CACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT
CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC
CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC
CCTGTCTCCGGGTGGAGGAGGA//(SEQ ID NO:399), encoding the amino acid
sequence MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCV
ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN
WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGGGG//(SEQ ID NO:400).
[0635] The vector fragment was then treated with Calf Intestine
Phosphatase (CIP) to remove the 5' Phosphate group and
Phenol/Chloroform extracted to prevent religation of the vector
upon itself. The insert came from pTT14-VH21SP-IgG2-Fc-ShK[2-35,
Q16K] encoding IgG2 Fc-L10-ShK(2-35, Q16K):
TABLE-US-00052 (SEQ ID NO: 346)
MEWSWVFLFFLSVTTGVHSERKVECPPCPAPPVAGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGG
SGGGGSSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC//,
and the insert was also digested out using BamHI/BamHI. The insert
ShK[2-35, Q16K] fragment was gel purified away from its vector and
cleaned up with Qiagen Gel Purification Kit. A purified DNA insert
containing the coding sequence
GGA TCC GGA GGA GGA GGA AGC AGC TGC ATC GAC ACC ATC CCC AAG AGC CGC
TGC ACC GCC TTC AAG TGC AAG CAC AGC ATG AAG TAC CGC CTG AGC TTC TGC
CGC AAG ACC TGC GGC ACC TGC TAA TGA//(SEQ ID NO:397),
[0636] encoding the amino acid sequence
GSGGGGSSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC (SEQ ID NO:398), was
ligated to the large vector fragment and transformed into OneShot
Top10 bacteria. DNAs from transformed bacterial colonies were
isolated and subjected to BamHI restriction enzyme digestion and
resolved on a one percent agarose gel. DNAs resulting in an
expected pattern were submitted for sequencing. Although, analysis
of several sequences of clones yielded a 100% percent match with
the above sequence, only one clone was selected for large scaled
plasmid purification. The final construct pTT5-aKLH-IgG2
HC-L10-ShK[2-35,Q16K] encoded an IgG2 HC-L10-ShK[2-35,Q16K] fusion
polypeptide:
TABLE-US-00053 (SEQ ID NO: 401)
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG
YTFTGYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRD
TSISTAYMELSRLRSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCV
ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN
WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSSCIDTIPKSRCTAFKCKH
SMKYRLSFCRKTCGTC//.
[0637] The Shk[1-35, R1A, I4A, Q16K] fragment was generated using
pTT5-aKLH 120.6-VK1SP-IgG2-HC-L10-ShK[1-35 Q16K] as a template and
the oligos:
TABLE-US-00054 (SEQ ID NO: 468) 5'- AGG AGG AGG AAG CGC CAG CTG CGC
CGA CAC CAT CCC C -3'//; and (SEQ ID NO: 469) 5'- GGG GAT GGT GTC
GGC GCA GCT GGC GCT TCC TCC TCC T -3'//.
Site-directed mutagenesis was performed using the Stratagene
Quikchange Multi site Directed Mutagenesis Kit, per manufacturer's
instructions. The final pTT5-aKLH120.6-VK1SP-IgG2-HC-L10-ShK[1-35
R1A, I4A, Q16K] construct encoded an IgG2-HC-L10-ShK[1-35, R1A,
I4A, Q16K] fusion polypeptide (SEQ ID NO:462).
[0638] The Shk[1-35, R1A, Q16K, K30E] fragment was generated as
described above using the following four oligos:
TABLE-US-00055 (SEQ ID NO: 470) 5'- GAG GAG GAG GAA GCG CCA GCT GCA
TCG ACA -3'//; (SEQ ID NO: 471) 5'- GAG CTT CTG CCG CGA GAC CTG CGG
CAC -37/; (SEQ ID NO: 472) 5'- CGA TGC AGC TGG CGC TTC CTC CTC CTC
-37/; and (SEQ ID NO: 473) 5'- GTG CCG CAG GTC TCG CGG CAG AAG CTC
-37/.
The final pTT5-aKLH120.6-VK1SP-IgG2-HC-L10-ShK[1-35 R1A, Q16K,
K30E] construct encoded an IgG2-HC-L10-ShK[1-35, R1A, Q16K, K30E]
fusion polypeptide (SEQ ID NO:463).
[0639] The ShK[1-35, R1H, I4A, Q16K] fragment was generated using
pTT5-aKLH120.6-VK1SP-IgG2-HC-L10-ShK[1-35 Q16K] as a template and
the oligos:
TABLE-US-00056 (SEQ ID NO: 474) 5'- GGA GGA GGA AGC CAC AGC TGC GCC
GAC ACC ATC CCC -3'//; and (SEQ ID NO: 475) 5'- GGG GAT GGT GTC GGC
GCA GCT GTG GCT TCC TCC TCC -3'//.
Site-directed mutagenesis was performed using the Stratagene
Quikchange Multi site Directed Mutagenesis Kit (Cat#200531), per
manufacturer's instructions. The final
pTT5-aKLH120.6-VK1SP-IgG2-HC-L10-ShK[1-35 R1H, I4A, Q16K] construct
encoded an IgG2-HC-L10-ShK[1-35, R1H, I4A, Q16K] fusion polypeptide
(SEQ ID NO:464).
[0640] The Shk[1-35, R1H, Q16K, K30E] fragment was generated as
described above using the following four oligos:
TABLE-US-00057 (SEQ ID NO: 476) and SEQ ID NO: 471 5'- GGA GGA GGA
AGC CAC AGC TGC ATC GAC -3'//; (SEQ ID NO: 477) and SEQ ID NO: 473
5'- GTC GAT GCA GCT GTG GCT TCC TCC TCC -3'//.
The final pTT5-aKLH120.6-VK1SP-IgG2-HC-L10-ShK[1-35 R1H, Q16K,
K30E] construct encoded an IgG2-HC-L10-ShK[1-35, R1H, Q16K, K30E]
fusion polypeptide (SEQ ID NO:465).
[0641] The Shk[1-35, R1K, I4A, Q16K] fragment was generated using
pTT5-aKLH 120.6-VK1SP-IgG2-HC-L10-ShK[1-35 Q16K] as a template and
the oligos:
TABLE-US-00058 (SEQ ID NO: 478) 5'- CCG GAG GAG GAG GAA GCA AGA GCT
GCG CCG ACA CCA TCC CCA AGA -3'//; and (SEQ ID NO: 479) 5'- TCT TGG
GGA TGG TGT CGG CGC AGC TCT TGC TTC CTC CTC CTC CGG -3'//.
Site-directed mutagenesis was performed using the Stratagene
Quikchange Multi site Directed Mutagenesis Kit (Cat#200531), per
manufacturer's instructions. The final
pTT5-aKLH120.6-VK1SP-IgG2-HC-L10-ShK[1-35 R1K, I4A, Q16K] construct
encoded an IgG2-HC-L10-ShK[1-35, R1K, I4A, Q16K] fusion polypeptide
(SEQ ID NO:466).
[0642] The Shk[1-35, R1K, Q16K, K30E] fragment was generated as
described above using the following four oligos:
TABLE-US-00059 (SEQ ID NO:480) and SEQ ID NO:471 5'-CGG AGG AGG AGG
AAG CAA GAG CTG CAT CGA CAC CA-3'//; (SEQ ID NO:481) and SEQ ID
NO:473 5'-TGG TGT CGA TGC AGC TCT TGC TTC CTC CTC CTC CG-3'//.
The final pTT5-aKLH 120.6-VK1SP-IgG2-HC-L10-ShK[1-35 R1H, Q16K,
K30E] construct encoded an IgG2-HC-L10-ShK[1-35, R1K, Q16K, K30E]
fusion polypeptide (SEQ ID NO:467).
[0643] Method for Isolating Monovalent Ab HC- and Monovalent,
Bivalent, and Trivalent Ab LC-Toxin Peptide Analog Fusions. Initial
purification of the conditioned media was done by affinity fast
protein liquid chromatography (FPLC) capture of the Fc region using
Protein A Sepharose (GE Healthcare) followed by a column wash with
Dulbecco's PBS without divalent cations (Invitrogen) and step
elution with 100 mM acetic acid, pH 3.5 at a flow rate of 2.5
cm/min. Protein containing fractions were pooled, and the pH was
adjusted to 5.0 using 10 N NaOH and further diluted with 5 volumes
of water. The material was filtered through a 0.45 .mu.m cellulose
acetate filter (Corning) and further purified by cation exchange
FPLC(SP Sepharose High Performance; GE Healthcare). Samples were
loaded onto a column equilibrated with 100% buffer A (50 mM acetic
acid, pH 5.0) and eluted with a gradient of 0 to 80% buffer B (50
mM acetic acid, 1 M NaCl, pH 5.0) over 30 column volumes at a
flowrate of 1.5 cm/min. Peaks containing target species were pooled
and formulated into 10 mM sodium acetate, 9% sucrose, pH 5.0.
Exemplary purifications of monovalent, bivalent and trivalent
immunoglobulin-toxin peptide analog fusion proteins are shown in
FIGS. 42-44A-B, 45-47A-B, 48-50A-B, and 51-53. The non-reducing
SDS-PAGE analysis (FIGS. 42, 46, 48 and 51) demonstrate that the
fully assembled antibody can be formed, and the reducing SDS-PAGE
analysis demonstrates that the desired components are present. The
size exclusion chromatograms (FIGS. 43, 46, 49 and 52) show that
the majority of the purified product is in the desired
non-aggregated state. Finally, the mass spectral analysis (FIGS.
44A-B, 47A-B, 50A-B and 53) demonstrates that the desired fusion
products are present. Taken together these examples demonstrate
that the aKLH 120.6 antibody can accept fusions in a wide variety
of configurations including species containing an even- or
odd-numbered valence of at least one to eight pharmacologically
active polypeptide moieties.
[0644] VH21SP-N-terminus ShK[1-35]Wild Type-IgG1-Fc mammalian
expression. A DNA sequence coding for a monomer of the Kv1.3
inhibitor peptide ShK[1-35] fused in-frame to the N-terminal Fc
region of human IgG1 was constructed as described below.
[0645] For construction of VH21 SP-ShK(1-35)-L10-IgG1 Fc expression
vector, a PCR strategy was employed to generate the VH21 signal
peptide ShK(1-35) gene linked to a four glycine and one serine
amino acid flanked by HindIII and BamHI restriction sites and a
four glycine and one serine amino acid linked to IgG1 Fc fragment
flanked by BamHI and NotI restriction sites was generated in a PCR
reaction using the Fc-L10-OSK1 in pcDNA3.1(+)CMVi as a template
(described in Example 41 and FIG. 42A-B of Sullivan et al., WO
2008/088422A2, incorporated by reference).
[0646] To generate VH21 SP-ShK(1-35)-G.sub.4S, two oligos with the
sequence as depicted below were used in a PCR reaction with
PfuTurbo HotStart DNA polymerase (Stratagene) at 95.degree. C.-30
sec, 55.degree. C.-30 sec, 75.degree. C.-45 sec for 35 cycles;
HindIII (aagctt) and BamHI (ggatcc) restriction sites are
underlined:
TABLE-US-00060 Forward primer: (SEQ ID NO: 361)
TGCAGAAGCTTCTAGACCACCATGGAATGGAGCTGGGTCTTTCTCTTCT
TCCTGTCAGTAACGACTGGTGTCCACTCCCGCAGCTGCATCGACACCA
TCCCCAAGAGCCGCTGCACCGCCTTCCAGT//; and Reverse primer: (SEQ ID NO:
362) CTCCGGATCCTCCTCCTCCGCAGGTGCCGCAGGTCTTGCGGCAGAAGC
TCAGGCGGTACTTCATGCTGTGCTTGCACTGGAAGGCGGTGCAGCGGC
TCTTGGGGATGGTGTCGAT//.
[0647] The resulting PCR products were resolved as the 202 bp bands
on a two percent agarose gel. The 202 bp PCR product was purified
using PCR Purification Kit (Qiagen), then digested with HindIII and
BamHI (Roche) restriction enzymes, and agarose gel was purified by
Gel Extraction Kit (Qiagen).
[0648] To generate G.sub.4S-IgG1 Fc, two oligos with the sequence
as depicted below were used in a PCR reaction with PfuTurbo
HotStart DNA polymerase (Stratagene) at 95.degree. C.-30 sec,
55.degree. C.-30 sec, 75.degree. C.-1 min for 30 cycles; BamHI
(ggatcc) and NotI (gcggccgc) restriction sites are underlined:
TABLE-US-00061 Forward primer: (SEQ ID NO: 363)
GTAGGATCCGGAGGAGGAGGAAGCGACAAAACTCACAC//; and Reverse primer: (SEQ
ID NO: 364) CGAGCGGCCGCTTACTATTTACCCGGAGACAGGGA//.
[0649] The resulting PCR products were resolved as the 721-bp bands
on a one percent agarose gel. The 721-bp PCR product was purified
using PCR Purification Kit (Qiagen), then digested with BamHI and
NotI (Roche) restriction enzymes, and agarose gel was purified by
Gel Extraction Kit (Qiagen).
[0650] The pcDNA3.1(+)CMVi-Fc-L10-OSK1 vector was digested with
BamHI and NotI restriction enzymes and the large fragment was
purified by Gel Extraction Kit. The gel purified 4GS-IgG1 Fc
fragment was ligated to the purified large fragment and transformed
into One Shot.RTM. Top10 (Invitrogen) to create a pCMVi-Fc-L10-IgG1
Fc vector. Subsequently, pCMVi-Fc-L10-IgG1 Fc vector was digested
with HindIII and BamHI restriction enzymes and the large fragment
was purified by Gel Extraction Kit. The gel purified VH21
SP-ShK(1-35)-4GS fragment was ligated to the purified large
fragment and transformed into One Shot.RTM. Top10 (Invitrogen)
resulting in a pCMV1-VH21 SP-ShK(1-35)-L10-IgG1 Fc construct. DNAs
from transformed bacterial colonies were isolated and digested with
BamHI and NotI restriction enzymes and resolved on a one percent
agarose gel. DNAs resulting in an expected pattern were submitted
for sequencing. Although, analysis of several sequences of clones
yielded a 100% percent match with the above sequences, only one
clone from each gene was selected for large scaled plasmid
purification. The DNA from VH21 SP-ShK(1-35)-L10-IgG1 Fc in pCMVi
vector was resequenced to confirm the Fc and linker regions and the
sequence was 100% identical to the above sequence. Fragment VH21
SP-ShK(1-35)-L10-IgG1 Fc contained the coding sequence
ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTG
TCCACTCCCGCAGCTGCATCGACACCATCCCCAAGAGCCGCTGCACCG
CCTTCCAGTGCAAGCACAGCATGAAGTACCGCCTGAGCTTCTGCCGCA
AGACCTGCGGCACCTGCGGAGGAGGAGGATCCGGAGGAGGAGGAAGC
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG
GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG
ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC
GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGT
GCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGT
ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG
GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTC
AGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCC
TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG
ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG
TCTCCGGGTAAATAGTAA//(SEQ ID NO:365), encoding amino acid sequence
MEWSWVFLFFLSVTTGVHSRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKT
CGTCGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//(SEQ ID NO:366).
[0651] Mammalian expression of N-terminus ShK[1-35, Q16K]-aKLH HC;
and N-terminus ShK[1-35Q16K]-aKLH LC. Using a construct encoding
N-terminus ShK[1-35]Wild Type-L10-IgG1-Fc, site directed
mutagenesis was performed using the following oligos to produce a
Q16K mutation in the ShK region:
TABLE-US-00062 (SEQ ID NO: 325) 5'-GCT GCA CCG CCT TCA AGT GCA AGC
ACA GC-3'//; and (SEQ ID NO: 326) 5'-GCT GTG CTT GCA CTT GAA GGC
GGT GCA GC-3'.
The Stratagene QuikChange Multi Site Directed Mutagenesis Kit was
used according to the manufacturer's instructions. The final
construct for pCMVi-N-terminus-ShK[1-35Q16K]-L10-IgG1-Fc encoded
the following Signal peptide-ShK[1-35, Q16K]-L10-IgG1-Fc fusion
polypeptide:
TABLE-US-00063 (SEQ ID NO: 402)
MEWSWVFLFFLSVTTGVHSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKT
CGTCGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT
PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//.
[0652] To generate the N-terminus ShK[1-35, Q16K]-aKLH HC
construct, a PCR product containing the Signal
peptide-ShK[1-35Q16K]-L10 linker was produced using the following
oligos:
5'-CAT TCT AGA CCA CCA TGG AAT GG-3' (SEQ ID NO:367);
[0653] 5'-CAG CTG CAC CTG GCT TCC TCC TCC TCC GG-3' (SEQ ID
NO:368); and template pCMVi-N-terminus-ShK[1-35Q16K]-L10-IgG1-Fc,
resulted in a fragment containing the coding sequence
ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTG
TCCACTCCCGCAGCTGCATCGACACCATCCCCAAGAGCCGCTGCACCG
CCTTCAAGTGCAAGCACAGCATGAAGTACCGCCTGAGCTTCTGCCGCA
AGACCTGCGGCACCTGCGGAGGAGGAGGATCCGGAGGAGGAGGAAGC//(SEQ ID
NO:369),
[0654] encoding the amino acid sequence
TABLE-US-00064 (SEQ ID NO: 370)
MEWSWVFLFFLSVTTGVHSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKT
CGTCGGGGSGGGGS//.
[0655] To generate the aKLH-HC fragment, a PCR product was created
using oligos:
5'-GGA GGA GGA AGC CAG GTG CAG CTG GTG CAG-3' (SEQ ID NO:371);
[0656] 5'-CAT GCG GCC GCT CAT TTA CCC-3' (SEQ ID NO:372); and
template pTT5-aKLH 120.6-HC, resulting in a DNA fragment containing
the coding sequence CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGC
CTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTA
CCACATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGAT
GGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTT
TCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTA
CATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTG
TGCGAGAGATCGTGGGAGCTACTACTGGTTCGACCCCTGGGGCCAGGG
AACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTC
CCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTG
GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGG
AACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTA
CAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCC
AGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGA
GTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCT
CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA
GGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCC
AGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACA
AAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTC
CTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGC
AAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCC
AAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC
ATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT
CAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG
GGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCG
ACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGT
GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGC
ACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA//(SEQ ID NO:373),
encoding amino acid sequence
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHMHWVRQAPGQGLEW
MGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYC
ARDRGSYYWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT
QTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK//(SEQ ID
NO:374).
[0657] The two PCR products were run out on a gel and the
appropriate sized band was punched for an agarose plug. The agarose
plugs were placed in a single new PCR reaction, and the fragments
were sewn together using outer most primers (SEQ ID NO:367) and
(SEQ ID NO:372). The PCR fragment was cut using XbaI and NotI and
cleaned with Qiagen PCR Cleanup Kit. At the same time, pTT5 vector
was also cut by XbaI and NotI and gel purified. The purified insert
was ligated to the large vector fragment and transformed into
OneShot Top10 bacteria. DNAs from transformed bacterial colonies
were isolated and subjected to XbaI and NotI restriction enzyme
digestions and resolved on a one percent agarose gel. DNAs
resulting in an expected pattern were submitted for sequencing.
Although, analysis of several sequences of clones yielded a 100%
percent match with the above sequence, only one clone was selected
for large scaled plasmid purification. The final construct
pTT5-N-terminus ShK[1-35Q16K]-L10-aKLH120.6-HC encoded a ShK[1-35,
Q16K]-L10-aKLH120.6-HC fusion polypeptide:
TABLE-US-00065 (SEQ ID NO: 403)
MEWSWVFLFFLSVTTGVHSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKT
CGTCGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHMHW
VRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRL
RSDDTAVYYCARDRGSYYWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRS
TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK
TKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK
TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK//.
[0658] Lastly, the N-terminus-ShK[1-35Q16K]-L10-aKLH120.6 Light
Chain (LC) was generated in the same manner as above. A PCR product
containing the signal peptide-ShK[1-35, Q16K]-L10 was created using
oligos: 5'-CAT TCT AGA CCA CCA TGG AAT GG-3' (SEQ ID NO:367); and
5'-CAT CTG GAT GTC GCT TCC TCC TCC TCC GG-3' (SEQ ID NO:375); and
template pCMVi-N-terminus-ShK[1-35Q16K]-L10-IgG1-Fc, resulting in a
DNA fragment containing the coding sequence
ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTG
TCCACTCCCGCAGCTGCATCGACACCATCCCCAAGAGCCGCTGCACCG
CCTTCAAGTGCAAGCACAGCATGAAGTACCGCCTGAGCTTCTGCCGCA
AGACCTGCGGCACCTGCGGAGGAGGAGGATCCGGAGGAGGAGGAAGC//(SEQ ID
NO:369),
[0659] encoding the amino acid sequence for a signal peptide (VH21
SP)-ShK(1-35, Q16K)-L10 linker:
TABLE-US-00066 (SEQ ID NO: 370)
MEWSWVFLFFLSVTTGVHSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKT
CGTCGGGGSGGGGS//.
Using template and oligos:
TABLE-US-00067 (SEQ ID NO: 378) 5'-GGA GGA GGA AGC GAC ATC CAG ATG
ACC CAG TC-3'; and (SEQ ID NO: 379) 5'-CAT CTC GAG CGG CCG CTC
AAC-3'.
[0660] The resulting cloned PCR fragment contained the coding
sequence
ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTG
TCCACTCCCGCAGCTGCATCGACACCATCCCCAAGAGCCGCTGCACCG
CCTTCAAGTGCAAGCACAGCATGAAGTACCGCCTGAGCTTCTGCCGCA
AGACCTGCGGCACCTGCGGAGGAGGAGGATCCGGAGGAGGAGGAAGC
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG
ACAGAGTCACCATCACTTGCC GGGCAAGTCAGGGCATTAGAAATGATT
TAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCTAAACGCCTGATCT
ATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCA
GTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTG
AAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCGCTCAC
TTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCAC
CATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAAC
TGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA
GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG
AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAG
CACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACG
CCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT
TCAACAGGGGAGAGTGTTGAP (SEQ ID NO:380) was generated, encoding the
amino acid sequence for N-terminus-ShK[1-35Q16K]-L10-aKLH120.6
Light Chain (LC) with an N-terminal signal peptide:
TABLE-US-00068 (SEQ ID NO: 381)
MEWSWVFLFFLSVTTGVHSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKT
CGTCGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY
QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATY
YCLQHNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL
NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY
EKHKVYACEVTHQGLSSPVTKSFNRGEC//.
[0661] Both PCR fragments (DNA fragment containing the coding
sequence SEQ ID NO:369 and aKLH 120.6 Light Chain LC fragment
containing the coding sequence SEQ ID NO:380) were run out on a
gel, and the appropriate sized band was punched for an agarose
plug. The agarose plugs were placed in a single new PCR reaction,
and the fragments were sewn together using outer most primers (SEQ
ID NO:367) and (SEQ ID NO:379). The resulting PCR fragment was cut
using XbaI and NotI and cleaned with Qiagen PCR Cleanup Kit.
[0662] At the same time, pTT14 vector (an Amgen vector containing a
CMV promoter, Poly A tail and a Puromycin resistance gene) was also
cut by XbaI and NotI and gel purified. The purified insert was
ligated to the large vector fragment and transformed into OneShot
Top10 bacteria. DNAs from transformed bacterial colonies were
isolated and subjected to XbaI and NotI restriction enzyme
digestions and resolved on a one percent agarose gel. DNAs
resulting in an expected pattern were submitted for sequencing. The
final construct pTT14-N-terminus ShK[1-35Q16K]-L10-aKLH120.6-LC
encoding a Signal Peptide-ShK[1-35, Q16K]-L10-aKLH120.6-LC fusion
polypeptide sequence (i.e., SEQ ID NO:381).
[0663] Mammalian expression of aDNP 3A4 (W101F) IgG2-Shk[1-35].
[0664] Making of Plasmid pTT5-aDNP 3A4 (W101F) IgG2-Shk[1-35Q16K]:
DNA sequences coding for the heavy chain of human
anti-2,4-dinitrophenyl (DNP) antibody fused in frame to a monomer
of the Kv1.3 inhibitor toxin peptide analog ShK[1-35, Q16K] (SEQ ID
NO:13) were constructed using standard cloning technology. Plasid
pTT5-aDNP 3A4 (W101F) IgG2-Shk[1-35Q16K] was generated by 3 way
ligation of the pTT5 vector with a portion of an anti-DNP 3A4
(W101F) IgG2 Heavy Chain (pDC324:aDNP 3A4 HC (W101F) having an
amino acid sequence:
MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGVVQPGRSLRLSCAASG
FTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCARYNFNYGMDVWGQGTTVTVSSASTK
GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE
CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNW
YVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSN
KGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVE
WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPG//(SEQ ID NO:405); and a portion from
IgG2Fc-Shk[1-35, Q16K]. The pTT5 vector was cut with SalI/NotI
releasing the multiple cloning site. The vector was then treated
with Calf Intestine Phosphatase (CIP) to reduce background. The
first insert came from pDC324:aDNP 3A4 HC (W101F) by cutting with
SalI/StuI, resulting in the a DNA fragment containing the coding
sequence ATGGACATGAGGGTGCCCGCTCAGCTCCTGGGGCTCCTGCTGCTGTGG
CTGAGAGGTGCGCGCTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGG
CGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGG
ATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGG
CAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATA
AATACTATGCAGACTCCGTGAAGGGCCGATTCACTATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG
ACACGGCTGTGTATTACTGTGCGAGGTATAACTTCAACTACGGTATGG
ACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTAGTGCCTCCACCA
AGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCG
AGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAAC
CGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACA
CCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGT
GGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAA
CGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGC
GCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAG
GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGAT
CTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGA
AGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA
TAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCC
GTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCA
AGGAGTACAAGTGCAAGGTCTCCAACAAAGGC//(SEQ ID NO:383), encoding amino
acid sequence MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGVVQPGRSLRLSCAASG
FTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCARYNFNYGMDVWGQGTTVTVSSASTK
GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE
CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNW
YVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSN KG//(SEQ ID
NO:384). The second insert was digested out using StuI/NotI and
contained the coding sequence
CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACC
AAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGC
GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA
CAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTAC
AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT
CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA
GAGCCTCTCCCTGTCTCCGGGTAAAGGAGGAGGAGGATCCGGAGGAG
GAGGAAGCCGCAGCTGCATCGACACCATCCCCAAGAGCCGCTGCACC
GCCTTCAAGTGCAAGCACAGCATGAAGTACCGCCTGAGCTTCTGCCGC
AAGACCTGCGGCACCTGCTAATGA//(SEQ ID NO:395), encoding the following
truncated IgG2 Fc-L10-ShK(1-35, Q16K) amino acid sequence
LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGKGGGGSGGGGSRSCIDTIPKSRCTAFKCKHSM
KYRLSFCRKTCGTC//(SEQ ID NO:396).
[0665] The vector and insert fragments were gel purified and
cleaned up with Qiagen Gel Purification Kit. The purified inserts
were ligated to the large vector fragment and transformed into
OneShot Top10 bacteria. DNAs from transformed bacterial colonies
were isolated and subjected to SalI/NotI restriction enzyme
digestion and resolved on a one percent agarose gel. DNAs resulting
in an expected pattern were submitted for sequencing. A clone
yielding a 100% percent match with the above sequence was selected
for large scale plasmid purification. The final pTT5-aDNP 3A4
(W101F) IgG2-Shk[1-35, Q16K] construct encoded a aDNP 3A4 (W101F)
IgG2-L10-Shk[1-35, Q16K] having the following amino acid
sequence:
TABLE-US-00069 (SEQ ID NO: 406)
MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGVVQPGRSLRLSCAASGFT
FSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNT
LYLQMNSLRAEDTAVYYCARYNFNYGMDVWGQGTTVTVSSASTKGPSVFP
LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP
APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG
VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAP
IEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPGGGGGSGGGGSRSCIDTIPKSRCTAFKCKHSMKYRL
SFCRKTCGTC//.
[0666] Mammalian expression of anti-DNP 3A4 Antibody Light Chain.
The XenoMouse.RTM. hybridoma expressing aDNP monoclonal antibody
3A4 was used as a source to isolate total RNA. One step RT-PCR with
multiplex gene-specific primers was done to obtain a variable
region product. This product was reamplified with a forward primer
to add a 5' BssHII restriction site 5'-TTT TTT TTG CGC GCT GTG ACA
TCC AGA TGA CCC AGT C-3' (SEQ ID NO:385) and a reverse primer to
add a 3' BsiWI restriction site 5'-AAA AAA CGT ACG TTT GAT ATC CAC
TTT GGT CC-3' (SEQ ID NO:386). The resulting PCR product was
cleaned by Qiagen PCR clean-up, digested with BssHII and BsiWI
restriction enzymes, cleaned by Qiagen nucleotide removal, and
ligated into a mammalian expression vector pTT5 containing a 5'
VK1/O12 signal peptide and a 3' human kappa constant region. The
amino acid sequence of the resulting anti-DNP 3A4 Antibody Light
Chain is the following:
TABLE-US-00070 (SEQ ID NO: 407)
MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSVSASVGDRVTITCRASQG
ISRRLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQQANSFPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC//.
[0667] Method for Isolating Monovalent Fc-Toxin Peptide Analog and
Ab HC- or Ab LC-Toxin Peptide Analog Fusions. Initial purification
of the conditioned media was done by affinity fast protein liquid
chromatography (FPLC) capture of the Fc region using Protein A
Sepharose (GE Healthcare) followed by a column wash with Dulbecco's
PBS without divalent cations (Invitrogen) and step elution with 100
mM acetic acid, pH 3.5 at a flow rate of 2.5 cm/min. Protein
containing fractions were pooled, and the pH was adjusted to 5.0
using 10 N NaOH and further diluted with 5 volumes of water. The
material was filtered through a 0.45 .mu.m cellulose acetate filter
(Corning) and further purified by cation exchange FPLC (SP
Sepharose High Performance; GE Healthcare). Samples were loaded
onto a column equilibrated with 100% buffer A (50 mM acetic acid,
pH 5.0) and eluted with a gradient of 0 to 80% buffer B (50 mM
acetic acid, 1 M NaCl, pH 5.0) over 30 column volumes at a flowrate
of 1.5 cm/min. Peaks containing monovalent species were pooled and
formulated into 10 mM sodium acetate, 9% sucrose, pH 5.0. Exemplary
purifications of monovalent Fc-toxin peptide analog fusion proteins
and immunoglobulin-toxin peptide analog fusion proteins are shown
in FIG. 13A-C, FIG. 15A-C, FIG. 16A-C, and FIG. 18A-C.
[0668] Method for Isolating Bivalent Fc-ShK and Ab HC- or LC-toxin
peptide analog fusions. Bivalent molecules were purified similar to
the monovalent molecules with the addition of a final polishing
step to remove higher molecular weight protein aggregates. After
cation exchange FPLC, peaks containing bivalent species were
polished by gel filtration FPLC (Superdex 200; GE Healthcare).
Samples were loaded onto a column (2.6.times.56.5 cm) equilibrated
with 10 mM sodium acetate, 0.5M NaCl, 9% Sucrose, pH 5.0 and eluted
with an isocratic gradient of buffer at a flowrate of 0.4 cm/min.
Peaks containing bivalent species were pooled and formulated into
10 mM sodium acetate, 9% sucrose, pH 5.0. Exemplary purifications
of bivalent Fc-toxin peptide analog fusion proteins and
immunoglobulin-toxin peptide analog fusion proteins are shown in
FIG. 14A-C and FIG. 17A-C.
Example 12
Monovalent Fc/Fc-L10-ShK[2-35] Heterodimers and Monovalent or
Bivalent Fc/Fc-ShK(1-35 Q16K)(IgG2) Heterodimers and Other
Immunoglobulin Fusion Proteins
[0669] Monovalent or bivalent Fc-L10-ShK[2-35], monovalent or
bivalent Fc-L10-ShK[1-35], monovalent or bivalent Fc-L10-ShK(1-35,
Q16K), monovalent or bivalent anti-KLH HC-ShK(1-35, Q16K) Ab,
monovalent or bivalent anti-KLH AbLoop-[Lys16]ShK fusion Ab
proteins, monovalent Fc-ShK(1-35 Q16K)/KLH Ab heterotrimer, and
other exemplary embodiments listed in Table 4H, were expressed,
isolated and purified by methods described in Example 11.
[0670] Generally, FIG. 12A and FIG. 12B show a schematic
representation of monovalent and bivalent Fc-toxin peptide (or
toxin peptide analog) fusion proteins (or "peptibodies"),
respectively. The bivalent Fc-ShK molecule is a homodimer
containing two Fc-ShK chains. The monovalent Fc-ShK toxin peptide
(or toxin peptide analog) molecule is a heterodimer containing one
Fc chain and one Fc-ShK (or analog) chain. Since the monovalent
Fc-ShK molecule contains just a single ShK peptide per dimer, it is
considered monovalent. Constructs or chains referred to as
Fc-(toxin peptide analog), contain an N-terminal Fc region and an
optional flexible linker sequence (e.g., L10 peptidyl linker
GGGGSGGGGS; SEQ ID NO:292) covalently attached to the toxin peptide
or toxin peptide analog, such that the orientation from N- to
C-terminus would be: Fc-linker-toxin peptide or toxin peptide
analog.
[0671] In Examples 1 and 2 of Sullivan et al., WO 2008/088422A2, we
described earlier the activity of bivalent Fc-ShK peptibodies,
Fc-L10-ShK(1-35) and Fc-L10-ShK(2-35) expressed from mammalian
cells. In Example 1 of WO 2008/088422A2, we also described
isolation of a monovalent Fc-L10-ShK(1-35) molecule, formed as a
small by-product during expression. The bivalent Fc-L10-ShK(1-35)
and Fc-L10-ShK(2-35) conjugates provided potent blockade of Kv1.3
and T cell cytokine secretion in human whole blood (see, Table 4H).
By whole cell patch clamp electrophysiology, the bivalent
Fc-L10-ShK(1-35) molecule had about 8-fold greater Kv1.3 activity
compared to the bivalent Fc-L10-ShK(2-35) molecule that is devoid
of Arg1 of ShK. Like N-terminal PEG conjugates of native ShK (see,
Example 5), both bivalent Fc-ShK conjugates showed little
selectivity for Kv1.3 versus Kv1.1. Thus, N-terminal conjugation of
native ShK alone (with either PEG or Fc-linker) does not
significantly improve its Kv1.3 versus Kv1.1 selectivity.
Pharmacokinetic (PK) studies in rats were performed on bivalent
Fc-L10-ShK(1-35) and Fc-L10-ShK(2-35) peptibodies to examine their
stability and half-life in vivo. As a control, PK was also
performed on CHO-derived recombinant human Fc (IgG1). All molecules
were delivered as a single, intravenous bolus dose. Using an ELISA
specific for the human Fc region (Example 8, "protocol 3"), the
bivalent Fc-L10-ShK(2-35) peptibody showed a long terminal
half-life and slow elimination rate in rats similar to CHO-Fc (FIG.
19A). Despite showing a slow elimination rate, the bivalent
Fc-L10-ShK(2-35) peptibody exhibited a large distribution phase
that was significantly greater than the CHO-Fc molecule. Similar
results were obtained from IV, single-dose rat PK studies on
bivalent Fc-L10-ShK(1-35) and bivalent Fc-L10-OSK1[K7S], two
peptibodies that have been described earlier (Example 1 and Example
41 of WO 2008/088422A2). To more fully understand what might be
causing the extensive distribution of the bivalent peptibodies in
vivo, we developed a solid-surface affinity capture/SDS-PAGE
western blot procedure which allowed us to isolate the peptibody
away from serum proteins for efficient SDS-PAGE and western blot
analysis of their molecular weight and integrity. Serum samples
from the intravenous, single-dose rat PK study were added to
microtiter plates coated with an anti-human Fc antibody to enable
affinity capture. Plates were then washed and captured samples were
released by addition of SDS/Laemalli sample buffer and run on a
polyacrylamide gel. After western blotting to a membrane, samples
were visualized by standard immunobloting techniques using an
anti-human Fc-specific antibody, secondary-HRP conjugate and
luminescent substrate. FIG. 19B indicates that during the extensive
distribution phase, at 0.25 hours after IV administration of
Fc-L10-ShK(2-35), the peptibody is intact. During the slow
elimination phase, from 1 to 48 hours after IV injection, two bands
are observed, the top or higher molecular weight band being
consistent with full-length Fc-L10-ShK[2-35] and the small band
being consistent with Fc alone. Since the peptibody is intact
during the large and extensive distribution phase, this implies the
bivalent peptibody was not proteolyzed but was sequestered within
the animal. In support of this, we do not see large serum
quantities of Fc alone, which would be expected to occur if the ShK
peptide portion of Fc-ShK were quickly cleaved off. Therefore, the
SDS-PAGE/western blot data (FIG. 19B) imply that during the rapid
distribution, the peptibody was intact and not proteolyzed, but
during the slow elimination phase it was a monovalent heterodimer
containing one Fc-ShK[2-35] chain and one Fc chain. Similar
findings were observed from huFc affinity capture, SDS-PAGE/western
blot analysis of serum samples from rat PK studies (intravenous) on
bivalent Fc-L10-ShK[1-35] and on bivalent Fc-L10-OSK1[K7S] (FIG.
19C). Although the exact reason for the rapid and extensive
distribution phase of bivalent Kv1.3 peptibodies in rats is
unknown, the avidity of bivalent peptibodies containing two toxin
units would be much greater than that of a monovalent peptibody.
Whether this is Kv1.3 target specific or non-specific is unknown.
It should be remembered that toxin peptides, such as ShK and OSK1
are highly basic and could be reacting in a non-specific manner.
Irrespective of the cause, because monovalent peptibodies were
present in the slow terminal phase, we investigated whether direct
IV injection of a monovalent peptibody would show less distribution
in rats. To address this, we first tested in rat IV PK studies a
purified monovalent Fc-L10-ShK(1-35) molecule isolated as a
by-product from conditioned media expressing the bivalent molecule
(Example 1, WO 2008/088422A2). FIG. 19D demonstrates by Fc affinity
capture/SDS-PAGE/western blot that the monovalent Fc-L10-ShK(1-35)
peptibody showed a distribution phase that was much less extensive
and had a slow elimination rate. Therefore, despite both bivalent
and monovalent peptibodies exhibiting a slow elimination rate, the
monovalent heterodimeric peptibody showed significantly less
distribution in rats compared to the bivalent homodimeric
forms.
[0672] Whereas, the original monovalent Fc-L10-ShK(1-35) molecule
was isolated as a small by-product during mammalian expression of
the bivalent molecule, Example 11 herein also describes cloning and
mammalian expression of the monovalent Fc-L10-ShK[2-35]
heterodimer. Briefly, to produce recombinant monovalent
Fc-L10-ShK[2-35], two recombinant polypeptides are co-expressed in
the same cell, those being a human Fc (IgG1) chain and a
Fc-L10-ShK[2-35] chain (also with human IgG1 Fc region). Under
these conditions it is possible to form three distinct dimers,
which include an Fc/Fc homodimer, a
Fc-L10-ShK(2-35)/Fc-L10-ShK(2-35) homodimer and a
Fc/Fc-L10-ShK(2-35) heterodimer. By optimizing expression
conditions, the monovalent Fc/Fc-L10-ShK(2-35) heterodimer (also
referred to as just monovalent Fc-L10-ShK(2-35)) was produced
efficiently and was readily purified to homogeneity (Example 11).
The monovalent Fc-L10-ShK(2-35) molecule had an IC50 of 2.1 nM
(Table 4H) in blocking IL-2 secretion from human whole blood. The
monovalent Fc-ShK/Fc heterodimer had an extended half-life in vivo
and exhibited significantly greater exposure than the bivalent
homodimers ShK-Fc/ShK-Fc (FIG. 23) and Fc-ShK/Fc-ShK. Since the
potency of this construct was about 10-fold less than the PEG-ShK
conjugate and conjugates of native ShK had poor Kv1.3/Kv1.1
selectivity, we developed additional monovalent peptibodies and
formed conjugates of ShK toxin peptide analogs that were identified
to have improved Kv1.3 versus Kv1.1 selectivity. The examples that
follow provide additional details of monovalent peptibodies with
improved selectivity and in vivo pharmacology. The results from
these studies indicated that monovalent ShK toxin peptide analog
molecules exhibited greater serum levels and exposure in rats when
compared to the bivalent forms of the same molecule, yet retain the
slow elimination rate observed in the original bivalent
peptibodies.
[0673] Monovalent Fc/Fc-ShK(1-35 Q16K) heterodimer (IgG2). Example
3 herein describes identification of the ShK[Lys16] toxin peptide
analog (SEQ ID NO:13) that shows improved Kv1.3 selectivity over
neuronal Kv1.1. To increase the stability of this toxin peptide
analog in vivo, we generated a monovalent Fc fusion construct that
from N- to C-terminus contained: human Fc(IgG2)-L10
linker-[Lys16]ShK molecule, that was co-expressed with the human
Fc(IgG2) chain alone to generate a monovalent heterodimer (see,
Example 11). A schematic representation of this monovalent
construct is provided in FIG. 12A. The monovalent
Fc/Fc-L10-ShK(1-35 Q16K) heterodimer [also referred to,
interchangeably, as monovalent Fc/Fc-ShK(1-35, Q16K) or monovalent
Fc-L10-ShK(1-35, Q16K]; (SEQ ID NOS: 337; 348)] potently blocked T
cell inflammation in whole blood, suppressing IL-2 secretion with
an IC50 of 0.16 nM (Table 4H). The monovalent Fc-L10-ShK(1-35,
Q16K) molecule also potently blocked proliferation of the rat
myelin-specific T effector memory cell line, PAS, with an IC50 of
0.244 nM as described in FIG. 32 and Example 9. Unexpectedly,
studies to examine the Kv1.3 versus Kv1.1 selectivity of the
molecule, revealed that the monovalent Fc-L10-ShK(1-35 Q16K)
conjugate had significantly better Kv1.3 selectivity than the
[Lys16]ShK peptide alone. Whereas the [Lys16]ShK (SEQ ID NO:13)
peptide alone showed about 18-fold selectivity for Kv1.3 versus
Kv1.1 (Table 4H), the monovalent Fc/Fc-L10-ShK(1-35 Q16K)
heterodimer was about 1225-fold more active in blocking Kv1.3
versus Kv1.1, as determined by PatchXpress.RTM. electrophysiology.
Therefore, the [Lys16]ShK peptide when conjugated shows a unique
pharmacology of enhanced selectivity. Since the N.alpha.-20
kDa-PEG-[Lys16]ShK conjugate (SEQ ID NO:16) also showed enhanced
Kv1.3 selectivity (Example 5, Table 4H) relative to the peptide
alone, the combined data suggests that the [Lys16]-ShK (SEQ ID
NO:13) peptide when fused at its N-terminus with either PEG or
Fc-linker exhibits a distinct pharmacology of improved Kv1.3 versus
Kv1.1 selectivity.
[0674] To expland our analysis of the Kv1.3 selectivity of the
monovalent Fc-L10-ShK(1-35, Q16K) molecule (SEQ ID NOS: 337; 348),
its activity was tested against the five remaining members of the
Kv1 family. The monovalent Fc-L10-ShK(1-35, Q16K) molecule blocked
Kv1.3 1225 times better than Kv1.1, about 1000 times better than
Kv1.2, >3663 times better than Kv1.4, >3663 times better than
Kv1.5, about 164 times better than Kv1.6, and >3663 times better
than Kv1.7, as determined by PatchXpress.RTM. electrophysiology
(Table 4H(b)). As whole cell patch clamp Kv1.3 electrophysiology
and the whole blood assay of T cell inflammation implies that the
true activity of the monovalent Fc-L10-ShK(1-35, Q16K) on Kv1.3 may
be even greater than determined by Kv1.3 PatchXpress.RTM. analysis
by a factor of 8 to 10 times (Table 4H(b)), its fold Kv1.3
selectivity over other Kv1 family members may be even greater.
[0675] To assess the pharmacokinetics and stability of the molecule
in vivo, single-dose PK studies were performed in rats. After a
single 6 mg/kg subcutaneous dose, the monovalent
Fc/Fc-L10-ShK(1-35, Q16K) heterodimer exhibited an extended
half-life in vivo (FIG. 20). Since the sandwich ELISA used to
measure serum levels of the molecule (Example 8, "protocol 2")
requires binding of two antibodies, one an antibody specific to
human Fc region and the other an antibody recognizing [Lys16]ShK
(SEQ ID NO:13), the data here indicate that the conjugate had
prolonged half-life and remained intact in vivo as a
Fc-L10-ShK(1-35 Q16K) fusion protein (FIG. 20, open squares; Table
41 below). The monovalent Fc/Fc-L10-ShK(1-35 Q16K) molecule
exhibited an extended half-life of about 56 hours, that was about
112 times longer than the ShK (SEQ ID NO:1) peptide alone that was
reported to have a half-life of 20-30 min (C. Beeton et al., PNAS
98:13942 (2001)).
[0676] The efficacy of the monovalent Fc/Fc-L10-ShK(1-35,Q16K)
molecule (SEQ ID NOS: 337; 348) in an animal model of multiple
sclerosis was determined using the adoptive-transfer (AT)-EAE
model, as described in Example 9. Monovalent Fc-L10-ShK(1-35,Q16K)
delayed disease onset and caused dose-dependent reduction in
disease severity (FIG. 35 and FIG. 36A-D). The molecule was highly
potent in this model and the estimated effective dose of monovalent
Fc-L10-ShK (1-35,Q16K) to cause 50% reduction in disease severity
(ED50), based on area under the curve of the EAE score, was a dose
of <2.5 nmol/kg or <138 .mu.g/kg. This ED50 compares
favorable to that activity of PEG-[Lys16]ShK in this model, since
the 2.5 nmol/kg dose of the larger monovalent Fc-L10-ShK(1-35,
Q16K) molecule corresponds to a 10 .mu.g/kg dose of the smaller
PEG-[Lys16]ShK molecule whose .mu.g/kg dose and dosing solution is
based on the molecular weight of the peptide portion alone.
[0677] Bivalent Fc-ShK(1-35 Q16K) homodimer (IgG2). The bivalent
Fc-ShK(1-35, Q16K) homodimer contains from N- to C-terminus: human
Fc (IgG2)-L10 linker-[Lys16]ShK (SEQ ID NO:348). A schematic
representation of this bivalent construct is provided in FIG. 12B.
The molecule (homodimer of SEQ ID NO:348) was cloned, expressed and
purified as described in Example 11. The purified molecule was
tested for activity in the human whole blood assay of inflammation
and found to have an IC50 of 1.850 nM in blocking IL-2 secretion
(Table 4H). The activity of this bivalent form was about 12 times
less than the monovalent form (above) which had an IC50 of 0.16 nM
in this same assay. The reason why the bivalent form was less
active than the monovalent is unknown. It is possible that the
bivalent molecule containing two positively charged [Lys16]ShK (SEQ
ID NO:13) peptides at its end, is less stable and/or interferes
with Kv1.3 channel binding to some extent.
[0678] Monovalent and bivalent aKLH HC-ShK(1-35, Q16K) Ab. The
monovalent anti-KLH Heavy Chain (HC) fusion antibody (Ab) construct
contained from N- to C-terminus: human anti-KLH Ab Heavy
Chain-peptidyl linker-[Lys16]ShK molecule (SEQ ID NO:342), that was
co-expressed with the human aKLH Heavy Chain alone (SEQ ID NO:339)
and the human aKLH light chain (SEQ ID NO:338) to form a monovalent
aKLH Ab-[Lys16]ShK molecule (heterotetramer of SEQ ID NO:338; SEQ
ID NO:339; SEQ ID NO:338; and SEQ ID NO:342). A schematic
representation of this monovalent construct is provided in FIG.
12F. The monovalent aKLH HC-ShK(1-35, Q16K) Ab (heterotetramer of
SEQ ID NO:338; SEQ ID NO:339; SEQ ID NO:338; and SEQ ID NO:342)
potently blocked T cell inflammation in whole blood, suppressing
IL-2 secretion with an IC50 of 0.274 nM (Table 4H). The monovalent
aKLH HC-ShK(1-35, Q16K) Ab molecule also potently blocked
proliferation of the rat myelin-specific T effector memory cell
line, PAS, with an IC50 of 0.839 nM as described in FIG. 32 and
Example 9. Unexpectedly, studies to examine the Kv1.3 versus Kv1.1
selectivity of the molecule, revealed that the monovalent aKLH
HC-ShK(1-35, Q16K) Ab (heterotetramer of SEQ ID NO:338; SEQ ID
NO:339; SEQ ID NO:338; and SEQ ID NO:342) had significantly better
Kv1.3 selectivity than the [Lys16]ShK (SEQ ID NO:13) peptide alone.
This monovalent Ab-ShK conjugate was about 1458-fold more active in
blocking Kv1.3 versus Kv1.1 (Table 4H and FIG. 6E-F), as determined
by PatchXpress.RTM. electrophysiology. To expand our analysis of
the Kv1.3 selectivity of the monovalent aKLH HC-ShK(1-35, Q16K) Ab
molecule, its activity was tested against the five remaining
members of the Kv1 family. The monovalent aKLH HC-ShK(1-35, Q16K)
Ab blocked Kv1.3 1458 times better than Kv1.1, 2013 times better
than Kv1.2, >2525 times better than Kv1.4, >2525 times better
than Kv1.5, .about.156 times better than Kv1.6, and >2525 times
better than Kv1.7, as determined by PatchXpress.RTM.
electrophysiology (Table 4H(a)). As whole cell patch clamp Kv1.3
electrophysiology and the whole blood assay of T cell inflammation
suggests the true activity of the monovalent aKLH HC-ShK(1-35,
Q16K) Ab (heterotetramer of SEQ ID NO:338; SEQ ID NO:339; SEQ ID
NO:338; and SEQ ID NO:342) on Kv1.3 may be even greater than
determined by Kv1.3 PatchXpress analysis by a factor of 11 to 13
times (Table 4H(a)), its fold Kv1.3 selectivity over other Kv1
family members may be even greater.
[0679] The efficacy of the monovalent aKLH HC-ShK(1-35,Q16K) Ab
molecule (heterotetramer of SEQ ID NO:338; SEQ ID NO:339; SEQ ID
NO:338; and SEQ ID NO:342) in an animal model of multiple sclerosis
was determined using the adoptive-transfer (AT)-EAE model, as
described in Example 9. The monovalent aKLH HC-ShK(1-35,Q16K) Ab
delayed disease onset and caused dose-dependent reduction in
disease severity (FIG. 33 and FIG. 34A-D). The molecule was highly
potent in this model and the estimated effective dose of monovalent
aKLH HC-ShK(1-35,Q16K) Ab to cause 50% reduction in disease
severity (ED50), based on area under the curve of the EAE score,
was a dose of 2.4 nmol/kg or 360 .mu.g/kg. The efficacy of the
monovalent aKLH HC-ShK(1-35,Q16K) Ab in reducing disease severity
compared favorably to that of PEG-[Lys16]ShK, since a 2.5 nmol/kg
(375 .mu.g/kg) dose of the larger monovalent aKLH HC-ShK(1-35,Q16K)
Ab provided similar disease amelioration to 10 .mu.g/kg (2.5
nmol/kg) of the smaller PEG-[Lys16]ShK (FIG. 33) molecule, whose
.mu.g/kg dose and dosing solution is based on the molecular weight
of the peptide portion alone.
[0680] To assess the pharmacokinetics and stability of the molecule
in vivo, single-dose PK studies were performed in rats. After a
single 6 mg/kg subcutaneous dose, the monovalent KLH HC-ShK(1-35
Q16K) Ab conjugate exhibited an extended half-life in vivo (FIG.
20, closed circles). Since the sandwich ELISA used to measure serum
levels of the molecule (Example 8, "protocol 2") requires binding
of two antibodies, one an antibody specific to human Ig region and
the other an antibody recognizing [Lys16]ShK (SEQ ID NO:13), the
data here indicates that conjugate has prolonged half-life and
remains intact in vivo as a monovalent aKLH HC-ShK(1-35 Q16K) Ab
fusion protein (FIG. 20, FIG. 21, and Table 4J). The bivalent aKLH
HC-ShK(1-35, Q16K) Ab molecule (schematically represented by FIG.
12G) given at the same 6 mg/kg dose, showed a similarly slow
elimination rate (FIG. 21), but provided about 37 times less
exposure (as measured by AUC0-t, Table 4J) relative to the
monovalent molecule (FIG. 21). The potent and selective monovalent
anti-KLH-Ab-[Lys16]ShK molecule exhibited very slow clearance in
rats (CL/F=10.9 mL h.sup.-1 kg.sup.-1) (Table 4J), a rate that was
about 188 times slower than ShK-L5 (SEQ ID NO:17; CL/F=2052 mL
h.sup.-1 kg.sup.-1).
[0681] Monovalent aKLH HC-ShK(2-35 Q16K) Ab. This monovalent aKLH
Heavy Chain (HC) fusion antibody (Ab) construct contained from N-
to C-terminus: human anti-KLH Ab Heavy Chain-linker-[desArg1,
Lys16]ShK molecule (SEQ ID NO:387), that was co-expressed with the
human aKLH Heavy Chain (SEQ ID NO:339) and the human aKLH light
chain (SEQ ID NO:338) to form a monovalent aKLH Ab-[desArg1,
Lys16]ShK molecule. A schematic representation of this monovalent
construct is provided in FIG. 12F. The monovalent aKLH HC-ShK(2-35,
Q16K) Ab (heterotetramer of SEQ ID NO:338; SEQ ID NO:339; SEQ ID
NO:338; and SEQ ID NO:387) potently blocked T cell inflammation in
whole blood, suppressing IL-2 secretion with an IC50 of 0.570 nM
(Table 4H) and unexpectedly was about 1576 fold more potent in
blocking the T-cell potassium channel Kv1.3 than the neuronal
channel Kv1.1.
[0682] Monovalent Fc-ShK(1-35 Q16K)/aKLH Ab Heterotrimer. The
monovalent Fc-ShK(1-35, Q16K)/aKLH Ab heterotrimer or hemibody
contained from N- to C-terminus: human Fc (IgG2)-L10
linker-[Lys16]ShK molecule (SEQ ID NO:348), that was co-expressed
with the human aKLH Heavy Chain (IgG2) (SEQ ID NO:339) and the
human aKLH light chain (SEQ ID NO:338). A schematic representation
of this monovalent construct is provided in FIG. 12E. The
monovalent Fc-ShK(1-35, Q16K)/aKLH Ab heterotrimer (SEQ ID NO:338;
SEQ ID NO:339; SEQ ID NO:348) potently blocked T cell inflammation
in whole blood, suppressing IL-2 secretion with an IC50 of 0.245 nM
(Table 4H). Surpisingly, studies examining the Kv1.3 versus Kv1.1
selectivity of the molecule revealed that the monovalent
Fc-ShK(1-35, Q16K)/aKLH Ab heterotrimer had significantly better
Kv1.3 selectivity than the [Lys16]ShK peptide alone (SEQ ID NO:13).
This monovalent heterotrimer was about 1935 fold more active in
blocking Kv1.3 versus Kv1.1 (Table 4H).
[0683] Although we haven't examined the pharmacokinetics (PK) of
the Kv1.3 selective monovalent Fc-ShK(1-35, Q16K)/aKLH Ab
heterotrimer or hemibody, we have examined the PK profile of a
similar hemibody, that being the Fc-ShK(2-35)/aKLH Ab heterotrimer.
A schematic of the structure of this molecule is provided in FIG.
12E, and the molecule from N- to C-terminus contains: human Fc
(IgG2)-ShK(2-35), which is coexpressed with the human aKLH heavy
chain and light chains. After a single 2 mg/kg subcutaneous dose,
the monovalent Fc-ShK(2-35)/aKLH Ab heterotrimer (also referred to
as monovalent Fc-ShK/aKLH Ab heterotrimer) exhibited an extended
half-life in rats (FIG. 23). Since the sandwich ELISA used to
measure serum levels of the molecule (Example 8, "protocol 2")
requires binding of two antibodies, one an antibody specific to
human Ig region and the other an antibody recognizing ShK(2-35),
the data here indicates that conjugate has prolonged half-life and
remains intact in vivo (FIG. 23, Table 4K). The large, about 103
kDa monovalent Fc-ShK(2-35)aKLH Ab heterotrimer or hemibody showed
greater exposure and about 2-fold less clearance than the about 56
kDa monovalent Fc/Fc-ShK heterodimer (FIG. 23, Table 4K). The very
small, about 4 kDa ShK-L5 peptide was cleared much more quickly,
having a clearance value in rats (CL/F=2052 mL h.sup.-1 kg.sup.-1,
Example 5) that was about 91 times faster than the large monovalent
Fc-ShK(2-35)/aKLH Ab heterotrimer (CL/F=22.6 mL h.sup.-1 kg.sup.-1)
molecule.
[0684] Monovalent and bivalent anti-KLH AbLoop-[Lys16]ShK fusion
proteins. Recombinant monovalent and bivalent anti-KLH
AbLoop-[Lys16]ShK fusion proteins were constructed as described in
Example 11 and U.S. Pat. No. 7,442,778 B2 to produce full
antibodies with [Lys16]ShK toxin peptide analog inserted into loop
regions of the Fc domain in one (monovalent) or both (bivalent) HC
monomers. The monovalent aKLH HC-loop-ShK(1-35, Q16K) Ab contained
three chains: a human aKLH Ab heavy chain, a human aKLH Ab light
chain and a human aKLH Ab heavy chain where the [Lys16]ShK peptide
was inserted into a loop within the Fc region of the heavy chain.
The [Lys16]ShK peptide within the Fc loop contained a flexible
linker sequence attached to its N- and C-terminus to allow for
independent folding and extension from the loop. A schematic
representation of this molecule is provided in provided in FIG.
12N. Linker sequences of differing amino acid composition and
length were examined. The monovalent anti-KLH AbLoop-[Lys16]ShK
fusion Ab protein was a selective inhibitor of Kv1.3 activity (over
Kv1.1; >121-fold more selective for Kv1.3; Table 4H and FIG.
6E-F). The monovalent aKLH-AbLoop-[Lys16]ShK molecule exhibited the
slowest clearance in rats of all the novel toxin-conjugates that we
have examined (FIG. 20 and FIG. 22 and Table 4L).
[0685] The bivalent aKLH HC-loop-ShK(1-35, Q16K) Ab contained two
chains: a human aKLH Ab light chain and a human aKLH Ab heavy chain
where the [Lys16]ShK peptide was inserted into a loop within the Fc
region of the heavy chain. A schematic representation of this
molecule is provided in provided in FIG. 12M. To compare the
pharmacokinetics and stability in vivo of this bivalent molecule to
the monovalent form, single 6 mg/kg subcutaneous doses of each
molecule were delivered to rats. Despite showing a slow elimination
rate, the bivalent aKLH HC-loop-ShK(1-35, Q16K) Ab gave profoundly
less exposure in rats than the monovalent form of the same molecule
(monovalent aKLH HC-loop-ShK(1-35, Q16) Ab) (see FIG. 22). Exposure
as measured by AUC0-t, was about 161 times less for the bivalent
aKLH HC-loop-ShK(1-35, Q16K) Ab molecule compared to the monovalent
aKLH HC-loop-ShK(1-35Q16K) Ab molecule (Table 4L). Therefore, our
novel monovalent forms show an unexpected and vastly better
pharmacokinetic profile in vivo compared to typical bivalent forms
of the same molecule.
[0686] Monovalent ShK(1-35, Q16K)-Fc/Fc heterodimer. The monovalent
ShK(1-35, Q16K)-Fc/Fc heterodimer contains two chains, one being a
human Fc(IgG2) chain and the other being ShK(1-35, Q16K) peptide
fused to Fc that contains from N- to C-terminus: [Lys16]ShK-L10
linker-human Fc (IgG2). This peptide-fusion protein contained from
N- to C-terminus: the 35 amino acid [Lys16]ShK peptide, a ten amino
acid GGGGSGGGGS (SEQ ID NO:292) L10 linker sequence and the human
Fc (IgG2) sequence. Therefore, the linker-Fc region was attached to
the C-terminus of [Lys16]ShK following Cys35. This molecule is also
referred to as monovalent ShK(1-35, Q16K)-Fc heterodimer. A
schematic representation of this monovalent construct is provided
in FIG. 12C. The molecule was cloned, expressed and purified as
described Example 11. The purified molecule was highly potent
having an IC50 of 0.11 nM in blocking IL-2 secretion in the human
whole blood assay of inflammation (Table 4H). Despite its excellent
potency, the monovalent ShK(1-35, Q16K)-Fc/Fc heterodimer showed
only a modest .about.10 fold selectivity for Kv1.3 versus Kv1.1
(Table 4H). Therefore, it would appear that this linker-Fc fusion
partner attached the C-terminus of [Lys16]ShK does not result in a
further enhancement of Kv1.3 selectivity. This contrasts with
N-terminal fusions to [Lys16]ShK, such as the monovalent
Fc/Fc-ShK(1-35, Q16K) heterodimer (Table 4H) which showed
.about.1225 fold selectivity and had the Fc-linker sequence
attached to the N-terminal Arg1 residue of [Lys16]ShK. An important
and notable exception, however, is the [Lys16]ShK-Ala peptide (SEQ
ID NO: 235, Example 3) which contains a single C-terminal Ala
residue adding following Cys35 of [Lys16]ShK. This molecule
exhibited an enhanced 262 fold improved selectivity for Kv1.3
versus Kv1.1 (Table 4H). Therefore, we envision that the specific
amino acid residue added after Cys35 at the C-terminus of
[Lys16]ShK, can alter the selectivity profile of the fusion
protein. For example, the monovalent ShK(1-35, Q16K)-L10-Fc
molecule described in this example contains the linker Gly residue
added after Cys35 of [Lys16]ShK. If an Ala residue was added
instead following Cys35, an enhanced Kv1.3 selectivity might be
observed. Indeed, we do see 262 fold improved Kv1.3 selectivity by
the [Lys16]ShK-Ala peptide. Thus, we anticipate that specific amino
acid residue at the fusion junction would alter the selectivity
profile. These residue can be readily incorporated into the linker
sequence between the [Lys16]ShK peptide and the human Fc to improve
the conjugates Kv1.3 selectivity.
[0687] Monovalent ShK(1-35, Q16K)-HC aKLH Ab. The monovalent
ShK(1-35, Q16K)-HC aKLH Ab contains three chains, one being the
human aKLH Ab light chain, another being the human aKLH Ab heavy
chain and the third being a peptide-aKLH Ab heavy chain fusion that
contained from N- to C-terminus: [Lys16]ShK-L10 linker-human aKLH
heavy chain. Therefore, this fusion contained the linker-heavy
chain region attached to the C-terminus of [Lys16]ShK following
Cys35. A schematic representation of the monovalent ShK(1-35,
Q16K)-HC aKLH Ab molecule is provided in FIG. 12I. The purified
molecule was highly potent having an IC50 of 0.214 nM in blocking
IL-2 secretion in the human whole blood assay of inflammation
(Table 4H). Despite being very large in size and fused to a human
Ig heavy chain, the monovalent [Lys16]-aKLH Ab molecule retained
high potency in blocking T cell responses.
[0688] Monovalent aDNP HC-ShK(1-35, Q16K) Ab. The monovalent aDNP
Heavy Chain (HC) fusion antibody (Ab) construct contained from N-
to C-terminus: human anti-DNP Ab Heavy Chain-linker-[Lys16]ShK
molecule, that was co-expressed with the human aDNP Heavy Chain and
the human aDNP light chain to form a monovalent aDNP Ab-[Lys16]ShK
molecule. A schematic representation of this monovalent construct
is provided in FIG. 12F. The monovalent aDNP HC-ShK(1-35, Q16K) Ab
potently blocked T cell inflammation in whole blood, suppressing
IL-2 secretion with an IC50 of 0.278 nM (Table 4H). Studies to
examine the Kv1.3 versus Kv1.1 selectivity of the molecule,
unexpectedly revealed that the monovalent aDNP HC-ShK(1-35, Q16K)
Ab conjugate had significantly better Kv1.3 selectivity than the
[Lys16]ShK peptide alone. This monovalent Ab-ShK conjugate was
>5806 fold more active in blocking Kv1.3 versus Kv1.1 (Table
4H).
TABLE-US-00071 TABLE 4H Data demonstrating conjugates of [Lys16]ShK
have improved Kv1.3 selectivity. Kv1.3 Kv1.1 WB WB Potency (PX)
(PX) Kv1.1/Kv1.3 (IL-2) (IFNg) Relative SEQ ID NO Conjugate IC50
IC50 Selectivity IC50 IC50 to ShK or citation Type Designation (nM)
(nM) Ratio by PX (nM) (nM) (WB, IL2) 1 none ShK(1-35) 0.062 0.087
1.40 0.067 0.078 1.00 317 none ShK-Dap22 0.012* 0.847* 70.58 3.763
3.112 56.16 17 none ShK-L5 0.221 0.214 0.97 0.031 0.046 0.46 13
none [Lys16]ShK 0.207 3.677 17.76 0.110 0.158 1.64 235 none
[Lys16]ShK-Ala 0.06 15.726 262.10 0.138 0.266 2.06 8 PEG 20
kDa-PEG-ShK 0.299* 1.628* 5.44 0.380 0.840 5.67 PEG 20
kDa-PEG-[Lys16]ShK 0.94 997 1060.64 0.092 0.160 1.37 316 PEG 20
kDa-PEG-[Lys16]ShK-Ala 0.596 2156 3617.45 0.754 1.187 11.25 277 PEG
30 kDa-PEG-[Lys16]ShK 1.204 1072 890.37 0.282 0.491 4.21 315 PEG 20
kDa-brPEG-[Lys16]ShK 2.095 1574 751.31 0.198 0.399 2.96 Example 1,
IgG1 Bivalent Fc-L10-ShK[1-35] 0.015* 0.067* 4.47 0.386 0.320 5.76
WO2008/088422A2 homodimer Example 2, IgG1 Bivalent Fc-L10-ShK[2-35]
0.116* 0.411* 3.54 0.585 2.285 8.73 WO2008/088422A2 homodimer
Example 2, IgG1 Monovalent Fc/Fc-L10-ShK[2-35] ND ND ND 2.149 5.199
32.07 WO2008/088422A2 heterodimer 337; 348 IgG2 Monovalent
Fc/Fc-ShK(1-35 Q16K) 2.73 3344 1224.91 0.160 0.499 2.39 heterodimer
348; 348 IgG2 Bivalent Fc-ShK(1-35 Q16K) ND ND ND 1.850 3.140 27.61
homodimer 338; 339; 348 IgG2 Monovalent Fc-ShK(1-35 Q16K)/KLH Ab
0.98 1896 1934.69 0.245 0.665 3.66 Heterotrimer 407; 406; 407; 405
IgG2 Monovalent aDNP HC-ShK(1-35 Q16K) Ab 0.574 >3333
>5806.62 0.278 0.660 4.15 338; 339; 338; 342 IgG2 Monovalent
aKLH HC-ShK(1-35 Q16K) Ab 3.96 5774 1458.08 0.274 0.657 4.09 338;
342; 338; 342 IgG2 Bivalent aKLH HC-ShK(1-35 Q16K) Ab ND ND ND
1.392 3.568 20.78 338; 339; 338; 387 IgG2 Monovalent aKLH
HC-ShK(2-35 Q16K) Ab 1.66 2617 1576.51 0.570 0.820 8.51 402; 278
IgG1 Monovalent ShK(1-35 Q16K)-Fc/Fc 1.45 14.77 10.19 0.110 0.120
1.64 heterodimer 338; 339; 338; 403 IgG2 Monovalent ShK(1-35
Q16K)-HC aKLH Ab ND ND ND 0.214 0.332 3.19 338; 344; 338; 343 IgG1
Monovalent aKLH HC-loop-ShK(1-35 Q16K) Ab 8.264 >1000 >121.01
1.604 5.386 23.94 338; 344; 338; 344 IgG1 Bivalent aKLH
HC-loop-ShK(1-35 Q16K) Ab ND ND ND 3.910 55.235 58.36 Toxin
peptides and toxin peptide analogs were PEGylated as described in
Example 4. Immunoglobulin-containing compounds were recombinantly
expressed and purified as described in Example 11.
Electrophysiology was by PatchXpress .RTM. (PX), except asterisks
indicate data from whole cell patch clamp.
TABLE-US-00072 TABLE 4H(a) Potency and selectivity of monovalent
aKLH HC-ShK(1- 35, Q16K) (heterotetramer of SEQ ID NO: 338; SEQ ID
NO: 339; SEQ ID NO: 338; and SEQ ID NO: 342). Assay IC50 nM (n)
Human whole blood 0.292 (2) (TG induced IL-2) Kv1.1 5774 (6) Kv1.2
7973 (4) Kv1.3 3.96 (4) *0.354 (4) Kv1.4 >10000 (3) Kv1.5
>10000 (3) Kv1.6 >616 (6) Kv1.7 >10000 (3) *= Whole cell
patch clamp (WCPC).
TABLE-US-00073 TABLE 4H(b) Selectivity of monovalent Fc-L10-ShK(1-
35, Q16K) (SEQ ID NO: 337; 348). Assay IC50 nM (n) Human whole
blood 0.262 (24) (TG induced IL-2) Kv1.1 3344 (5) Kv1.2 2725 (4)
Kv1.3 2.73 (7) *0.336 (5) Kv1.4 >10000 (3) Kv1.5 >10000 (3)
Kv1.6 448 (11) Kv1.7 >10000 (4) *= Whole cell patch clamp
(WCPC).
TABLE-US-00074 TABLE 4I Pharmacokinetics of monovalent
Fc/Fc-[Lys16]ShK (SEQ ID NO: 337; 348) in prague-Dawley rats (n =
3). Tmax Cmax AUC0-t AUC0-inf CL/F HL CMPD (h) (ng/ml) (ng hr
mL.sup.-1) (ng hr mL.sup.-1) (mL hr.sup.-1 kg.sup.-1) (h)
Monovalent Fc/Fc- 4 .+-. 3.46 1530 .+-. 1230 39600 .+-. 13900 43900
.+-. 14600 146 .+-. 47.1 56.3 .+-. 19.3 [Lys16]ShK
TABLE-US-00075 TABLE 4J Pharmacokinetic data for recombinant
monovalent and bivalent anti-KLH Ab-[Lys16]ShK fusion proteins
administered by subcutaneous injection (dose = 6 mg/kg) to
Sprague-Dawley rats (n = 3). Tmax Cmax AUC0-t AUC0-inf CL/F HL CMPD
(h) (ng/ml) (ng hr mL.sup.-1) (ng hr mL.sup.-1) (mL hr.sup.-1
kg.sup.-1) (h) Monovalent 32 .+-. 13.9 5890 .+-. 1770 481000 .+-.
157000 594000 .+-. 182000 10.9 .+-. 3.47 32 .+-. 13.9 Bivalent 60
.+-. 50.5 126 .+-. 83.4 12900 .+-. 9750 17800 .+-. 17100 655 .+-.
551 60 .+-. 50.5
TABLE-US-00076 TABLE 4K Pharmacokinetic data for recombinant
monovalent Fc/Fc-ShK heterodimer, monovalent Fc- ShK/aKLH Ab
heterotrimer and bivalent ShK-Fc/ShK-Fc homodimer fusion proteins
administered by subcutaneous injection (dose = 2 mg/kg) to
Sprague-Dawley rats (n = 3). Tmax Cmax AUC0-t AUC0-inf CL/F MRT
CMPD (h) (ng/ml) (ng hr mL.sup.-1) (ng hr mL.sup.-1) (mL hr.sup.-1
kg.sup.-1) (h) Monovalent Fc/Fc-ShK 18.7 .+-. 9.2 .sup. 728 .+-.
64.6 .sup. 42469 .+-. 6566 .sup. 44012 .+-. 7484 46.4 .+-. 8.6 46.8
.+-. 6.6 (heterodimer) Monovalent Fc-ShK/KLH Ab 32.0 .+-. 13.9 1107
.+-. 26.2 83355.2 .+-. 5673 89158.6 .+-. 7915 22.6 .+-. 1.9 63.0
.+-. 8.7 (heterotrimer) Bivalent ShK-Fc/ShK-Fc 18.7 .+-. 9.2 27.0
.+-. 4.7 1418.8 .+-. 232 1460.7 .+-. 238 1395.0 .+-. 239.sup. 43.6
.+-. 2.3 (homodimer)
TABLE-US-00077 TABLE 4L Pharmacokinetic data for recombinant
monovalent and bivalent anti-KLH AbLoop-[Lys16]ShK fusion proteins
administered by subcutaneous injection (dose = 6 mg/kg) to
Sprague-Dawley rats (n = 3). Tmax Cmax AUC0-t AUC0-inf CL/F HL CMPD
(h) (ng/ml) (ng hr mL.sup.-1) (ng hr mL.sup.-1) (mL hr.sup.-1
kg.sup.-1) (h) monovalent 40.7 .+-. 35.6 7870 .+-. 605 878000 .+-.
259000 2730000 .+-. 2060000 3.11 .+-. 2.28 245 .+-. 151 bivalent
3.33 .+-. 1.15 .sup. 102 .+-. 41.3 5460 .+-. 3930 6070 .+-. 4510
1440 .+-. 985 49.5 .+-. 13.1
Example 13
Single-Chain (sc) Fc-Toxin Peptide Analog Fusion Proteins
[0689] The fusion of immunoglobulin Fc domains with therapeutic
proteins has proven to be a powerful drug delivery platform with
broad utility. Traditionally, this platform allows recombinant
fusion of therapeutic peptides and proteins to either the
N-terminus or C-terminus of an immunoglobulin Fc domain. An
extension of this technology involves fusing select therapeutic
polypeptides as internal sequences within the Fc sequence (U.S.
Pat. No. 7,442,778 B2). These insertions are termed FcLoop
constructs because they target elements of secondary structure
predicted to be loop domains. In vivo, these Fc fusion proteins
typically manifest prolonged pharmacokinetic lifetimes due to Fc
recycling pathways which result in very efficacious therapeutic
drugs. However, Fc fusion proteins, whether they're N- or
C-terminal or FcLoop fusions are typically bivalent due to the
dimeric nature of Fc (see, Example 12).
[0690] As described in Example 12, in some instances, it may be
useful for an Fc fusion to be monovalent with respect to the
therapeutic warhead, i.e., a toxin peptide analog moiety. In the
case of Shk toxin peptide analog fusions it was observed that the
bivalent forms of Fc-Shk or antibody FcLoop-Shk were cleared in
vivo much more rapidly than their monovalent counterparts (see,
Table 4H, Table 4I, Table 4J, Table 4K, and Table 4L). This example
demonstrates the design, purification and efficacy of a
single-chain Fc-Shk molecule using either an FcLoop insertion or
C-terminal fusion to attach a single copy of the Shk toxin peptide
analog to an Fc domain, which can be expressed recombinantly with a
polypeptide linker of the correct composition and length to connect
the C-terminus of one Fc subunit to the N-terminus of the other Fc
subunit, thus creating a single-chain Fc (scFc). Initial linker
design occurred in silico by examining crystal structures of huFc
domains and determining the approximate distance between N- and
C-termini. Next, FcLoop-Shk fusions with different length linkers
using either 20 or 25 amino acids and FcLoop-Shk insertions in
either the first sequential monomeric subunit of Fc or the second
were constructed as illustrated schematically in FIG. 24. Also
shown is the C-terminal fusion of scFc with a 20 amino acid linker
between the Fc monomer portions of the molecule and another 10
amino acid linker before the ShK portion. FIG. 25A-C gives the
polypeptide sequences of these three constructs. These constructs
were expressed in E. coli and were refolded and purified (FIG.
26A-B). Finally, the purified molecules were tested for activity in
vitro and found to be efficacious (Table 4M, below).
[0691] Preparation of Scfc-Shk.
[0692] Cloning and Expression of scFc-Shk: Two distinct nucleotide
sequences (79% identical) encoding human Fc were used to create a
scFc. The first sequence had minor modifications from wild-type Fc
sequence to remove rare codons and minimize secondary structure
near the 5'-end of the mRNA. The second was fully optimized for
expression in bacteria by Verdezyne (Laguna Hills, Calif.). The two
Fc sequences were first separated by 45 nucleotides encoding three
repeats of a Gly-Gly-Gly-Gly-Ser (G.sub.4S; SEQ ID NO:291) linker.
Later constructs would add one or two additional five-residue
repeats to the linker.
[0693] Formation of the expression construct was multi-fold. The
expression vector (pET30, Novagen/EMD Biosciences, San Diego,
Calif.) and a first Fc domain coding sequence, along with 20
nucleotides of linker were amplified from a pre-existing expression
construct, using primers
5'-TAATGAATTCGAGCTCCGTCGACAAGCT-3' (SEQ ID NO:420) and
[0694] 5'-CCACCGGATCCACCACCACCTTTACCCGGAGACAGGGAGAGGCT-3' (SEQ ID
NO:421). This PCR and all other amplifications in this section used
PfuUltra II DNA polymerase (Stratagene, La Jolla Calif.) according
to manufacturer's instructions. A Touchdown PCR profile was used
throughout with the annealing temperature dropping from 60.degree.
C. to 51.degree. C. over ten cycles, followed by 20 cycles at
55.degree. C. A second reaction amplified: 15 nucleotides of linker
homologous to the 3'-end of the first reaction; the remaining 25
nucleotides of the linker; the second Fc; and another 16
nucleotides homologous to the 5'-end of the first reaction which
also included the stop codon, using primers
5'-TGGTGGATCCGGTGGTGGTGGCTCCGGT-3' (SEQ ID NO:422) and
[0695] 5'-GAGCTCGAATTCATTATTTACCCGGAGACAGAGACAGGGA-3' (SEQ ID
NO:423). The two PCR products were joined by Polymerase Chain
Reaction "Splicing by Overlap Extension" (SOE) reaction at the
15-nucleotide homologous linker region, using primers
(5'-TGGTGGATCCGGTGGTGGTGGCTCCGGT-3' (SEQ ID NO:422) and
5'-CCACCGGATCCACCACCACCTTTACCCGGAGACAGGGAGAGGCT-3' (SEQ ID NO:421).
This final PCR product was then treated with DpnI (New England
Biolabs, NEB, Ipswich Mass.) and transformed into competent cells.
The stop codon containing 16-nucleotide homologous region served as
a site for in vivo recircularization of the vector.
[0696] Linker length was extended from 15 residues to 20, by
amplifying the entire construct with two primers designed to insert
15 nucleotides encoding another G.sub.4S in front of the previous
three repeats: 5'-GGTGGCGGTGGCTCTGGTGGTGGTGGATCCGGTGGT-3' (SEQ ID
NO:424) and
5'-AGAGCCACCGCCACCTTTACCCGGAGACAGGGAGAGGCT-3' (SEQ ID NO:425). This
PCR product was treated with DpnI(NEB) and transformed into
competent cells. During the screening process, one clone was
sequenced to reveal a repeat of this newly inserted 15-nucleotide
region and yielded the 25-residue linker sequence.
[0697] ShK (SEQ ID NO:1), flanked by Gly-Gly dipeptide linkers, was
cloned into the first Fc domain of the Fc-L25-Fc chain by a method
similar to that used to create the original scFc. One PCR amplified
the entire vector, adding 6 nucleotides at the 5'- and 3'-ends
encoding the flanking Gly-Gly dipeptide linkers, using primers
5'-GGCGGTACCAAGAACCAGGTCAGCCTGA-3' (SEQ ID NO:426) and
5'-ACCGCCCAGCTCATCACGGGATGGGGGCA-3' (SEQ ID NO:427). Another
reaction amplified Shk (SEQ ID NO:1), with flanking Gly-Gly
dipeptide linkers and a total of 18 nucleotides at it's 5'-end and
20 nucleotides at it's 3-end that were homologous to the
corresponding termini of the first reaction, using primers
5'-CGTGATGAGCTGGGCGGTCGTTCTTGCATCGATACAATCCCT-3' (SEQ ID NO:428)
and
[0698] 5'-ACCTGGTTCTTGGTACCGCCACAGGTGCCACAGGTTTTACGA-3' (SEQ ID
NO:429). A PCR SOE reaction using primers
5'-GGCGGTACCAAGAACCAGGTCAGCCTGA-3' (SEQ ID NO:426) and
[0699] 5'-ACCTGGTTCTTGGTACCGCCACAGGTGCCACAGGTTTTACGA-3' (SEQ ID
NO:429) gave a product that was transformed into competent cells
after treatment with DpnI (NEB). Unfortunately, the only clone was
found to have a mutation that needed to be removed by a subsequent
round of mutagenesis, using primers
5'-CACTCAATGAAATACCGTCTCAGTTTCTGTCGT-3'(SEQ ID NO:430) and
5'-GTATTTCATTGAGTGTTTGCATTGAAAGGCA-3' (SEQ ID NO:431).
[0700] The same DNA sequence encoding Gly-Gly-ShK-Gly-Gly (SEQ ID
NO:360) was inserted into the second Fc domain of the DNA encoding
the Fc-L25-Fc polypeptide chain of the following amino acid
sequence:
MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGG
GGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK//SEQ ID NO:413, by two rounds of
PCR. Three overlapping plus-strand primers and three overlapping
minus strand primers covered the region. The first pair of
primers:
5'-AAACCTGTGGCACCTGTGGCGGTACCAAAAACCAGGTGTCCCTGA-3' (SEQ ID
NO:414); and
[0701] 5'-TATCGATGCAAGAACGACCGCCCAGTTCGTCACGAGACGGCGGCA-3' (SEQ ID
NO:415), was added to reaction 1.1; this first pair and a second
pair of primers:
5'-AAATACCGTCTCAGTTTCTGTCGTAAAACCTGTGGCACCTGTGGC-3' (SEQ ID
NO:416); and
[0702] 5'-CAGTACAGCGGGACTTAGGGATTGTATCGATGCAAGAACGACCGC-3' (SEQ ID
NO:417), were both added to reaction 1.2; and primer pair 1 (SEQ ID
NO:414 and SEQ ID NO:415) and primer pair 2 (SEQ ID NO:416 and SEQ
ID NO:417) and the third pair of primers:
5'-CAATGCAAACACTCAATGAAATACCGTCTCAGTTTCTGTCGTAAA-3' (SEQ ID
NO:418); and
[0703] 5'-TGAGTGTTTGCATTGAAAGGCAGTACAGCGGGACTTAGGGATT-3' (SEQ ID
NO:419), were all added to reaction 1.3. A second round of PCRs
used the first's rounds product as template. Reaction 2.1 added the
second primer pair (SEQ ID NO:416 and SEQ ID NO:417) to the product
from reaction 1.1. The second and third primer pairs (SEQ ID NO:416
and SEQ ID NO:417; SEQ ID NO:418 and SEQ ID NO:419, respectively)
were also added to reaction 1.1's product in reaction 2.2.
Reactions 2.3 and 2.4 had the third primer pair (SEQ ID NO:418 and
SEQ ID NO:419) being added to the reaction 1.2 and 1.3 products.
All four of these second round reaction products were then mixed
and used to transform competent cells. A single clone was found
with the correct insert, but the 25-residue linker was found to
have lost one G.sub.4S repeat.
[0704] The Fc-L20-Fc DNA construct encoding the following:
(a) the Fc-L20-Fc amino acid sequence:
MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK//SEQ ID NO:408, and (b) the
CH2.L10.[Lys16]Shk-Ala DNA construct encoding an amino acid
sequence containing L10 linker (SEQ ID NO:292; underlined) and
[Lys16]ShK-Ala (SEQ ID NO:235; in boldface type):
MKPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
ISGGGGSGGGGSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTCA//SEQ ID NO:409,
were used as templates for creating Fc-L20-Fc-L10-[Lys16]Shk-Ala
(amino acid sequence shown in FIG. 25C; SEQ ID NO:410). One PCR
amplified the Fc-L20-Fc vector, using primers:
5'-TAATGAATTCGAGCTCCGTCGACAAGCT-3' 9SEQ ID NO:432) and
5'-CCCGGAGACAGAGACAGGGA-3' (SEQ ID NO:433),
[0705] while another amplified 15 nucleotides homologous to the
3'-end of the first amplicon, the L10-[Lys16]ShK-Ala, and 15
nucleotides homologous to the 5'-end of the first amplicon, using
primers:
5'-GTCTCTGTCTCCGGGTAAAGGCGGCGGCGGCA-3' (SEQ ID NO:434) and
[0706] 5'-AGCTCGAATTCATTAAGCACAGGTGCCACAGGTT-3' (SEQ ID NO:435).
Clontech's (a Takara company, Shiga, Japan) In-Fusion.TM.
technology was used to fuse the homologous regions.
[0707] All expression constructs were transformed into
chemically-competent BL21Star (Invitrogen, Carlsbad, Calif.).
Autoinduction (AI) medium was inoculated and grown overnight at
37.degree. C. Harvest was generally 16-24 h after inoculation. The
AI medium was prepared by adding 20 mL of 50.times. Master Mix (10
g Glucose; 500 mL Glycerol; 100 g Lactose; 5 g Aspartate; 100 mM
MgSO.sub.4; dH.sub.2O to 1 L) to 1 L TB (Teknova).
[0708] Purification of scFc-Shk: Cell pastes containing expressed
scFc-Shk constructs were resuspended in water at 0.1 g/mL and lysed
with 3 passes through a microfluidizer. The lysate was centrifuged
to pellet the inclusion bodies, which were then washed once with 1%
deoxycholate, followed by a second water wash. The washed inclusion
bodies were then solubilized in 7.2 M guanidine hydrochloride, 10
mM dithiothreitol (DTT), pH8.5 at 0.1 g/mL. The solubilized
inclusion bodies were then refolded by 1:25 dilution at 4.degree.
C. into a refolding buffer containing: 2 M urea, 150 mM arginine,
50 mM Tris, 3 mM cysteine and 1 mM cystamine at pH 8.5. The diluted
refold reaction was gently stirred about 48 hrs at 4.degree. C.,
then clarified by centrifugation followed by filtration. Refolded
scFc-Shk fusions were purified by affinity chromatography using
Protein A Sepharose resin (GE Healthcare) in PBS and eluted with
0.1 N acetic acid, pH 3.5. The eluted peak was neutralized and
dialyzed into 10 mM sodium acetate, 50 mM NaCl, pH5. The Protein
A-purified scFc-Shk constructs were further purified by ion
exchange chromatography using SP Sepharose HP resin (GE Healthcare)
in 10 mM NaOAc, 50 mM NaCl, pH5 using a linear 50 mM-1M NaCl
gradient. The eluted peaks were evaluated by SDS-PAGE and pooled
based on size. The scFc-Shk constructs underwent a final
purification step using hydrophobic interaction chromatography on
Phenyl Sepharose HP (GE Healthcare) in 10 mM NaOAc, 50 mM NaCl, pH
5 and eluted with a linear 1 M (NH.sub.4).sub.2SO.sub.4 with eluted
peaks further evaluated by SDS-PAGE. Final pools were concentrated,
dialyzed into 10 mM sodium acetate, 9% sucrose, pH 5 and sterile
filtered. The finished product was characterized by SDS-PAGE (FIG.
26A), RP-HPLC (FIG. 26B) and in vitro whole blood activity assay
was run as described in Example 2 (Table 4M).
TABLE-US-00078 TABLE 4M In vitro whole blood assay (inhibition of
IL-2) average results demonstrating equipotence with conventional
FcLoop-Shk bivalent peptibody. Construct In Vitro IC50 (nM)
FcLoopShk/FcLoopShk 2.0 (bivalent Fc) Fc.L20FcLoopShk 2.01 (scFc)
FcLoopShk.L25.Fc 1.91 (scFc)
TABLE-US-00079 TABLE 5 Reference Peptides WB WB (IL- (IFNg) Kv1.3
Kv1.1 Kv1.1/ WB WB 2) Po- Po- (IWQ) (IWQ) Kv1.3 (IL-2) (IFNg) tency
tecny SEQ IC50 IC50 Selec- IC50 IC50 Rela- Rela- ID Desig- (nM,
(nM, tivity (nM, (nM, tive tive NO: Amino Acid Sequence nation Avg)
Avg) Ratio Avg) Avg) to ShK to ShK 1
RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK(1-35) 0.146 0.018 0.123
0.043 0.052 1.00 1.00 (bench mark compound) 8
RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC 20 kDa 1.208 0.110 0.091 0.387
0.855 9.00 16.44 PEG-ShK 9 GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCAC
[Ala34] 0.100 1.071 10.710 0.052 0.074 1.21 1.42 TPK Osk-1 2
RTCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC HmK 0.900 0.015 0.017 0.85
1.195 19.77 22.98 10 RTCKDLIPVSECTDIKCRTS[Nva] [Lys16, 23.030 0.580
0.025 6.387 9.826 148.53 188.96 KYRLNLCRKTCGSC Nva21] HmK-amide 11
RSCIDTIPKSRCTAF[1-Nal] [1-Nal16] 0.699 169.177 242.027 28.86 9.552
671.16 183.69 CKHSMKYRLSFCRKTCGTC ShK 12
RSCIDTIPKSRCTAFQCKHSMKYRL[1-Nal] [1-Nal26] 0.352 1.869 5.310 0.357
0.573 8.30 11.02 FCRKTCGTC ShK 13
RSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC [Lys16] 0.220 2.085 9.477 0.113
0.194 2.63 3.73 ShK 14 RSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC [Lys16]
0.174 0.600 3.448 0.223 0.278 5.19 5.35 ShK- amide 15
RSCIDTIPKSRCTAFKCKHS[Nle] [Lys16, 0.153 13.220 86.405 0.823 1.099
19.14 21.13 KYRLSFCRKTCGTC Nle21] ShK-amide 16
RSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC 20 kDa 0.665 1082.214 1627.389
0.104 0.215 2.42 4.13 PEG- [Lys16] ShK 17 [pY][Aeea] ShK(L5) 0.085
0.015 0.176 0.031 0.046 0.72 0.88
RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC (bench mark compound) 18
[pY][Aeea]RSCIDTIPKSRCTAFQCKHS[Nle] [Nle21] 0.170 0.030 0.176 0.16
0.492 3.72 9.46 KYRLSFCRKTCGTC ShK(L5)- amide (bench mark
compound)
TABLE-US-00080 TABLE 6 Arginine Scan WB WB (IL- (IFNg) Kv1.3 Kv1.1
Kv1.1/ WB WB 2) Po- Po- (IWQ) (IWQ) Kv1.3 (IL- (IFNg) tency tency
SEQ IC50 IC50 Selec- 2) IC50 IC50 Rela- Rela- ID Desig- (nM, (nM,
tivity (nM, (nM, tive tive NO: Amino Acid Sequence nation Avg) Avg)
Ratio Avg) Avg) to ShK to ShK 19
RRCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC [Arg2] 0.074 0.004 0.054 0.097
0.087 2.26 1.67 ShK 20 RSCRDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC [Arg4]
0.110 0.006 0.055 0.051 0.081 1.19 1.56 ShK 21
RSCIRTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC [Arg5] 3.300 2.050 0.621 47.255
91.733 1098.95 1764.10 ShK 22 RSCIDRIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
[Arg6] 0.178 0.051 0.287 0.246 0.4 5.72 7.69 ShK 23
RSCIDTRPKSRCTAFQCKHSMKYRLSFCRKTCGTC [Arg7] 0.301 0.021 0.070 6.391
11.111 148.63 213.67 ShK 24 RSCIDTIRKSRCTAFQCKHSMKYRLSFCRKTCGTC
[Arg8] 0.070 0.004 0.057 0.187 1.422 4.35 27.35 ShK 25
RSCIDTIPRSRCTAFQCKHSMKYRLSFCRKTCGTC [Arg9] 0.096 0.004 0.042 0.108
0.116 2.51 2.23 ShK 26 RSCIDTIPKRRCTAFQCKHSMKYRLSFCRKTCGTC [Arg10]
0.070 0.004 0.057 0.029 0.068 0.67 1.31 ShK 27
RSCIDTIPKSRCRAFQCKHSMKYRLSFCRKTCGTC [Arg13] 0.149 0.024 0.161 0.503
0.629 11.70 12.10 ShK 28 RSCIDTIPKSRCTRFQCKHSMKYRLSFCRKTCGTC
[Arg14] 0.077 0.004 0.052 0.266 0.307 6.19 5.90 ShK 29
RSCIDTIPKSRCTARQCKHSMKYRLSFCRKTCGTC [Arg15] 0.088 0.022 0.250 0.162
0.204 3.77 3.92 ShK 30 RSCIDTIPKSRCTAFRCKHSMKYRLSFCRKTCGTC [Arg16]
0.164 0.066 0.402 0.652 0.818 15.16 15.73 ShK 31
RSCIDTIPKSRCTAFQCRHSMKYRLSFCRKTCGTC [Arg18] 0.110 0.005 0.045 0.012
0.023 0.28 0.44 ShK 32 RSCIDTIPKSRCTAFQCKRSMKYRLSFCRKTCGTC [Arg19]
0.100 0.004 0.040 0.039 0.116 0.91 2.23 ShK 33
RSCIDTIPKSRCTAFQCKHRMKYRLSFCRKTCGTC [Arg20] 3.842 100.000 26.028
36.906 31.291 858.28 601.75 ShK 34
RSCIDTIPKSRCTAFQCKHSRKYRLSFCRKTCGTC [Arg21] 0.283 0.045 0.159 1.76
4.655 40.93 89.52 ShK 35 RSCIDTIPKSRCTAFQCKHSMRYRLSFCRKTCGTC
[Arg22] 18.737 33.330 1.779 100 100 2325.58 1923.08 ShK 36
RSCIDTIPKSRCTAFQCKHSMKRRLSFCRKTCGTC [Arg23] 0.154 3.300 21.429 100
100 2325.58 1923.08 ShK 37 RSCIDTIPKSRCTAFQCKHSMKYRRSFCRKTCGTC
[Arg25] 0.107 0.001 0.009 0.095 0.2 2.21 3.85 ShK 38
RSCIDTIPKSRCTAFQCKHSMKYRLRFCRKTCGTC [Arg26] 0.279 0.089 0.319 0.208
0.572 4.84 11.00 ShK 39 RSCIDTIPKSRCTAFQCKHSMKYRLSRCRKTCGTC [Arg27]
100.000 5.037 0.050 12.958 59.73 301.35 1148.65 ShK 40
RSCIDTIPKSRCTAFQCKHSMKYRLSFCRRTCGTC [Arg30] 0.040 0.006 0.150 0.014
0.027 0.33 0.52 ShK 41 RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKRCGTC [Arg31]
3.300 0.597 0.181 11.978 54.791 278.56 1053.67 ShK 42
RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCRTC [Arg33] 0.102 0.004 0.039 0.068
0.232 1.58 4.46 ShK 43 RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGRC [Arg34]
0.182 0.008 0.044 0.418 0.486 9.72 9.35 ShK
TABLE-US-00081 TABLE 7 Glutamate Scan WB WB Kv1.3 Kv1.1 Kv1.1/ WB
WB (IL-2) (IFNg) (IWQ) (IWQ) Kv1.3 (IL-2) (IFNg) Potency Potency
SEQ IC50 IC50 Selec- IC50 IC5- Rel- Rel- ID Desig- (nM, (nM, tivity
(nM, (nM, ative ative NO: Amino Acid Sequence nation Avg) Avg)
Ratio Avg) Avg) to ShK to ShK 44
ESCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC [Glu1]ShK 0.144 0.010 0.069
0.213 0.249 4.95 4.79 45 RECIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
[Glu2]ShK 0.111 0.002 0.018 0.029 0.039 0.67 0.75 46
RSCEDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC [Glu4]ShK 0.070 0.006 0.086
0.031 0.049 0.72 0.94 47 RSCIETIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
[Glu5]ShK 3.300 1.186 0.359 16.91 24.557 393.26 472.25 48
RSCIDEIPKSRCTAFQCKHSMKYRLSFCRKTCGTC [Glu6]ShK 0.170 0.026 0.153
0.073 0.109 1.70 2.10 49 RSCIDTEPKSRCTAFQCKHSMKYRLSFCRKTCGTC
[Glu7]ShK 1.253 0.034 0.027 4.999 19.512 116.26 375.23 50
RSCIDTIEKSRCTAFQCKHSMKYRLSFCRKTCGTC [Glu8]ShK 0.097 0.007 0.072
0.072 0.238 1.67 4.58 51 RSCIDTIPESRCTAFQCKHSMKYRLSFCRKTCGTC
[Glu9]ShK 0.128 0.006 0.047 0.036 0.056 0.84 1.08 52
RSCIDTIPKERCTAFQCKHSMKYRLSFCRKTCGTC [Glu10]ShK 0.134 0.007 0.052
0.023 0.055 0.53 1.06 53 RSCIDTIPKSECTAFQCKHSMKYRLSFCRKTCGTC
[Glu11]ShK 0.969 0.031 0.032 1.322 4.542 30.74 87.35 54
RSCIDTIPKSRCEAFQCKHSMKYRLSFCRKTCGTC [Glu13]ShK 0.157 0.002 0.013
0.785 1.099 18.26 21.13 55 RSCIDTIPKSRCTEFQCKHSMKYRLSFCRKTCGTC
[Glu14]ShK 0.110 0.008 0.073 0.035 0.132 0.81 2.54 56
RSCIDTIPKSRCTAEQCKHSMKYRLSFCRKTCGTC [Glu15]ShK 0.266 0.112 0.421
0.312 0.575 7.26 11.06 57 RSCIDTIPKSRCTAFECKHSMKYRLSFCRKTCGTC
[Glu16]ShK 0.206 0.058 0.282 0.351 0.373 8.16 7.17 58
RSCIDTIPKSRCTAFQCEHSMKYRLSFCRKTCGTC [Glu18]ShK 0.117 0.004 0.034
0.047 0.083 1.09 1.60 59 RSCIDTIPKSRCTAFQCKESMKYRLSFCRKTCGTC
[Glu19]ShK 0.256 0.018 0.070 0.202 0.215 4.70 4.13 60
RSCIDTIPKSRCTAFQCKHEMKYRLSFCRKTCGTC [Glu20]ShK 100.000 13.426 0.134
92 92 2139.53 1769.23 61 RSCIDTIPKSRCTAFQCKHSEKYRLSFCRKTCGTC
[Glu21]ShK 3.333 0.071 0.021 18.468 28.089 429.49 540.17 62
RSCIDTIPKSRCTAFQCKHSMEYRLSFCRKTCGTC [Glu22]ShK 33.300 33.300 1.000
100 100 2325.58 1923.08 63 RSCIDTIPKSRCTAFQCKHSMKERLSFCRKTCGTC
[Glu23]ShK 33.300 33.300 1.000 100 100 2325.58 1923.08 64
RSCIDTIPKSRCTAFQCKHSMKYELSFCRKTCGTC [Glu24]ShK 0.260 0.014 0.054
1.11 4.073 25.81 78.33 65 RSCIDTIPKSRCTAFQCKHSMKYRESFCRKTCGTC
[Glu25]ShK 0.240 0.031 0.129 0.494 0.678 11.49 13.04 66
RSCIDTIPKSRCTAFQCKHSMKYRLEFCRKTCGTC [Glu26]ShK 0.317 0.039 0.123
0.202 0.4 4.70 7.69 67 RSCIDTIPKSRCTAFQCKHSMKYRLSECRKTCGTC
[Glu27]ShK 0.000 0.000 8.155 34.03 189.65 654.42 68
RSCIDTIPKSRCTAFQCKHSMKYRLSFCEKTCGTC [Glu29]ShK 0.142 0.009 0.063
0.73 1.955 16.98 37.60 69 RSCIDTIPKSRCTAFQCKHSMKYRLSFCRETCGTC
[Glu30]ShK 0.272 0.002 0.007 0.019 0.043 0.44 0.83 70
RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKECGTC [Glu31]ShK 0.220 0.006 0.027
0.242 0.309 5.63 5.94 71 RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCETC
[Glu33]ShK 0.060 0.005 0.083 0.054 0.176 1.26 3.38 72
RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGEC [Glu34]ShK 0.067 0.008 0.119
0.073 0.123 1.70 2.37
TABLE-US-00082 TABLE 8 1-Nal Scan WB WB (IL-2) (IFNg) Po- Po- ten-
ten- Kv1.3 Kv1.1 Kv1.1/ WB WB cy cy (IWQ) (IWQ) Kv1.3 (IL-2) (IFNg)
Rela- Rela- SEQ IC50 IC50 Selec- IC50 IC50 tive tive ID (nM, (nM,
tivity (nM, (nM, to to NO: Amino Acid Sequence Designation Avg)
Avg) Ratio Avg) Avg) ShK ShK 73 [1-Nal]SCIDTIPKSRCTAFQCKHSMKY
[1-Nal1]ShK 0.225 0.005 0.022 0.222 0.247 5.16 4.75 RLSFCRKTCGTC 74
R[1-Nal]CIDTIPKSRCTAFQCKHSMKY [1-Nal2]ShK 0.250 0.002 0.008 0.223
0.258 5.19 4.96 RLSFCRKTCGTC 75 RSC[1-Nal]DTIPKSRCTAFQCKHSMKY
[1-Nal4]ShK 0.150 0.002 0.013 0.274 0.317 6.37 6.10 RLSFCRKTCGTC 76
RSCI[1-Nal]TIPKSRCTAFQCKHSMKY [1-Nal5]ShK 3.300 3.300 1.000 70.4
31.937 1637.21 614.17 RLSFCRKTCGTC 77 RSCID[1-Nal]IPKSRCTAFQCKHSMKY
[1-Nal6]ShK 0.150 0.034 0.227 0.86 0.962 20.00 18.50 RLSFCRKTCGTC
78 RSCIDT[1-Nal]PKSRCTAFQCKHSMKY [1-Nal7]ShK 0.699 0.036 0.052
11.623 18.007 270.30 346.29 RLSFCRKTCGTC 79
RSCIDTI[1-Nal]KSRCTAFQCKHSMKY [1-Nal8]ShK 0.000 0.000 0 0
RLSFCRKTCGTC 80 RSCIDTIP[1-Nal]SRCTAFQCKHSMKY [1-Nal9]ShK 0.473
0.011 0.023 0.367 0.666 8.53 12.81 RLSFCRKTCGTC 81
RSCIDTIPK[1-Nal]RCTAFQCKHSMKY [1-Nal10]ShK 0.206 0.004 0.019 0.205
0.255 4.77 4.90 RLSFCRKTCGTC 82 RSCIDTIPKS[1-Nal]CTAFQCKHSMKY
[1-Nal11]ShK 0.922 0.027 0.029 1.137 1.645 26.44 31.63 RLSFCRKTCGTC
83 RSCIDTIPKSRC[1-Nal]AFQCKHSMKY [1-Nal13]ShK 0.235 0.016 0.068
3.563 4.856 82.86 93.38 RLSFCRKTCGTC 84
RSCIDTIPKSRCT[1-Nal]FQCKHSMKY [1-Nal14]ShK 0.000 0.000 0.089 0.11
2.07 2.12 RLSFCRKTCGTC 85 RSCIDTIPKSRCTA[1-Nal]QCKHSMKY
[1-Nal15]ShK 0.072 0.009 0.125 0.184 0.291 4.28 5.60 RLSFCRKTCGTC
11 RSCIDTIPKSRCTAF[1-Nal]CKHSMKY [1-Nal16]ShK 0.699 169.177 242.027
28.86 9.552 671.16 183.69 RLSFCRKTCGTC 87
RSCIDTIPKSRCTAFQC[1-Nal]HSMKY [1-Nal18]ShK 0.471 0.004 0.008 0.419
0.375 9.74 7.21 RLSFCRKTCGTC 88 RSCIDTIPKSRCTAFQCK[1-Nal]SMKY
[1-Nal19]ShK 0.277 0.001 0.004 0.153 0.255 3.56 4.90 RLSFCRKTCGTC
89 RSCIDTIPKSRCTAFQCKH[1-Nal]MKY [1-Nal20]ShK 2.110 3.300 1.564 100
100 2325.58 1923.08 RLSFCRKTCGTC 90 RSCIDTIPKSRCTAFQCKHS[1-Nal]KY
[1-Nal21]ShK 0.669 0.038 0.057 6.586 8.131 153.16 156.37
RLSFCRKTCGTC 91 RSCIDTIPKSRCTAFQCKHSM[1-Nal]Y [1-Nal22]ShK 33.300
33.330 1.001 100 100 2325.58 1923.08 RLSFCRKTCGTC 92
RSCIDTIPKSRCTAFQCKHSMK[1-Nal] [1-Nal23]ShK 3.300 3.300 1.000 100
100 2325.58 1923.08 RLSFCRKTCGTC 93 RSCIDTIPKSRCTAFQCKHSMKY[1-
[1-Nal24]ShK 0.000 0.000 0 0 Nal]LSFCRKTCGTC 94
RSCIDTIPKSRCTAFQCKHSMKYR[1- [1-Nal25]ShK 0.106 0.012 0.113 0.061
0.084 1.42 1.62 Nal]SFCRKTCGTC 12 RSCIDTIPKSRCTAFQCKHSMKYRL[1-
[1-Nal26]ShK 0.352 1.869 5.310 0.357 0.573 8.30 11.02 Nal]FCRKTCGTC
95 RSCIDTIPKSRCTAFQCKHSMKYRLS[1- [1-Nal27]ShK 0.144 2.027 14.076
1.072 2.84 24.93 54.62 Nal]CRKTCGTC 96 RSCIDTIPKSRCTAFQCKHSMKYRLSFC
[1-Nal29]ShK 3.300 3.300 1.000 4.717 8.082 109.70 155.42
[1-Nal]KTCGTC 97 RSCIDTIPKSRCTAFQCKHSMKYRLSFCR [1-Nal30]ShK 0.160
0.005 0.031 0.082 0.148 1.91 2.85 [1-Nal]TCGTC 98
RSCIDTIPKSRCTAFQCKHSMKYRLSFCR [1-Nal31]ShK 0.120 0.004 0.033 0.032
0.129 0.74 2.48 K[1-Nal]CGTC 99 RSCIDTIPKSRCTAFQCKHSMKYRLSFCR
[1-Nal33]ShK 0.182 0.006 0.033 0.439 1.429 10.21 27.48 KTC[1-Nal]TC
100 RSCIDTIPKSRCTAFQCKHSMKYRLSFCR [1-Nal34]ShK 0.077 0.004 0.052
0.018 0.063 0.42 1.21 KTCG[1-Nal]C
TABLE-US-00083 TABLE 9 Alanine Scan WB WB (IL-2) (IFNg) Po- Po-
ten- ten- Kv1.3 Kv1.1 Kvl.l/ WB WB cy cy SEQ (IWQ) (IWQ) Kv1.3
(IL-2) (IFNg) Rel- Rel- IC50 IC50 Selec- IC50 IC50 ative ative ID
Desig- (nM, (nM, tivity (nM, (nM, to to NO: Amino Acid Sequence
nation Avg) Avg) Ratio Avg) Avg) ShK ShK 101
ASCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC [Ala1]ShK 0.168 0.007 0.042
0.073 0.102 1.70 1.96 102 RACIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
[Ala2]ShK 0.156 0.003 0.019 0.054 0.087 1.26 1.67 103
RSCADTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC [Ala4]ShK 0.176 0.005 0.028
0.012 0.008 0.28 0.15 104 RSCIATIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
[Ala5]ShK 0.000 0.000 0 0 105 RSCIDAIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
[Ala6]ShK 0 208 0.012 0.058 0.065 0.032 1.51 0.62 106
RSCIDTAPKSRCTAFQCKHSMKYRLSFCRKTCGTC [Ala7]ShK 0.188 0.005 0.027
0.886 2.223 20.60 42.75 107 RSCIDTIAKSRCTAFQCKHSMKYRLSFCRKTCGTC
[Ala8]ShK 0.078 0.004 0.051 0.039 0.061 0.91 1.17 108
RSCIDTIPASRCTAFQCKHSMKYRLSFCRKTCGTC [Ala9]ShK 0.116 0.010 0.086
0.053 0.103 1.23 1.98 109 RSCIDTIPKARCTAFQCKHSMKYRLSFCRKTCGTC
[Ala10]ShK 0.193 0.440 2.280 1.211 1.779 28.16 34.21 110
RSCIDTIPKSACTAFQCKHSMKYRLSFCRKTCGTC [Ala11]ShK 0.431 0.001 0.002
1.237 1.899 28.77 36.52 111 RSCIDTIPKSRCAAFQCKHSMKYRLSFCRKTCGTC
[Ala13]ShK 0.156 0.006 0.038 0.582 0.954 13.53 18.35 112
RSCIDTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC [Ala15]ShK 0.084 0.002 0.024
0.032 0.048 0.74 0.92 113 RSCIDTIPKSRCTAFACKHSMKYRLSFCRKTCGTC
[Ala16]ShK 0.196 0.001 0.005 0.058 0.078 1.35 1.50 114
RSCIDTIPKSRCTAFQCAHSMKYRLSFCRKTCGTC [Ala18]ShK 0.160 0.001 0.006
0.025 0.025 0.58 0.48 115 RSCIDTIPKSRCTAFQCKASMKYRLSFCRKTCGTC
[Ala19]ShK 0.237 0.006 0.025 0.379 0.472 8.81 9.08 116
RSCIDTIPKSRCTAFQCKHAMKYRLSFCRKTCGTC [Ala20]ShK 0.195 0.017 0.087
0.3 0.825 6.98 15.87 117 RSCIDTIPKSRCTAFQCKHSAKYRLSFCRKTCGTC
[Ala21]ShK 0.227 0.001 0.004 0.275 0.641 6.40 12.33 118
RSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC [Ala22]ShK 0.454 0.573 1.262
2.326 5.487 54.09 105.52 119 RSCIDTIPKSRCTAFQCKHSMKARLSFCRKTCGTC
[Ala23]ShK 0.621 2.095 3.374 3.752 5.44 87.26 104.62 120
RSCIDTIPKSRCTAFQCKHSMKYALSFCRKTCGTC [Ala24]ShK 0.062 0.005 0.081
0.043 0.216 1.00 4.15 121 RSCIDTIPKSRCTAFQCKHSMKYRASFCRKTCGTC
[Ala25]ShK 0.116 0.006 0.052 0.195 0.408 4.53 7.65 122
RSCIDTIPKSRCTAFQCKHSMKYRLAFCRKTCGTC [Ala26]ShK 0.137 0.001 0.007
0.068 0.114 1.58 2.19 123 RSCIDTIPKSRCTAFQCKHSMKYRLSACRKTCGTC
[Ala27]ShK 0.207 0.008 0.039 1.569 8.777 36.49 168.79 124
RSCIDTIPKSRCTAFQCKHSMKYRLSFCAKTCGTC [Ala29]ShK 4.771 0.490 0.103
4.28 7.815 99.53 150.29 125 RSCIDTIPKSRCTAFQCKHSMKYRLSFCRATCGTC
[Ala30]ShK 0.178 0.005 0.028 0.258 0.394 6.00 7.58 126
RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKACGTC [Ala31]ShK 0.039 0.004 0.103
0.043 0.284 1.00 5.46 127 RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCATC
[Ala33]ShK 0.075 0.005 0.067 0.049 0.1 1.14 1.92 128
RSCIDTIPKSRCTAFOCKHSMKYRLSFCRKTCGAC [Ala34]ShK 0.123 0.004 0.033
0.063 0.075 1.47 1.44
TABLE-US-00084 TABLE 10 Lysine Scan Kv1.3 Kv1.1 Kv1.1/ WB WB WB WB
(IWQ) (IWQ) Kv1.3 (IL-2) (IFNg) (IL-2) (IFNg) SEQ IC50 IC50 Selec-
IC50 IC50 Potency Potency ID Desig- (nM, (nM, tivity (nM, (nM,
Relative Relative NO: Amino Acid Sequence nation Avg) Avg) Ratio
Avg) Avg) to ShK to ShK 129 KSCIDTIPKSRCTAFQCKHSMKYRLS [Lys1]ShK
0.158 0.006 0.038 0.035 0.073 0.81 1.40 FCRKTCGTC 130
RKCIDTIPKSRCTAFQCKHSMKYRLS [Lys2]ShK 0.149 0.006 0.040 0.099 0.093
2.30 1.79 FCRKTCGTC 131 RSCKDTIPKSRCTAFQCKHSMKYRLS [Lys4]ShK 0.153
0.004 0.026 0.023 0.054 0.53 1.04 FCRKTCGTC 132
RSCIKTIPKSRCTAFQCKHSMKYRLS [Lys5]ShK 3.300 1.579 0.478 100 100
2325.58 1923.08 FCRKTCGTC 133 RSCIDKIPKSRCTAFQCKHSMKYRLS [Lys6]ShK
0.085 0.002 0.024 0.117 0.21 2.72 4.04 FCRKTCGTC 134
RSCIDTKPKSRCTAFQCKHSMKYRLS [Lys7]ShK 0.792 8.689 10.971 4.671
13.468 108.63 259.00 FCRKTCGTC 135 RSCIDTIKKSRCTAFQCKHSMKYRLS
[Lys8]ShK 0.083 0.005 0.060 0.154 0.974 3.58 18.73 FCRKTCGTC 136
RSCIDTIPKKRCTAFQCKHSMKYRLS [Lys10]ShK 0.174 0.004 0.023 0.052 0.082
1.21 1.58 FCRKTCGTC 137 RSCIDTIPKSKCTAFQCKHSMKYRLS [Lys11]ShK 0.137
0.002 0.015 0.095 0.252 2.21 4.85 FCRKTCGTC 138
RSCIDTIPKSRCKAFQCKHSMKYRLS [Lys13]ShK 0.157 0.005 0.032 0.257 0.363
5.98 6.98 FCRKTCGTC 139 RSCIDTIPKSRCTKFQCKHSMKYRLS [Lys14]ShK 0.073
0.005 0.068 0.075 0.202 1.74 3.88 FCRKTCGTC 140
RSCIDTIPKSRCTAKQCKHSMKYRLS [Lys15]ShK 0.295 0.046 0.156 0.465 0.991
10.81 19.06 FCRKTCGTC 13 RSCIDTIPKSRCTAFKCKHSMKYRLS [Lys16]ShK
0.220 2.085 1 1 t: 0.113 0.194 2.63 3.73 FCRKTCGTC 141
RSCIDTIPKSRCTAFQCKKSMKYRLS [Lys19]ShK 0.189 0.004 0.021 0.041 0.089
0.95 1.71 FCRKTCGTC 142 RSCIDTIPKSRCTAFQCKHKMKYRLS [Lys20]ShK 1.332
15.408 11.568 0.108 0.192 2.51 3.69 FCRKTCGTC 143
RSCIDTIPKSRCTAFQCKHSKKYRLS [Lys21]ShK 0.822 0.048 0.058 3.589 4.822
83.47 92.73 FCRKTCGTC 144 RSCIDTIPKSRCTAFQCKHSMKKRLS [Lys23]ShK
0.000 0.000 0 0 FCRKTCGTC 145 RSCIDTIPKSRCTAFQCKHSMKYKLS [Lys24]ShK
0.067 0.004 0.060 0.044 0.108 1.02 2.08 FCRKTCGTC 146
RSCIDTIPKSRCTAFQCKHSMKYRKS [Lys25]ShK 0.101 0.030 0.297 0.226 0.366
5.26 7.04 FCRKTCGTC 147 RSCIDTIPKSRCTAFQCKHSMKYRLK [Lys26]ShK 0.115
0.037 0.322 0.273 0.495 6.35 9.52 FCRKTCGTC 148
RSCIDTIPKSRCTAFQCKHSMKYRLS [Lys27]ShK 0.928 0.136 0.147 3.485
11.751 81.05 225.98 KCRKTCGTC 149 RSCIDTIPKSRCTAFQCKHSMKYRLS
[Lys29]ShK 0.158 0.181 1.146 0.04 0.071 0.93 1.37 FCKKTCGTC 150
RSCIDTIPKSRCTAFQCKHSMKYRLS [Lys31]ShK 1.417 0.174 0.123 5.984
27.906 139.16 536.65 FCRKKCGTC 151 RSCIDTIPKSRCTAFQCKHSMKYRLS
[Lys33]ShK 0.122 0.005 0.041 0.218 0.317 5.07 6.10 FCRKTCKTC 152
RSCIDTIPKSRCTAFQCKHSMKYRLS [Lys34]ShK 0.150 0.006 0.040 0.067 0.136
1.56 2.62 FCRKTCGKC
TABLE-US-00085 TABLE 11 Position 16 Analogs WB WB Kv1.3 Kv1.1
Kv1.1/ WB WB (IL-2) (IFNg) (IWQ) (IWQ) Kv1.3 (IL-2) (IFNg) Potency
Potency SEQ IC50 IC50 Selec- IC50 IC50 Rela- Rela- ID (nM, (nM,
tivity (nM, (nM, tive tive NO: Amino Acid Sequence Designation Avg)
Avg) Ratio Avg) Avg) to ShK to ShK 153 RSCIDTIPKSRCTAFOCKHSMKYRLSFC
[Orn16]ShK 0.140 0.740 5.286 0.138 0.16 3.21 3.08 RKTCGTC 154
RSCIDTIPKSRCTAF[Dab]CKHSMKYR [Dab16]ShK 0.082 0.011 0.134 0.086
0.223 2.00 4.29 LSFCRKTCGTC 155 RSCIDTIPKSRCTAF[Ahp]CKHS[Nle]
[Ahp16,Nle21] 1.100 3.300 3.000 7.202 10.352 167.49 199.08
KYRLSFCRKTCGTC ShK-amide 156 RSCIDTIPKSRCTAFNCKHS[Nle]KYRL
[Asn16,Nle21] 0.171 0.009 0.053 0.689 1.075 16.02 20.67 SFCRKTCGTC
ShK-amide 157 RSCIDTIPKSRCTAFHCKHS[Nle]KYRL [His, Nle21] 0.139
0.016 0.115 0.24 0.148 5.58 2.85 SFCRKTCGTC ShK-amide
TABLE-US-00086 TABLE 12 PEGylated Toxin Peptide Analogs WB WB Kv1.3
Kv1.1 Kv1.1/ WB WB (IL-2) (IFNg) (IWQ) (IWQ) Kv1.3 (IL-2) (IFNg)
Potency Potency SEQ IC50 IC50 Selec- IC50 IC50 Rela- Rela- ID (nM,
(nM, tivity (nM, (nM, tive tive NO: Amino Acid Sequence Designation
Avg) Avg) Ratio Avg) Avg) to ShK to ShK 277 RSCIDTIPKSRCTAFQCKHS
30kDaPEG-ShK 1.931 0.182 0.094 0.372 2.425 8.65 46.63
MKYRLSFCRKTCGTC 16 RSCIDTIPKSRCTAFKCKHS 20kDaPEG-[Lys16]ShK 0.665
1082.214 1627.389 0.104 0.215 2.42 4.13 MKYRLSFCRKTCGTC 158
RSCIDTIPKSRCTAFKCKHS 30kPEG-[Lys16]ShK 0.454 33.300 73.348 0.282
0.491 6.56 9.44 MKYRLSFCRKTCGTC 159 RSCIDTIPKSRCTAF[1-Nal]
20kDaPEG-[-Nal16] 39.080 333.000 8.521 100 100 2325.58 1923.08
CKHSMKYRLSFCRKTCGTC ShK 160 RSCIDTIPKSRCTAFQCKHSM
20kDaPEG-[1-Na127] 8.301 33.300 4.012 6.416 11.037 149.21 212.25
KYRLS[1-Nal]CRKTCGTC ShK 161 RSCIDTIPKSRCTAFKCKHSA 20kDaPEG-[Lys16,
4.573 33.300 7.282 4.802 7.922 111.67 152.35 KYRLSFCRKTCGTC
A1a21]ShK 162 RSCIDTIPKSRCTAFKCKHSM 20kDaPEG-[Lys16, 3.410 33.300
9.765 1.659 1.127 38.58 21.67 KYRLS[1-Nal]CRKTCGTC 1-Na127]ShK 163
RSCIDTIPKSRCTAFKCKHSM 20kDaPEG-[Lys16] 0.605 219.863 363.410 0.089
0.164 2.07 33.73 KYRLSFCRKTCGTC ShK-amide 164 RSCIDTIPKSRCTAFKCKHS
20kDaPEG-[Lys16, 1.429 33.300 23.303 0.707 1.754 16.44 33.73
[Nle]KYRLSFCRKTCGTC Nle21]ShK-amide 165 RSCIDTIPKSRCTAFKCKHS
20kDaPEG-[Lys16, 1.610 1000.000 621.118 100 100 2325.58 1923.08
[Nva]KYRLSFCRKTCGTC Nva21]ShK-amide 166 RSCIDTIPKSRCTAFKCKHS
20kDaPEG-[Lys16, 1.740 536.944 308.589 0.398 0.942 9.26 18.12
QKYRLSFCRKTCGTC Gln21]ShK-amide 167 RSCIDTIPKSRCTAAKCKHS 20kDaPEG-
2.691 3.300 1.226 0.631 1.618 14.67 31.12 [Nle]KYRLSFCRKTCGTC
[Ala15,Lys16, Nle21]ShK-amide 168 RSCIDTIPKSRCTAFKCEHS 20kDaPEG-
9.022 33.300 3.691 2.888 8.846 67.16 170.12 [Nle]KYRLSFCRKTCGTC
[Lys16,Glu18, N1e21]ShK-amide 169 RSCEDTIPKSRCTAFKCKHS
20kDaPEG-[G1u4, 2.372 3.300 1.391 1.731 3.782 40.26 72.73
[Nle]KYRLSFCRKTCGTC Lys16,Nle21] ShK-amide 170 RSCIDTIPKSRCTAFKCKHS
20kDaPEG-[Lys16, 2.435 1000.000 410.678 0.505 0.78 11.74 15.00
[Nle]KYRLSFCRKTCGTCA Nle21]ShK-Ala 171 RSCIDTIPKSRCTAFKCKHS
20kDaPEG-[Lys16, 5.970 1000.000 167.504 0.655 0.693 15.23 13.33
[Nva]KYRLSFCRKTCGTCA Nva21]ShK-Ala 172 RSCEDTIPKSRCTAFKCKHS
20kDaPEG- 5.830 33.300 5.712 3 241. 9.463 75.37 181.98
[Nva]KYRLSFCRKTCGTCA [Glu4,Lys16,Nva21] ShK-Ala 173
RSCEDTIPKSRCTAFKCKHS 20kDaPEG- 7.780 33.300 4.280 2.781 14.023
64.67 269.67 QKYRLCSFCRKTGTCA [Glu4,Lys16,Gln21] ShK-Ala 174
RSCIDTIPKSRCTAFKCKHS [Lys16(20KPeg)]ShK 6.900 33.300 4.826 1.126
1.485 26.19 28.56 MKYRLSFCRKTCGTC
TABLE-US-00087 TABLE 13 Combination Toxin Peptide Analogs {Free
Acid} Kv1.3 Kv1.1 Kv1.1/ WB WB WB WB (IWQ) (IWQ) Kv1.3 (IL-2)
(IFNg) (IL-2) (IFNg) SEQ IC50 IC50 Selec- IC50 IC50 Potency Potency
ID (nM, (nM, tivity (nM, (nM, Relative Relative NO: Amino Acid
Sequence Designation Avg) Avg) Ratio Avg) Avg) to ShK to ShK 175
RSCIDTIPKSRCTAFQCKHS[Nle] [N1e21]ShK 0.040 0.015 0.375 0.153 0.303
3.56 5.83 KYRLSFCRKTCGTC 176 RSCIDTIPKSRCTAAKCKHSMKYRL [Ala15,
Lys16]ShK 0.960 9.081 9.459 0.057 0.052 1.33 1.00 SFCRKTCGTC 177
RSCIDTIPKSRCTAFKCRHSMKYRL [Lys16,Arg18]ShK 0.425 1.090 2.565 0.077
0.097 1.79 1.87 SFCRKTCGTC 178 RSCIDTIPKSRCTAFKCKHSAKYRL
[Lys16,A1a21]ShK 0.076 4.690 61.711 0.872 1.334 20.28 25.65
SFCRKTCGTC 179 RSCIDTIPKSRCTAFKCKHS[Nle] [Lys16,N1e21]ShK 0.130
29.258 225.062 0.249 0.848 5.79 16.31 KYRLSFCRKTCGTC 180
RSCIDTIPKSRCTAFKCKHSMKYRL [Lys16,1-Na127]ShK 0.113 2.806 24.832
0.924 5.678 21.49 109.19 S[1-Nal]CRKTCGTC 181
RSCIDTIPKSRCTAFKCKHSMKYRL [Lys16, Lys29]ShK 0.071 1.172 16.507
0.112 0.143 2.60 2.75 SFCKKTCGTC 182 RSCIDTIPKSRCTAFKCKHSMKYRL
[Lys16,Arg30]ShK 0.092 0.837 9.098 0.169 0.189 3.93 3.63 SFCRRTCGTC
183 RSCIDTIPKSRCTAFKCKHSMKYRL [Lys16, Glu30]ShK 0.073 3.300 45.205
0.032 0.081 0.74 1.56 SFCRETCGTC 184 RSCIDTIPKSRCTAFKCKHSMKYRL
[Lys16,29; 1-Na127; 0.110 3.383 30.755 3.372 3.831 78.42 73.67
S[1-Nal]CKRTCGTC Arg30]ShK 185 RSCEDTIPKRRCTAAKCKHSMKYRL
[Glu4,Arg10,30,A1a15, 0.140 8.857 63.264 0.209 0.305 4.86 5.87
SFCRRTCGTC Lys16]ShK
TABLE-US-00088 TABLE 14 Combination Toxin Peptide Analogs
{C-Terminal Amide} WB WB (IL-2) (IFNg) Po- Po- ten- ten- Kv1.3
Kv1.1 Kv1.1/ WB WB cy cy SEQ (IWQ) (IWQ) Kv1.3 (IL-2) (IFNg) Rel-
Rel- IC50 IC50 Selec- IC50 IC50 ative ative ID (nM, (nM, tivity
(nM, (nM, to to NO: Amino Acid Sequence Designation Avg) Avg) Ratio
Avg) Avg) ShK ShK 14 RSCIDTIPKSRCTAFKCKHSM [Lys16]ShK-amide 0.174
0.600 3.448 0.223 0.278 5.19 5.35 KYRLSFCRKTCGTC 186
RSCIDTIPKSRCTAAKCKHSM [Ala15,Lys16]ShK- 0.091 1.662 18.264 0.167
0.193 3.88 3.71 KYRLSFCRKTCGTC amide 187 RSCIDTIPKSRCTAFKCKHSM
[Lys16,1-Na127]ShK- 0.299 2.247 7.515 3.948 2.943 91.81 56.60
KYRLS[1-Nal]CRKTCGTC amide 188 RSCEDTIPKSRCTAFKCKHSM
[Glu4,Lys16]ShK- 0.127 4.523 35.614 0.071 0.068 1.65 1.31
KYRLSFCRKTCGTC amide 189 RSCIDTIPKSRCTAFKCKHSM [Lys16,Arg30]ShK-
0.129 0.689 5.341 0.151 0.236 3.51 4.54 KYRLSFCRRTCGTC amide 190
RSCIDTIPKSRCTAFKCKHSM [Lys16, Glu30]ShK- 0.028 1.412 50.429 0.105
0.144 2.44 2.77 KYRLSFCRETCGTC amide 191 RSCEDTIPKSRCTAFKCKHSM
[Glu4; Lys16,29; 0.384 33.300 86.719 0.712 0.598 16.56 11.50
KYRL[1-Na1]FCKRTCGTC 1-Na126;Arg30]ShK- amide 192
RSCIDTIPKSRCTAFKCKHSM [Lys16,29; 0.084 33.300 396.429 1.04 0.754
24.19 14.50 KYRLYFCKKTCGTC Tyr26]ShK-amide 193
RSCIDTIPKSRCTAFKCKHSM [Lys16,29;2-Na126] 0.762 33.300 43.701 0.222
0.148 5.16 2.85 KYRL[2Nal]FCKKTCGTC ShK-amide 194
RSCIDTIPKSRCTAFKCKHS [Lys16, Nle21, 1.882 33.300 17.694 3.183 5.027
74.02 96.67 [Nle]KYRLSFCRETCGTC Glu30]ShK-amide 195
RSCIDTIPKSRCTAFKCEHS [Lys16, Glu18]ShK- 0.065 3.436 52.862 0.119
0.234 2.77 4.50 MKYRLSFCRKTCGTC amide 196 RSCIDTIPKSRCTAFKCKHS
[Lys16, Nle21, 0.423 12.665 29.941 0.953 1.904 22.16 36.62
[Nle]KYRLSFCRRTCGTC Arg30]ShK-amide 15 RSCIDTIPKSRCTAFKCKHS
[Lys16,N1e21] 0.153 13.220 86.405 0.823 1.099 19.14 21.13
[Nle]KYRLSFCRKTCGTC ShK-amide 197 RSCEDTIPKSRCTAFKCKHS
[Glu4;Lys16,29;1- 1.655 3.300 1.994 10.683 17.391 248.44 334.44
[Nle]KYRL[1-Nal]FCKR Nal26;Arg30;Nle21] TCGTC ShK-amide 198
RSCEDTIPESRCTAFKCRHS [Glu4,9;Lys16,29; 0.062 3.300 53.226 0.151
0.345 248.44 334.44 MKYRLSFCKRTCGTC Arg18,30]ShK-amide 199
RSCEDTIPKERCTAFKCRHS [Glu4,10; Lys16,29; 0.064 3.300 51.563 0.114
0.217 2.65 4.17 MKYRLSFCKRTCGTC Arg18,30]ShK-amide 200
RSCEDTIPEERCTAFKCRHS [Glu4,9,10; Lys16,29; 0.059 3.300 55.932 0.204
0.513 4.74 9.87 MKYRLSFCKRTCGTC Arg18, 30]ShK-amide 201
RSCEDTIPKSRCTAFKCKHS [Glu4; Lys16,29; 0.104 3.300 31.731 0.067 0.28
1.56 5.38 MKYRLSFCKRTCGTC Arg30]ShK-amide 202 RSCIDTIPKSRCTAFKCKHS
[Lys16,29; 0.121 3.300 27.273 0.264 0.606 6.14 11.65
MKYRLSWCKKTCGTC Trp27]ShK-amide 203 RSCIDTIPKSRCTAFKCKHS [Lys16,29;
Nle21, 0.484 33.300 68.802 1.721 2.838 40.02 54.58
[Nle]KYRLSFCKRTCGTC Arg30]ShK-amide 204 RSCIDTIPKSRCTAFKCEHS
[Lys16,Glu18, 0.280 33.300 118.929 0.477 0.929 11.09 17.87
[Nle]KYRLSFCRKTCGTC Nle21]ShK-amide 205 RSCEDTIPKSRCTAFKCKHS [Glu4,
Lys16, 0.149 33.300 223.490 0.57 0.965 13.26 18.56
[Nle]KYRLSFCRKTCGTC Nle21]ShK-amide 206 RSCIDTIPKSRCTAAKCKHS
[Ala15, Lys16, 0.127 13.944 109.759 0.952 1.866 22.14 35.88
[Nle]KYRLSFCRKTCGTC Nle21]ShK-amide 207 RSCIDTIPKSRCTAFKCKHS
[Lys16, 29;2- 10.108 33.300 3.294 13.112 24.747 304.93 475.90
MKYRLS[2Nal]CKKTCGTC Nal27]ShK-amide 208 RSCIDTIPKSRCTAFKCRHS
[Lys16, 29; 0.401 1.896 4.728 0.26 0.242 6.05 4.65 MKYRLSFCKRTCGTC
Arg18,30]ShK-amide 209 RSCIDTIPKSRCTAFKCKHS [Lys16, 29;Phe26]ShK-
0.542 3.300 6.089 1.417 1.363 32.95 26.21 MKYRLFFCKKTCGTC amide 210
RSCIDTIPKSRCTAFKCKHS [Lys16, 29;A1a30]ShK- 0.197 3.300 16.751 0.186
0.289 4.33 5.56 MKYRLSFCKATCGTC amide 211 RSCEDTIPKSRCTAFKCRHS
[Glu4; Lys16,29;Arg18, 0.269 3.300 12.268 1.961 1.331 45.60 25.60
[Nle]KYRLSFCKRTCGTC 30; Nle21]ShK-amide 212 RSCEDTIPKSRCTAFKCRHS
[Glu4; Lys16, 29; 0.192 3.300 17.188 0.203 0.353 4.72 6.79
MKYRLSFCKRTCGTC Arg18, 30]ShK-amide 213 RSCIDTIPKSRCTAFKCKHS
[Lys16,29; Arg30]ShK- 0.131 2.469 18.847 0.339 0.281 7.88 5.40
MKYRLSFCKRTCGTC amide 214 RSCEDTIPKSRCTAAKCEHS
[Glu4,18,A1a15,Lys16, 0.346 3.300 9.538 1.215 1.597 28.26 30.71
[Nle]KYRLSFCRKTCGTC Nle21]ShK-amide 215 RSCEDTIPKSRCTAFKCEHS
[Glu4,18, Lys16, 0.239 3.300 13.808 0.603 0.934 14.02 17.96
[Nle]KYRLSFCRKTCGTC Nle21]ShK-amide 216 RSCEDTIPKSRCTAAKCKHS
[Glu4,A1a15,Lys16, 0.168 3.300 19.643 0.856 0.901 19.91 17.33
[Nle]KYRLSFCRKTCGTC Nle21]ShK-amide 217 RSCIDTIPKSRCTAAKCEHS
[Glu18,Ala15,Lys16, 0.202 3.300 16.337 1.355 1.291 31.51 24.83
[Nle]KYRLSFCRKTCGTC Nle21]ShK-amide 218 RSCEDTIPKSRCTAFKCKHS [Glu4,
Lys16, Nle21, 0.132 33.300 252.273 1.181 1.341 27.47 25.79
[Nle]KYRLSFCRRTCGTC Arg30]ShK-amide 219 RSCIDTIPKSRCTAFKCEHS
[Glu18, Lys16, Nle21, 0.226 33.300 147.345 1.22 0.969 28.37 'kl 4
[Nle]KYRLSFCRRTCGTC Arg30]ShK-amide 220 RSCEDTIPKSRCTAFKCEHS
[Glu4,18, Lys16, 0.260 3.300 101.216 0.97 1.985 22.56 38.17
[Nle]KYRLSFCRRTCGTC Nle21, Arg30]ShK- amide 221
RSCIDTIPKSRCTAAKCKHS [Ala15, Lys16, Nle21, 0.329 33.300 101.216
1.348 1.714 31.35 32.96 [Nle]KYRLSFCRRTCGTC Arg30]ShK-amide 222
RSCIDTIPESRCTAFKCKHS [Glu9, Lys16, 0.263 33.300 126.616 0.257 0.342
5.98 6.58 [Nle]KYRLSFCRKTCGTC Nle21]ShK-amide 223
RSCIDTIPKSRCTAAKCEHS [Glu18,Ala15,Lys16, 0.398 33.300 83.668 1.321
2.028 30.72 39.00 [Nle]KYRLSFCRRTCGTC Nle21, Arg30]ShK- amide 224
RSCEDTIPKSRCTAAKCKHS [Glu4,Ala15,Lys16, 0.118 3.300 27.966 0.647
0.816 15.05 15.69 [Nle]KYRLSFCRRTCGTC Nle21, Arg30]ShK- amide 225
RSCIDTIPKERCTAFKCKHS [Glu10,Lys16, 0.127 3.300 25.984 0.776 1.257
18.05 24.17 [Nle]KYRLSFCRKTCGTC Nle21]ShK-amide 226
RSCIDTIPKSRCTAFKCKHS [Lys16,29; 1- 0.341 3.300 9.677 0.808 1.975
18.79 37.98 MKYRL[1-Nal]FCKKTCGTC Nal26]ShK-amide 227
RSCIDTIPKSRCTAFKCKHS [Lys16,29; 1- 2.334 3.300 1.414 9.442 8.222
219.58 158.12 [Nle]KYRL[1-Nal]FCKR Nal26;Arg30; TCGTC
Nle21]ShK-amide 228 RSCIDTIPKSRCTAFKCKHS [Lys16,29; 0.042 3.300
78.571 0.738 1.172 17.16 22.54 MKYRLSYCKKTCGTC Tyr27]ShK-amide 229
RSCEDTIPKSRCTAFKCKHS [Glu4, Lys16, 0.085 3.300 38.824 1.384 3.942
32.19 75.81 TKYRLSFCRKTCGTC Thr21]ShK-amide 230
RSCEDTIPKSRCTAFKCKHS [Glu4, Lys16, 0.098 3.300 33.673 0.86 1.994
20.00 38.35 SKYRLSFCRKTCGTC Ser21]ShK-amide 231
RSCIDTIPKSRCTAFKCKHS [Lys16, 29; Ala30, 0.057 24.070 422.281 0.791
1.7 18.40 32.69 [Nle]KYRLSFCKATCGTC Nle21]ShK-amide 232
RSCEDTIPKSRCTAFKCKHS [Glu4, Lys16, 0.086 3.300 38.372 0.405 0.427
9.42 8.21 [Nva]KYRLSFCRKTCGTC Nva21]ShK-amide 233
RSCEDTIPKSRCTAFKCKHS [Glu4, Lys16, 0.330 33.000 100.000 1.115 1.765
25.93 33.94 [Abu]KYRLSFCRKTCGTC Abu21]ShK-amide 234
RSCEDTIPKSRCTAFKCKHS [Glu4, Lys16, 0.000 0.000 0.23 0.423 5.35 8.13
QKYRLSFCRKTCGTC Gln21]ShK-amide
TABLE-US-00089 TABLE 15 Toxin Peptide Analogs with C-Terminal
Extension Kv1.1 Kv1.3 Kv1.1/ WB WB WB WB (IWQ) (IWQ) Kv1.3 (IL-2)
(IFNg) (IL-2) (IFNg) SEQ IC50 IC50 Selec- IC50 IC50 Potency Potency
ID (nM, (nM, tivity (nM, (nM, Relative Relative NO: Amino Acid
Sequence Designation Avg) Avg) Ratio Avg) Avg) to ShK to ShK 235
RSCIDTIPKSRCTAFKCKHSMKYRLSFCRK [Lys16]ShK-Ala 0.060 9.500 158.333
0.138 0.266 3.21 5.12 TCGTCA 236 RSCIDTIPKSRCTAFKCKHS[Nle]KYRLS
[Lys16,N1e21]ShK- 0.071 33.300 469.014 0.305 0.515 7.09 9.90
FCRKTCGTCA Ala 237 RSCIDTIPKSRCTAFKCKHSNKYRLSFCRK [Lys16,Asn21]ShK-
0.360 33.000 91.667 0.891 0.693 20.72 13.33 TCGTCA Ala 238
RSCIDTIPKSRCTAFKCKHS[Nva]KYRLS [Lys16,Nva21]ShK- 0.540 13.490
24.981 0.164 0.307 3.81 5.90 FCRKTCGTCA Ala 239
RSCIDTIPKSRCTAFKCKHS[Nva]KYRLS [Lys16,Nva21]ShK- 0.080 7.606 95.075
0.431 1.455 10.02 27.98 FCRKTCGTCA Ala-amide 240
RSCIDTIPKSRCTAFKCKHSQKYRLSFCRK [Lys16,Gln21]ShK- 0.350 4.010 11.457
0.168 0.206 3.91 5.10 TCGTCA Ala 241 RSCEDTIPKSRCTAFKCKHSQKYRLSFCRK
[Glu4,Lys16,Gln21] 0.145 26.350 181.724 0.137 0.265 3.19 5.10
TCGTCA ShK-Ala 242 RSCEDTIPKSRCTAFKCKHS[Nva]KYRLS
[Glu4,Lys16,Nva21] 0.133 33.300 250.376 0.208 0.368 4.84 7.08
FCRKTCGTCA ShK-Ala 243 RSCEDTIPKSRCTAFKCKHS[Nle]KYRLS
[Glu4,Lys16,N1e21] 0.060 33.300 555.000 0.327 0.987 7.60 18.98
FCRKTCGTCA ShK-Ala 244 RSCIDTIPKSRCTAFKCKHS[Nle]KYRLS
[Lys16,N1e21]ShK- 0.090 22.490 249.889 0.386 0.621 8.98 11.94
FCRKTCGTCE Glu 245 RSCIDTIPKSRCTAFKCKHS[Nle]KYRLS [Lys16,N1e21]ShK-
0.215 109.305 508.395 0.418 0.485 9.72 9.33 FCRKTCGTCY Tyr 246
RSCIDTIPKSRCTAFKCKHS[Nle]KYRLS [Lys16,N1e21]ShK- 0.000 0.000 2.046
5.647 47.58 108.60 FCRKTCGTCV Val 247
RSCIDTIPKSRCTAFKCKHS[Nva]KYRLS [Lys16,Nva21]ShK- 0.260 12.580
48.385 0.304 0.231 7.07 4.44 FCRKTCGTC[bAla] betaAla
TABLE-US-00090 TABLE 16 Toxin Peptide Analogs with C-Terminal
Extension {and C-Terminal Amide} Kv1.3 Kv1.1 Kv1.1/ WB WB WB WB
(IWQ) (IWQ) Kv1.3 (IL-2) (IFNg) (IL-2) (IFNg) SEQ IC50 IC50 Selec-
IC50 IC50 Potency Potency ID (nM, (nM, tivity (nM, (nM, Relative
Relative NO: Amino Acid Sequence Designation Avg) Avg) Ratio Avg)
Avg) to ShK to ShK 248 RSCIDTIPKSRCTAFKCKHS[Nva]KYRLSFC
[Lys16,Nva21]ShK- 0.280 12.670 45.250 0.255 0.349 5.93 6.71
RKTCGTCD Asp-amide 249 RSCIDTIPKSRCTAFKCKHS[Nva]KYRLSFC [Lys16,
Nva21]ShK- 0.210 7.970 37.952 0.218 0.353 5.07 6.79 RKTCGTCE
Glu-amide 250 RSCIDTIPKSRCTAFKCKHS[Nle]KYRLSFC [Lys16, Nle21]ShK-
0.390 16.600 42.564 0.295 0.403 6.86 7.75 RKTCGTCD Asp-amide 251
RSCIDTIPKSRCTAFKCKHS[Nva]KYRLSFC [Lys16,Nva21]ShK- 0.090 10.730
119.222 0.467 0.662 10.86 12.73 RKTCGTCS Ser-amide 252
RSCIDTIPKSRCTAFKCKHS[Nva]KYRLSFC [Lys16,Nva21]ShK- 0.030 4.430
147.667 0.385 0.702 8.95 13.50 RKTCGTCT Thr-amide 253
RSCIDTIPKSRCTAFKCKHS[Nva]KYRLSFC [Lys16,Nva21]ShK- 0.030 5.730
191.000 0.483 0.722 11.23 13.88 RKTCGTC[Aib] Aib-amide 254
RSCIDTIPKSRCTAFKCKHS[Nva]KYRLSFC [Lys16,Nva21]ShK- 0.080 7.606
95.075 0.431 1.455 10.02 27.98 RKTCGTCA Ala-amide
TABLE-US-00091 TABLE 17 Position 21 Toxin Peptide Analogs WB WB
(IL-2) (IFNg) Kv1.3 Kv1.1 Kv1.1/ WB WB Poten- Poten- SEQ (IWQ)
(IWQ) Kv1.3 (IL-2) (IFNg) cy cy IC50 IC50 Selec- IC50 IC50 Rel-
Rel- ID (nM, (nM, tivity (nM, (nM, ative ative NO: Amino Acid
Sequence Designation Avg) Avg) Ratio Avg) Avg) to ShK to ShK 255
RSCIDTIPKSRCTAFQCKHS[Nle]KYR [Nle21]ShK- 0.040 0.015 0.375 0.325
1.133 7.56 21.79 LSFCRKTCGTC amide 15 RSCIDTIPKSRCTAFKCKHS[Nle]KYR
[Lys16, Nle21] 0.153 13.220 86.405 0.823 1.099 19.14 21.13
LSFCRKTCGTC ShK-amide 257 RSCIDTIPKSRCTAFKCKHS[Met(O)] [Lys16,
Met(O) 0.106 2.106 19.868 0.24 0.289 5.58 5.56 KYRLSFCRKTCGTC
21]ShK-amide 258 RSCIDTIPKSRCTAFKCKHSSKYRLSF [Lys16, Ser21] 0.068
3.300 48.529 1.448 1.705 33.67 32.79 CRKTCGTC ShK-amide 259
RSCIDTIPKSRCTAFKCKHSTKYRLSF [Lys16, Thr21] 0.058 3.300 56.897 1.554
2.978 36.14 57.27 CRKTCGTC ShK-amide 260 RSCIDTIPKSRCTAFKCKHS[Nva]K
[Lys16, Nva21] 0.470 7.374 15.689 0.486 0.932 11.30 17.92
YRLSFCRKTCGTC ShK-amide 261 RSCIDTIPKSRCTAFKCKHSQKYRLS [Lys16,
Gln21] 0.161 3.151 19.571 0.42 0.812 9.77 15.62 FCRKTCGTC ShK-amide
262 RSCIDTIPKSRCTAFKCKHSHKYRLS [Lys16, His21] 0.219 3.300 15.068
5.498 9.248 127.86 177.85 FCRKTCGTC ShK-amide 263
RSCIDTIPKSRCTAFKCKHSYKYRLS [Lys16, Tyr21] 0.246 3.300 13.415 2.835
3.19 65.93 61.35 FCRKTCGTC ShK-amide 264 RSCIDTIPKSRCTAFKCKHSNKYRLS
[Lys16, Asn21] 0.167 3.300 19.760 3.236 2.727 75.26 52.44 FCRKTCGTC
ShK-amide 265 RSCIDTIPKSRCTAFKCKHSVKYRLS [Lys16, VaI21] 0.344
33.300 96.802 4.801 6.654 111.65 127.96 FCRKTCGTC ShK-amide 266
RSCIDTIPKSRCTAFKCKHSLKYRLS [Lys16, Leu21] 0.213 23.808 111.775
0.889 0.636 20.67 12.33 FCRKTCGTC ShK-amide 267
RSCIDTIPKSRCTAFKCKHSWKYRLS [Lys16, Trp21] 0.782 3.300 4.220 3.864
3.195 89.86 61.44 FCRKTCGTC ShK-amide 268
RSCIDTIPKSRCTAFKCKHS[Abu]K [Lys16, Abu21] 0.246 3.300 13.415 1.202
1.526 27.95 29.35 YRLSFCRKTCGTC ShK-amide 269
RSCIDTIPKSRCTAFKCKHS[Cha]K [Lys16, Cha21] 0.499 3.300 6.613 1.739
1.325 40.44 25.48 YRLSFCRKTCGTC ShK-amide 270
RSCIDTIPKSRCTAFKCKHS[Chg]K [Lys16, Chg21] 0.205 3.300 16.098 0.875
0.727 20.35 13.98 YRLSFCRKTCGTC ShK-amide 271
RSCIDTIPKSRCTAFKCKHS[Hyp]K [Lys16, Hyp21] 3.300 3.300 1.000 100 100
2325.58 1923.08 YRLSFCRKTCGTC ShK-amide 272
RSCIDTIPKSRCTAFKCKHSFKYRLS [Lys16, Phe21] 0.246 3.300 13.415 1.474
2.041 34.28 39.25 FCRKTCGTC ShK-amide 273
RSCIDTIPKSRCTAFKCKHSIKYRLS [Lys16, Ile21] 0.140 33.300 237.657
2.494 5.772 58.00 111.00 FCRKTCGTC ShK-amide 274
RSCIDTIPKSRCTAFKCKHSDKYRLS [Lys16, Asp21] 0.350 33.300 95.143 2.305
3.634 53.60 69.88 FCRKTCGTC ShK-amide
ABBREVIATIONS
[0709] Abbreviations used throughout this specification are as
defined below, unless otherwise defined in specific
circumstances.
[0710] Ac acetyl (used to refer to acetylated residues)
[0711] AcBpa acetylated p-benzoyl-L-phenylalanine
[0712] ACN acetonitrile
[0713] AcOH acetic acid
[0714] ADCC antibody-dependent cellular cytotoxicity
[0715] Aib aminoisobutyric acid
[0716] bA beta-alanine
[0717] Bpa p-benzoyl-L-phenylalanine
[0718] BrAc bromoacetyl (BrCH.sub.2C(O)
[0719] BSA Bovine serum albumin
[0720] Bzl Benzyl
[0721] Cap Caproic acid
[0722] CBC complete blood count
[0723] COPD Chronic obstructive pulmonary disease
[0724] CTL Cytotoxic T lymphocytes
[0725] DCC Dicylcohexylcarbodiimide
[0726] Dde 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyl
[0727] DNP 2,4-dinitrophenol
[0728] DOPC 1,2-Dioleoyl-sn-Glycero-3-phosphocholine
[0729] DOPE 1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine
[0730] DPPC 1,2-Dipalmitoyl-sn-Glycero-3-phosphocholine
[0731] DSPC 1,2-Distearoyl-sn-Glycero-3-phosphocholine
[0732] DTT Dithiothreitol
[0733] EAE experimental autoimmune encephalomyelitis
[0734] ECL enhanced chemiluminescence
[0735] ESI-MS Electron spray ionization mass spectrometry
[0736] FACS fluorescence-activated cell sorting
[0737] Fmoc fluorenylmethoxycarbonyl
[0738] HOBt 1-Hydroxybenzotriazole
[0739] HPLC high performance liquid chromatography
[0740] HSL homoserine lactone
[0741] IB inclusion bodies
[0742] KCa calcium-activated potassium channel (including IKCa,
BKCa, SKCa)
[0743] KLH Keyhole Limpet Hemocyanin
[0744] Kv voltage-gated potassium channel
[0745] Lau Laurie acid
[0746] LPS lipopolysaccharide
[0747] LYMPH lymphocytes
[0748] MALDI-MS Matrix-assisted laser desorption ionization mass
spectrometry
[0749] Me methyl
[0750] MeO methoxy
[0751] MeOH methanol
[0752] MHC major histocompatibility complex
[0753] MMP matrix metalloproteinase
[0754] MW Molecular Weight
[0755] MWCO Molecular Weight Cut Off
[0756] 1-Nap 1-napthylalanine
[0757] NEUT neutrophils
[0758] Nle norleucine
[0759] NMP N-methyl-2-pyrrolidinone
[0760] OAc acetate
[0761] PAGE polyacrylamide gel electrophoresis
[0762] PBMC peripheral blood mononuclear cell
[0763] PBS Phosphate-buffered saline
[0764] Pbf 2,2,4,6,7-pendamethyldihydrobenzofuran-5-sulfonyl PCR
polymerase chain reaction
[0765] PD pharmacodynamic
[0766] Pec pipecolic acid
[0767] PEG Poly(ethylene glycol)
[0768] pGlu pyroglutamic acid
[0769] Pic picolinic acid
[0770] PK pharmacokinetic
[0771] pY phosphotyrosine
[0772] RBS ribosome binding site
[0773] RT room temperature (25.degree. C.)
[0774] Sar sarcosine
[0775] SDS sodium dodecyl sulfate
[0776] STK serine-threonine kinases
[0777] t-Boc tert-Butoxycarbonyl
[0778] tBu tert-Butyl
[0779] TCR T cell receptor
[0780] TFA trifluoroacetic acid
[0781] THF thymic humoral factor
[0782] Trt trityl
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 482 <210> SEQ ID NO 1 <211> LENGTH: 35 <212>
TYPE: PRT <213> ORGANISM: Stichodactyla helianthus
<400> SEQUENCE: 1 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg
Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu
Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ
ID NO 2 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: Radianthus magnifica <400> SEQUENCE: 2 Arg Thr Cys
Lys Asp Leu Ile Pro Val Ser Glu Cys Thr Asp Ile Arg 1 5 10 15 Cys
Arg Thr Ser Met Lys Tyr Arg Leu Asn Leu Cys Arg Lys Thr Cys 20 25
30 Gly Ser Cys 35 <210> SEQ ID NO 3 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: Anemonia erythraea
<400> SEQUENCE: 3 Arg Ala Cys Lys Asp Tyr Leu Pro Lys Ser Glu
Cys Thr Gln Phe Arg 1 5 10 15 Cys Arg Thr Ser Met Lys Tyr Lys Tyr
Thr Asn Cys Lys Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ
ID NO 4 <211> LENGTH: 38 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Toxin peptide analog <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION:
Carboxy-terminal residue is optionally amidated <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(2) <223> OTHER INFORMATION: Xaa 1 and Xaa 2 are absent,
or Xaa 1 is absent and Xaa 2 is Glu, Ser, Ala, or Thr, or Xaa 1 is
Arg or Ala and Xaa 2 is Glu, Ser, Ala, or Thr <220> FEATURE:
<221> NAME/KEY: DISULFID <222> LOCATION: (3)..(35)
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa 4 is an
alkyl, basic, or acidic amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (6)..(6)
<223> OTHER INFORMATION: Xaa 6 is Thr, Tyr, Ala, or Leu
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (7)..(7) <223> OTHER INFORMATION: Xaa 7 is Leu,
Ile, Ala, or Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (8)..(8) <223> OTHER
INFORMATION: Xaa 8 is Pro, Ala, Arg, Lys, 1-Nal, or Glu <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(9)..(9) <223> OTHER INFORMATION: Xaa 9 is Lys, Ala, Val or
or an acidic amino acid residue <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (10)..(10) <223>
OTHER INFORMATION: Xaa 10 is Ser, Glu, Arg, or Ala <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(11)..(11) <223> OTHER INFORMATION: Xaa 11 is Arg, Glu, or
Ala <220> FEATURE: <221> NAME/KEY: DISULFID <222>
LOCATION: (12)..(28) <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (13)..(13) <223> OTHER
INFORMATION: Xaa 13 is Thr, Ala, Arg, Lys, 1-Nal, or Glu
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (14)..(14) <223> OTHER INFORMATION: Xaa 14 is Gln,
Ala or an acidic amino acid reside <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (15)..(15) <223>
OTHER INFORMATION: Xaa 15 is an alkyl or aromatic amino acid
residue <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (16)..(16) <223> OTHER INFORMATION: Xaa
16 is a basic, alkyl, or aromatic amino acid residue other than
Ala, Gln, Glu or Arg <220> FEATURE: <221> NAME/KEY:
DISULFID <222> LOCATION: (17)..(32) <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (18)..(18)
<223> OTHER INFORMATION: Xaa 18 is an Ala or an acidic or
basic amino acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: Xaa 19 is Thr, Ala or a basic amino acid residue
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (20)..(20) <223> OTHER INFORMATION: Xaa 20 is Ser,
Ala or a basic amino acid residue <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Xaa 21 is an alkyl or aromatic amino acid
residue, other than Ala or Met <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (22)..(22) <223>
OTHER INFORMATION: Xaa 22 is Lys or Ala <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (23)..(23)
<223> OTHER INFORMATION: Xaa 23 is Tyr or Ala <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(24)..(24) <223> OTHER INFORMATION: Xaa 24 is Arg, Lys, or
Ala <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (25)..(25) <223> OTHER INFORMATION: Xaa
25 is Tyr, Leu, or Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (26)..(26) <223> OTHER
INFORMATION: Xaa 26 is Ser, Thr, Asn, Ala, or an aromatic amino
acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (27)..(27) <223> OTHER
INFORMATION: Xaa 27 is Leu, Ala, Asn, or an aromatic amino acid
residue <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (29)..(29) <223> OTHER INFORMATION: Xaa
29 is 1-Nal, 2-Nal, Ala, or an basic amino acid residue <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(30)..(30) <223> OTHER INFORMATION: Xaa 30 is Ala or an
acidic or basic amino acid residue <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (31)..(31) <223>
OTHER INFORMATION: Xaa 31 is Thr, Ala or an aromatic amino acid
residue <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (33)..(33) <223> OTHER INFORMATION: Xaa
33 is Gly, Ala, Arg, Lys, 1-Nal, or Glu <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (34)..(34)
<223> OTHER INFORMATION: Xaa 34 is Thr, Ser, Ala, Lys, or an
aromatic amino acid residue <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (36)..(38) <223>
OTHER INFORMATION: Xaa in positions 36-38 are each independently
absent are independently a neutral, basic, acidic, or N-alkylated
amino acid residue <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (38)..(38) <223> OTHER
INFORMATION: Carboxy-terminal residue is optionally amidated
<400> SEQUENCE: 4 Xaa Xaa Cys Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Cys 20 25 30 Xaa Xaa Cys Xaa Xaa Xaa 35
<210> SEQ ID NO 5 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: Actinia equina <400> SEQUENCE: 5
Gly Cys Lys Asp Asn Phe Ser Ala Asn Thr Cys Lys His Val Lys Ala 1 5
10 15 Asn Asn Asn Cys Gly Ser Gln Lys Tyr Ala Thr Asn Cys Ala Lys
Thr 20 25 30 Cys Gly Lys Cys 35 <210> SEQ ID NO 6 <211>
LENGTH: 36 <212> TYPE: PRT <213> ORGANISM: Anemonia
sulcata <400> SEQUENCE: 6 Ala Cys Lys Asp Asn Phe Ala Ala Ala
Thr Cys Lys His Val Lys Glu 1 5 10 15 Asn Lys Asn Cys Gly Ser Gln
Lys Tyr Ala Thr Asn Cys Ala Lys Thr 20 25 30 Cys Gly Lys Cys 35
<210> SEQ ID NO 7 <211> LENGTH: 37 <212> TYPE:
PRT <213> ORGANISM: Bunodosoma granulifera <400>
SEQUENCE: 7 Val Cys Arg Asp Trp Phe Lys Glu Thr Ala Cys Arg His Ala
Lys Ser 1 5 10 15 Leu Gly Asn Cys Arg Thr Ser Gln Lys Tyr Arg Ala
Asn Cys Ala Lys 20 25 30 Thr Cys Glu Leu Cys 35 <210> SEQ ID
NO 8 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: Stichodactyla helianthus <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: Pegylated
<400> SEQUENCE: 8 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg
Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu
Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ
ID NO 9 <211> LENGTH: 38 <212> TYPE: PRT <213>
ORGANISM: Orthochirus scrobiculosus <400> SEQUENCE: 9 Gly Val
Ile Ile Asn Val Lys Cys Lys Ile Ser Arg Gln Cys Leu Glu 1 5 10 15
Pro Cys Lys Lys Ala Gly Met Arg Phe Gly Lys Cys Met Asn Gly Lys 20
25 30 Cys Ala Cys Thr Pro Lys 35 <210> SEQ ID NO 10
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: HmK peptide <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norvaline <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 10 Arg Thr Cys Lys Asp
Leu Ile Pro Val Ser Glu Cys Thr Asp Ile Lys 1 5 10 15 Cys Arg Thr
Ser Xaa Lys Tyr Arg Leu Asn Leu Cys Arg Lys Thr Cys 20 25 30 Gly
Ser Cys 35 <210> SEQ ID NO 11 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (16)..(16) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 11 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Xaa 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 12 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (26)..(26) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 12 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Xaa Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 13 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK[1-35, Q16K]
polypeptide <400> SEQUENCE: 13 Arg Ser Cys Ile Asp Thr Ile
Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met
Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 14 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination toxin peptide analog
(C-Terminal Amide) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 14 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 15 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 15 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His
Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 16 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK PEGylated
Toxin Peptide <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <400> SEQUENCE: 16 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 17 <211> LENGTH: 36
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK reference
peptide <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION:
L-phosphonotyrosine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: Between residues 1 and 2 is: {2-[2-Aminoethoxy]ethoxy
}acetic acid <400> SEQUENCE: 17 Xaa Arg Ser Cys Ile Asp Thr
Ile Pro Lys Ser Arg Cys Thr Ala Phe 1 5 10 15 Gln Cys Lys His Ser
Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr 20 25 30 Cys Gly Thr
Cys 35 <210> SEQ ID NO 18 <211> LENGTH: 36 <212>
TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: ShK reference peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(2) <223> OTHER INFORMATION: Between residues
1 and 2 is: {2-[2-Aminoethoxy]ethoxy }acetic acid <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: L-phosphonotyrosine
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (22)..(22) <223> OTHER INFORMATION: Norleucine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (36)..(36) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 18 Xaa Arg Ser Cys Ile Asp Thr Ile Pro Lys
Ser Arg Cys Thr Ala Phe 1 5 10 15 Gln Cys Lys His Ser Xaa Lys Tyr
Arg Leu Ser Phe Cys Arg Lys Thr 20 25 30 Cys Gly Thr Cys 35
<210> SEQ ID NO 19 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 19
Arg Arg Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 20 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 20 Arg Ser Cys Arg Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 21 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 21
Arg Ser Cys Ile Arg Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 22 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 22 Arg Ser Cys Ile Asp Arg Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 23 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 23
Arg Ser Cys Ile Asp Thr Arg Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 24 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 24 Arg Ser Cys Ile Asp Thr Ile Arg
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 25 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 25
Arg Ser Cys Ile Asp Thr Ile Pro Arg Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 26 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 26 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Arg Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 27 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 27
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Arg Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 28 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 28 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Arg Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 29 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 29
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Arg Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 30 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 30 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Arg 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 31 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 31
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Arg His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 32 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 32 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys Arg Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 33 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 33
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Arg Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 34 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 34 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Arg Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 35 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 35
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Arg Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 36 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 36 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Arg Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 37 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 37
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Arg Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 38 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 38 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Arg Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 39 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 39
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Arg Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 40 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 40 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Arg Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 41 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 41
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Arg
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 42 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 42 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Arg Thr Cys 35
<210> SEQ ID NO 43 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 43
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Arg Cys 35 <210> SEQ ID NO 44 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 44 Glu Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 45 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 45
Arg Glu Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 46 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 46 Arg Ser Cys Glu Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 47 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 47
Arg Ser Cys Ile Glu Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 48 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 48 Arg Ser Cys Ile Asp Glu Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 49 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 49
Arg Ser Cys Ile Asp Thr Glu Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 50 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 50 Arg Ser Cys Ile Asp Thr Ile Glu
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 51 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 51
Arg Ser Cys Ile Asp Thr Ile Pro Glu Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 52 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 52 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Glu Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 53 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 53
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Glu Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 54 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 54 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Glu Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 55 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptdie <400> SEQUENCE: 55
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Glu Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 56 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 56 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Glu Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 57 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 57
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Glu 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 58 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 58 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Glu His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 59 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 59
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys Glu Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 60 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 60 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Glu Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 61 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Shk Peptide <400> SEQUENCE: 61
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Glu Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 62 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 62 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Glu
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 63 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 63
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Glu Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 64 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 64 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Glu Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 65 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 65
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Glu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 66 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 66 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Glu Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 67 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 67
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Glu Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 68 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 68 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Glu Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 69 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 69
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Glu Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 70 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 70 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Glu Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 71 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 71
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Glu Thr Cys 35 <210> SEQ ID NO 72 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 72 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Glu Cys 35
<210> SEQ ID NO 73 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: 3-(1-naphthyl)alanine <400>
SEQUENCE: 73 Xaa Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
74 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: ShK peptide <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (2)..(2) <223>
OTHER INFORMATION: 3-(1-naphthyl)alanine <400> SEQUENCE: 74
Arg Xaa Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 75 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (4)..(4) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 75 Arg Ser Cys Xaa Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 76 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 76 Arg Ser Cys Ile Xaa
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 77 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (6)..(6) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 77 Arg Ser Cys Ile Asp
Xaa Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 78 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (7)..(7) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 78 Arg Ser Cys Ile Asp
Thr Xaa Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 79 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (8)..(8) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 79 Arg Ser Cys Ile Asp
Thr Ile Xaa Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 80 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (9)..(9) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 80 Arg Ser Cys Ile Asp
Thr Ile Pro Xaa Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 81 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (10)..(10) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 81 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Xaa Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 82 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (11)..(11) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 82 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Xaa Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 83 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (13)..(13) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 83 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Xaa Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 84 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (14)..(14) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 84 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Xaa Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 85 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (15)..(15) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 85 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Xaa Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 86 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Linker sequence
<400> SEQUENCE: 86 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210>
SEQ ID NO 87 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (18)..(18)
<223> OTHER INFORMATION: 3-(1-naphthyl)alanine <400>
SEQUENCE: 87 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Gln 1 5 10 15 Cys Xaa His Ser Met Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
88 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: ShK peptide <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 3-(1-naphthyl)alanine <400> SEQUENCE: 88
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys Xaa Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 89 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (20)..(20) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 89 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Xaa Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 90 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptdie
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 90 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 91 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (22)..(22) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 91 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Xaa Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 92 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (23)..(23) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 92 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Xaa Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 93 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (24)..(24) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 93 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Xaa Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 94 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (25)..(25) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 94 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Xaa Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 95 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (27)..(27) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 95 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Xaa Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 96 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (29)..(29) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 96 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Xaa Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 97 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (30)..(30) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 97 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Xaa Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 98 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShKpeptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (31)..(31) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 98 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Xaa Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 99 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (33)..(33) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 99 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Xaa
Thr Cys 35 <210> SEQ ID NO 100 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (34)..(34) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 100 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Xaa Cys 35 <210> SEQ ID NO 101 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 101 Ala Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 102 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
102 Arg Ala Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 103
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 103 Arg Ser Cys Ala
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 104 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 104 Arg Ser Cys Ile Ala Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 105 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
105 Arg Ser Cys Ile Asp Ala Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 106
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 106 Arg Ser Cys Ile
Asp Thr Ala Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 107 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 107 Arg Ser Cys Ile Asp Thr Ile Ala Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 108 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
108 Arg Ser Cys Ile Asp Thr Ile Pro Ala Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 109
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 109 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ala Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 110 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 110 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Ala Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 111 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Shk peptide <400> SEQUENCE:
111 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Ala Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 112
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 112 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 113 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Shk peptide
<400> SEQUENCE: 113 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Ala 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 114 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
114 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Ala His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 115
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 115 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
Ala Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 116 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 116 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ala Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 117 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
117 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Ala Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 118
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 118 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Ala Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 119 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 119 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Ala Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 120 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
120 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Ala Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 121
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 121 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Ala Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 122 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 122 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ala Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 123 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
123 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Ala Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 124
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 124 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Ala Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 125 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 125 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Ala Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 126 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
126 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Ala Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 127
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 127 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Ala Thr Cys 35 <210> SEQ ID NO 128 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 128 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Ala Cys 35 <210>
SEQ ID NO 129 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
129 Lys Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 130
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 130 Arg Lys Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 131 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 131 Arg Ser Cys Lys Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 132 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
132 Arg Ser Cys Ile Lys Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 133
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 133 Arg Ser Cys Ile
Asp Lys Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 134 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 134 Arg Ser Cys Ile Asp Thr Lys Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 135 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
135 Arg Ser Cys Ile Asp Thr Ile Lys Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 136
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 136 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Lys Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 137 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 137 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Lys Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 138 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
138 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Lys Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 139
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptdie <400> SEQUENCE: 139 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Lys Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 140 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 140 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Lys Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 141 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
141 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys Lys Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 142
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 142 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Lys Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 143 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 143 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Lys Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 144 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
144 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Lys Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 145
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 145 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Lys Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 146 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 146 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Lys Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 147 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
147 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Lys Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 148
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 148 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Lys Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 149 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 149 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Lys Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 150 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
150 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Lys Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 151
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 151 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Lys Thr Cys 35 <210> SEQ ID NO 152 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 152 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Lys Cys 35 <210>
SEQ ID NO 153 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Shk peptide <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (16)..(16)
<223> OTHER INFORMATION: Ornithine <400> SEQUENCE: 153
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Xaa 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 154 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (16)..(16) <223> OTHER INFORMATION:
alpha, gamma-diaminobutyric acid <400> SEQUENCE: 154 Arg Ser
Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Xaa 1 5 10 15
Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20
25 30 Gly Thr Cys 35 <210> SEQ ID NO 155 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (16)..(16) <223> OTHER INFORMATION:
3-amino-6-hydroxy-2-piperidone <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norleucine <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (35)..(35) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 155 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Xaa 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 156 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norleucine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 156 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Asn 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 157 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Norleucine <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 157
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe His 1 5
10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 158 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
PEGylated toxin peptide analog <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1) <223>
OTHER INFORMATION: PEGylated <400> SEQUENCE: 158 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 159 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK PEGylated
toxin peptide analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (16)..(16) <223> OTHER
INFORMATION: 3-(1-naphthyl)alanine <400> SEQUENCE: 159 Arg
Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Xaa 1 5 10
15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys
20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 160 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
PEGylated toxin peptide analog <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1) <223>
OTHER INFORMATION: PEGylated <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (27)..(27) <223>
OTHER INFORMATION: 3-(1-naphthyl)alanine <400> SEQUENCE: 160
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Xaa Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 161 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
PEGylated toxin peptide analog <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1) <223>
OTHER INFORMATION: PEGylated <400> SEQUENCE: 161 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Ala Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 162 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (27)..(27) <223> OTHER
INFORMATION: 3-(1-naphthyl)alanine <400> SEQUENCE: 162 Arg
Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10
15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Xaa Cys Arg Lys Thr Cys
20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 163 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
PEGylated Toxin Peptide Analog <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1) <223>
OTHER INFORMATION: PEGylated <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (35)..(35) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 163 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 164 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 164 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 165 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norvaline <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 165 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 166 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 166 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Gln Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 167 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 167 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 168 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 168 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Glu
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 169 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 169 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 170 <211> LENGTH: 36
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <400> SEQUENCE: 170 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys Ala 35 <210> SEQ ID NO 171 <211> LENGTH: 36
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norvaline <400> SEQUENCE: 171 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys Ala 35 <210> SEQ ID NO 172 <211> LENGTH: 36
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norvaline <400> SEQUENCE: 172 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys Ala 35 <210> SEQ ID NO 173 <211> LENGTH: 36
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <400> SEQUENCE: 173 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Gln Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys Ala 35 <210> SEQ ID NO 174 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (16)..(16) <223> OTHER
INFORMATION: PEGylated <400> SEQUENCE: 174 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 175 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
toxin peptide analog (free acid) <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norleucine <400> SEQUENCE: 175 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 176 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
toxin peptide analog (free acid) <400> SEQUENCE: 176 Arg Ser
Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Lys 1 5 10 15
Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20
25 30 Gly Thr Cys 35 <210> SEQ ID NO 177 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
toxin peptide analog (free acid) <400> SEQUENCE: 177 Arg Ser
Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15
Cys Arg His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20
25 30 Gly Thr Cys 35 <210> SEQ ID NO 178 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
toxin peptide analog (free acid) <400> SEQUENCE: 178 Arg Ser
Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15
Cys Lys His Ser Ala Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20
25 30 Gly Thr Cys 35 <210> SEQ ID NO 179 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
toxin peptide analog (free acid) <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norleucine <400> SEQUENCE: 179 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 180 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
toxin peptide analog (free acid) <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (27)..(27) <223>
OTHER INFORMATION: 3-(1-naphthyl)alanine <400> SEQUENCE: 180
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Xaa Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 181 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination toxin peptide analog (free acid) <400> SEQUENCE:
181 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Lys Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 182
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Combination toxin peptide analog (free acid)
<400> SEQUENCE: 182 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Arg Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 183 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination toxin peptide analog
(free acid) <400> SEQUENCE: 183 Arg Ser Cys Ile Asp Thr Ile
Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met
Lys Tyr Arg Leu Ser Phe Cys Arg Glu Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 184 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination toxin peptide analog
(free acid) <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (27)..(27) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 184 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Xaa Cys Lys Arg Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 185 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
toxin peptide analog (free acid) <400> SEQUENCE: 185 Arg Ser
Cys Glu Asp Thr Ile Pro Lys Arg Arg Cys Thr Ala Ala Lys 1 5 10 15
Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Arg Thr Cys 20
25 30 Gly Thr Cys 35 <210> SEQ ID NO 186 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 186
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 187 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(27)..(27) <223> OTHER INFORMATION: 3-(1-naphthyl)alanine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 187 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Xaa Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 188 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide} <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 188 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 189 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 189
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Arg Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 190 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide} <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 190 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe
Cys Arg Glu Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
191 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (26)..(26) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 191 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Xaa Phe Cys Lys Arg Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 192 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 192
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Tyr Phe Cys Lys Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 193 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(26)..(26) <223> OTHER INFORMATION: 3-(2-naphthyl)alanine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 193 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Xaa Phe Cys Lys Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 194 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 194 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Glu Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 195 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 195
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Glu His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 196 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Norleucine <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 196 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe
Cys Arg Arg Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
197 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (26)..(26) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 197 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Xaa Phe Cys Lys Arg Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 198 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 198
Arg Ser Cys Glu Asp Thr Ile Pro Glu Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Arg His Ser Met Lys Tyr Arg Leu Ser Phe Cys Lys Arg Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 199 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 199 Arg Ser Cys Glu Asp Thr Ile Pro Lys Glu Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Arg His Ser Met Lys Tyr Arg Leu Ser Phe
Cys Lys Arg Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
200 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 200 Arg Ser Cys Glu Asp Thr Ile Pro
Glu Glu Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Arg His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Lys Arg Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 201 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 201 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Lys Arg Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 202 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 202
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Trp Cys Lys Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 203 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Norleucine <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 203 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe
Cys Lys Arg Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
204 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 204 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Glu His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 205 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 205 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 206 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Norleucine <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 206
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Lys 1 5
10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 207 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(27)..(27) <223> OTHER INFORMATION: 3-(2-naphthyl)alanine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 207 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Xaa Cys Lys Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 208 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 208 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Arg
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Lys Arg Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 209 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 209
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Phe Phe Cys Lys Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 210 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 210 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe
Cys Lys Ala Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
211 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 211 Arg Ser Cys Glu Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Arg His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Lys Arg Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 212 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 212 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Arg
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Lys Arg Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 213 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 213
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Lys Arg Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 214 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Norleucine <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 214 Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Ala Lys 1 5 10 15 Cys Glu His Ser Xaa Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
215 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 215 Arg Ser Cys Glu Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Glu His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 216 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 216 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 217 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Norleucine <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 217
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Lys 1 5
10 15 Cys Glu His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 218 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Norleucine <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 218 Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe
Cys Arg Arg Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
219 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 219 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Glu His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Arg Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 220 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 220 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Glu
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Arg Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 221 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Norleucine <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 221
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Lys 1 5
10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Arg Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 222 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Norleucine <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 222 Arg Ser Cys Ile Asp Thr Ile Pro Glu Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
223 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 223 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Ala Lys 1 5 10 15 Cys Glu His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Arg Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 224 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 224 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Arg Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 225 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Norleucine <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 225
Arg Ser Cys Ile Asp Thr Ile Pro Lys Glu Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 226 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(26)..(26) <223> OTHER INFORMATION: 3-(1-naphthyl)alanine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 226 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Xaa Phe Cys Lys Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 227 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (26)..(26) <223> OTHER
INFORMATION: 3-(1-naphthyl)alanine <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (35)..(35) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 227 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Xaa Phe Cys Lys Arg Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 228 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 228
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Tyr Cys Lys Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 229 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 229 Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Thr Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
230 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 230 Arg Ser Cys Glu Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Ser Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 231 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 231 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Lys Ala Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 232 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Norvaline <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 232
Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 233 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Aminobutyric acid
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 233 Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 234 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 234 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Gln Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 235 <211> LENGTH: 36
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension <400> SEQUENCE: 235 Arg Ser
Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15
Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20
25 30 Gly Thr Cys Ala 35 <210> SEQ ID NO 236 <211>
LENGTH: 36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Toxin
Peptide Analog with C-Terminal extension <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Norleucine <400> SEQUENCE: 236
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys Ala 35 <210> SEQ ID NO 237
<211> LENGTH: 36 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Toxin Peptide Analog with C-Terminal extension
<400> SEQUENCE: 237 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Asn Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Ala 35
<210> SEQ ID NO 238 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norvaline <400> SEQUENCE: 238 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Ala 35
<210> SEQ ID NO 239 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norvaline <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (36)..(36) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 239 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Ala 35
<210> SEQ ID NO 240 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension <400> SEQUENCE: 240 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Gln Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Ala 35
<210> SEQ ID NO 241 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension <400> SEQUENCE: 241 Arg Ser Cys Glu Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Gln Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Ala 35
<210> SEQ ID NO 242 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norvaline <400> SEQUENCE: 242 Arg Ser Cys Glu Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Ala 35
<210> SEQ ID NO 243 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <400> SEQUENCE: 243 Arg Ser Cys Glu Asp Thr Ile
Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa
Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys
Ala 35 <210> SEQ ID NO 244 <211> LENGTH: 36 <212>
TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Toxin Peptide Analog with
C-Terminal extension <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <400> SEQUENCE: 244 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys Glu 35 <210> SEQ ID NO 245 <211> LENGTH: 36
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norleucine <400> SEQUENCE: 245 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys Tyr 35 <210> SEQ ID NO 246 <211> LENGTH:
36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norleucine <400> SEQUENCE: 246 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys Val 35 <210> SEQ ID NO 247 <211> LENGTH:
36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norvaline <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (36)..(36) <223>
OTHER INFORMATION: Beta Ala <400> SEQUENCE: 247 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys Xaa 35 <210> SEQ ID NO 248 <211> LENGTH:
36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension {and C-Terminal Amide }
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norvaline
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (36)..(36) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 248 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Asp 35
<210> SEQ ID NO 249 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension {and C-Terminal Amide } <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norvaline <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (36)..(36) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 249 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys Glu 35 <210> SEQ ID NO 250 <211> LENGTH:
36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension {and C-Terminal Amide }
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norleucine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (36)..(36) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 250 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Asp 35
<210> SEQ ID NO 251 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension {and C-Terminal Amide } <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norvaline <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (36)..(36) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 251 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys Ser 35 <210> SEQ ID NO 252 <211> LENGTH:
36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension {and C-Terminal Amide }
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norvaline
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (36)..(36) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 252 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Thr 35
<210> SEQ ID NO 253 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension {and C-Terminal Amide } <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norvaline <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (36)..(36) <223>
OTHER INFORMATION: Aminoisobutyric acid <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (36)..(36)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 253
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys Xaa 35 <210> SEQ ID NO 254
<211> LENGTH: 36 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Toxin Peptide Analog with C-Terminal extension {and
C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norvaline <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (36)..(36) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 254 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys Ala 35 <210> SEQ ID NO 255 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 255 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 256 <400> SEQUENCE: 256
000 <210> SEQ ID NO 257 <211> LENGTH: 35 <212>
TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Position 21 Toxin Peptide
Analog <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Methinine oxide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 257 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 258 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 258 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Ser Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 259 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 259 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Thr Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 260 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norvaline
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 260 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 261 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 261 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Gln Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 262 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 262 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser His Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 263 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 263 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Tyr Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 264 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 264 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Asn Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 265 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 265 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Val Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 266 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 266 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Leu Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 267 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 267 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Trp Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 268 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Aminobutyric
acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 268 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 269 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION:
Cyclohexylalanine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 269 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 270 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Cyclohexylglycine <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (35)..(35) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 270 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 271 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: 4-hydroxyproline (or hydroxyproline) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 271 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
272 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Position 21 Toxin Peptide Analog <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 272 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Phe Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
273 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Position 21 Toxin Peptide Analog <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 273 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Ile Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
274 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Position 21 Toxin Peptide Analog <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 274 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Asp Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
275 <211> LENGTH: 83 <212> TYPE: PRT <213>
ORGANISM: Anemonia erythraea <220> FEATURE: <221>
NAME/KEY: SIGNAL <222> LOCATION: (1)..(83) <223> OTHER
INFORMATION: Putative signal sequence <400> SEQUENCE: 275 Met
Lys Gly Gln Met Ile Ile Cys Leu Val Leu Ile Ala Leu Cys Met 1 5 10
15 Ser Val Val Val Met Ala Gln Asn Leu Arg Ala Glu Glu Leu Glu Lys
20 25 30 Ala Asn Pro Lys Asp Glu Arg Val Arg Ser Phe Glu Arg Asn
Gln Lys 35 40 45 Arg Ala Cys Lys Asp Tyr Leu Pro Lys Ser Glu Cys
Thr Gln Phe Arg 50 55 60 Cys Arg Thr Ser Met Lys Tyr Lys Tyr Thr
Asn Cys Lys Lys Thr Cys 65 70 75 80 Gly Thr Cys <210> SEQ ID
NO 276 <211> LENGTH: 74 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Hmk peptide sequence <400> SEQUENCE: 276
Met Lys Ser Gln Met Ile Ala Ala Val Leu Leu Ile Ala Phe Cys Leu 1 5
10 15 Cys Val Val Val Thr Ala Arg Met Glu Leu Gln Asp Val Glu Asp
Met 20 25 30 Glu Asn Gly Phe Gln Lys Arg Arg Thr Cys Lys Asp Leu
Ile Pro Val 35 40 45 Ser Glu Cys Thr Asp Ile Arg Cys Arg Thr Ser
Met Lys Tyr Arg Leu 50 55 60 Asn Leu Cys Arg Lys Thr Cys Gly Ser
Cys 65 70 <210> SEQ ID NO 277 <211> LENGTH: 738
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(738) <400> SEQUENCE: 277 atg gaa tgg agc tgg
gtc ttt ctc ttc ttc ctg tca gta acg act ggt 48 Met Glu Trp Ser Trp
Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 gtc cac tcc
gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa 96 Val His Ser
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30 ctc
ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 144 Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40
45 acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac
192 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
50 55 60 gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg
gac ggc 240 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly 65 70 75 80 gtg gag gtg cat aat gcc aag aca aag ccg cgg gag
gag cag tac aac 288 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn 85 90 95 agc acg tac cgt gtg gtc agc gtc ctc acc
gtc ctg cac cag gac tgg 336 Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp 100 105 110 ctg aat ggc aag gag tac aag tgc
aag gtc tcc aac aaa gcc ctc cca 384 Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro 115 120 125 gcc ccc atc gag aaa acc
atc tcc aaa gcc aaa ggg cag ccc cga gaa 432 Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140 cca cag gtg tac
acc ctg ccc cca tcc cgg gat gag ctg acc aag aac 480 Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 145 150 155 160 cag
gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 528 Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170
175 gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc
576 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
180 185 190 acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac
agc aag 624 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys 195 200 205 ctc acc gtg gac aag agc agg tgg cag cag ggg aac
gtc ttc tca tgc 672 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys 210 215 220 tcc gtg atg cat gag gct ctg cac aac cac
tac acg cag aag agc ctc 720 Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu 225 230 235 240 tcc ctg tct ccg ggt aaa 738
Ser Leu Ser Pro Gly Lys 245 <210> SEQ ID NO 278 <211>
LENGTH: 246 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 278 Met Glu Trp Ser Trp Val Phe Leu
Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30 Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45 Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 65
70 75 80 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn 85 90 95 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp 100 105 110 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro 115 120 125 Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140 Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 145 150 155 160 Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175 Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185
190 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
195 200 205 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys 210 215 220 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu 225 230 235 240 Ser Leu Ser Pro Gly Lys 245
<210> SEQ ID NO 279 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Linker sequence <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(2)
<223> OTHER INFORMATION: Xaa in positions 1-2 are each
independently any amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: Xaa in positions 4-5 are each
independently any amino acid residue <400> SEQUENCE: 279 Xaa
Xaa Asn Xaa Xaa Gly 1 5 <210> SEQ ID NO 280 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Peptidyl linker <400> SEQUENCE: 280 Gly Gly Gly Gly Gly Gly
Gly Gly 1 5 <210> SEQ ID NO 281 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptidyl linker
<400> SEQUENCE: 281 Gly Gly Gly Gly Gly 1 5 <210> SEQ
ID NO 282 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Peptidyl linker <400> SEQUENCE: 282 Gly
Gly Gly Gly Gly Gly Gly 1 5 <210> SEQ ID NO 283 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Peptidyl linker <400> SEQUENCE: 283 Gly Gly Gly Gly Ser 1 5
<210> SEQ ID NO 284 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptidyl linker <400>
SEQUENCE: 284 Gly Gly Gly Gly Gly Lys 1 5 <210> SEQ ID NO 285
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Peptidyl linker <400> SEQUENCE: 285 Gly Gly Gly
Gly Gly Lys Arg 1 5 <210> SEQ ID NO 286 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptidyl linker
<400> SEQUENCE: 286 Gly Gly Gly Lys Gly Gly Gly Gly 1 5
<210> SEQ ID NO 287 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptidyl linker <400>
SEQUENCE: 287 Gly Gly Gly Asn Gly Ser Gly Gly 1 5 <210> SEQ
ID NO 288 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Peptidyl linker <400> SEQUENCE: 288 Gly
Gly Gly Cys Gly Gly Gly Gly 1 5 <210> SEQ ID NO 289
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Peptidyl linker <400> SEQUENCE: 289 Gly Pro Asn
Gly Gly 1 5 <210> SEQ ID NO 290 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptidyl linker
<400> SEQUENCE: 290 Gly Gly Gly Lys Gly Gly Gly Gly 1 5
<210> SEQ ID NO 291 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptidyl linker <400>
SEQUENCE: 291 Gly Gly Gly Gly Ser 1 5 <210> SEQ ID NO 292
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Peptidyl linker <400> SEQUENCE: 292 Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> SEQ ID NO 293
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Peptidyl linker <400> SEQUENCE: 293 Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly
Gly Gly Ser Gly Gly Gly Gly Ser 20 25 <210> SEQ ID NO 294
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Peptide linker <400> SEQUENCE: 294 Gly Gly Glu
Gly Gly Gly 1 5 <210> SEQ ID NO 295 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptide linker
<400> SEQUENCE: 295 Gly Gly Glu Glu Glu Gly Gly Gly 1 5
<210> SEQ ID NO 296 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptide linker <400> SEQUENCE:
296 Gly Glu Glu Glu Gly 1 5 <210> SEQ ID NO 297 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Peptide linker <400> SEQUENCE: 297 Gly Glu Glu Glu 1
<210> SEQ ID NO 298 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptide linker <400> SEQUENCE:
298 Gly Gly Asp Gly Gly Gly 1 5 <210> SEQ ID NO 299
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Peptide linker <400> SEQUENCE: 299 Gly Gly Asp
Asp Asp Gly Gly 1 5 <210> SEQ ID NO 300 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptide linker
<400> SEQUENCE: 300 Gly Asp Asp Asp Gly 1 5 <210> SEQ
ID NO 301 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Peptide linker <400> SEQUENCE: 301 Gly Asp
Asp Asp 1 <210> SEQ ID NO 302 <211> LENGTH: 21
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptide linker
<400> SEQUENCE: 302 Gly Gly Gly Gly Ser Asp Asp Ser Asp Glu
Gly Ser Asp Gly Glu Asp 1 5 10 15 Gly Gly Gly Gly Ser 20
<210> SEQ ID NO 303 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptide linker <400> SEQUENCE:
303 Trp Glu Trp Glu Trp 1 5 <210> SEQ ID NO 304 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Peptide linker <400> SEQUENCE: 304 Phe Glu Phe Glu Phe 1 5
<210> SEQ ID NO 305 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptide linker <400> SEQUENCE:
305 Glu Glu Glu Trp Trp Trp 1 5 <210> SEQ ID NO 306
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Peptide linker <400> SEQUENCE: 306 Glu Glu Glu
Phe Phe Phe 1 5 <210> SEQ ID NO 307 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptide linker
<400> SEQUENCE: 307 Trp Trp Glu Glu Glu Trp Trp 1 5
<210> SEQ ID NO 308 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptide linker <400> SEQUENCE:
308 Phe Phe Glu Glu Glu Phe Phe 1 5 <210> SEQ ID NO 309
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Peptide linker <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(2) <223>
OTHER INFORMATION: Xaa in positions 1-2 are each independently any
amino acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: Xaa in positions 4-5 are each independently any amino
acid residue <400> SEQUENCE: 309 Xaa Xaa Tyr Xaa Xaa Gly 1 5
<210> SEQ ID NO 310 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptide linker <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(2)
<223> OTHER INFORMATION: Xaa in positions 1-2 are each
independently any amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: Xaa in positions 4-5 are each
independently any amino acid residue <400> SEQUENCE: 310 Xaa
Xaa Ser Xaa Xaa Gly 1 5 <210> SEQ ID NO 311 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Peptide linker <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: Xaa in positions 1-2 are each independently any amino
acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: Xaa in positions 4-5 are each independently any amino
acid residue <400> SEQUENCE: 311 Xaa Xaa Thr Xaa Xaa Gly 1 5
<210> SEQ ID NO 312 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Flexible peptide linker <400>
SEQUENCE: 312 Gly Ser Gly Ser Ala Thr Gly Gly Ser Gly Ser Thr Ala
Ser Ser Gly 1 5 10 15 Ser Gly Ser Ala Thr His 20 <210> SEQ ID
NO 313 <211> LENGTH: 22 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Flexible peptide linker <400> SEQUENCE:
313 His Gly Ser Gly Ser Ala Thr Gly Gly Ser Gly Ser Thr Ala Ser Ser
1 5 10 15 Gly Ser Gly Ser Ala Thr 20 <210> SEQ ID NO 314
<211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Flexible peptide linker <400> SEQUENCE: 314 Ala
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10
15 Ala Gly Gly <210> SEQ ID NO 315 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Reference
peptide <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION:
Nalpha branched PEGylated <400> SEQUENCE: 315 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 316 <211> LENGTH: 36
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: 20kDa PEG-[Lys16]Shk-Ala <400> SEQUENCE: 316 Arg
Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10
15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys
20 25 30 Gly Thr Cys Ala 35 <210> SEQ ID NO 317 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (22)..(22) <223> OTHER INFORMATION:
Diaminopropionic acid <400> SEQUENCE: 317 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Xaa Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 318 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: modifed
VH21signal peptide <400> SEQUENCE: 318 Met Glu Trp Ser Trp
Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser
<210> SEQ ID NO 319 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 319 catgaattcc ccaccatgga atggagctgg 30 <210> SEQ
ID NO 320 <211> LENGTH: 38 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 320
cacggtgggc actcgacttt gcgctcggag tggacacc 38 <210> SEQ ID NO
321 <211> LENGTH: 132 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: ShK[2-35] with N-terminal linker extension
<400> SEQUENCE: 321 ggaggaggag gatccggagg aggaggaagc
agctgcatcg acaccatccc caagagccgc 60 tgcaccgcct tccagtgcaa
gcacagcatg aagtaccgcc tgagcttctg ccgcaagacc 120 tgcggcacct gc 132
<210> SEQ ID NO 322 <211> LENGTH: 44 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK[2-35] with N-terminal linker
extension <400> SEQUENCE: 322 Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Ser Cys Ile Asp Thr Ile 1 5 10 15 Pro Lys Ser Arg Cys Thr
Ala Phe Gln Cys Lys His Ser Met Lys Tyr 20 25 30 Arg Leu Ser Phe
Cys Arg Lys Thr Cys Gly Thr Cys 35 40 <210> SEQ ID NO 323
<211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 323 gtccactccg
agcgcaaagt cgagtgccca ccgtgcc 37 <210> SEQ ID NO 324
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 324 tcctcctcct
ttacccggag acagggagag 30 <210> SEQ ID NO 325 <211>
LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 325 gctgcaccgc cttcaagtgc aagcacagc
29 <210> SEQ ID NO 326 <211> LENGTH: 29 <212>
TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 326 gctgtgcttg cacttgaagg cggtgcagc 29 <210> SEQ ID
NO 327 <211> LENGTH: 132 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: ShK[2-35, K16] with N-terminal linker extension
<400> SEQUENCE: 327 ggaggaggag gatccggagg aggaggaagc
agctgcatcg acaccatccc caagagccgc 60 tgcaccgcct tcaagtgcaa
gcacagcatg aagtaccgcc tgagcttctg ccgcaagacc 120 tgcggcacct gc 132
<210> SEQ ID NO 328 <211> LENGTH: 44 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK[2-35, Q16K] with N-terminal
linker extension <400> SEQUENCE: 328 Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Ser Cys Ile Asp Thr Ile 1 5 10 15 Pro Lys Ser Arg
Cys Thr Ala Phe Lys Cys Lys His Ser Met Lys Tyr 20 25 30 Arg Leu
Ser Phe Cys Arg Lys Thr Cys Gly Thr Cys 35 40 <210> SEQ ID NO
329 <211> LENGTH: 28 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 329
ccgggtaaag gaggaggagg atccggag 28 <210> SEQ ID NO 330
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 330 catgcggccg
ctcattagca ggtg 24 <210> SEQ ID NO 331 <211> LENGTH:
648 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: Coding sequence of fragment of immunoglobulin Fc
domain of human IgG2 <400> SEQUENCE: 331 gcaccacctg
tggcaggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 60
atgatctccc ggacccctga ggtcacgtgc gtggtggtgg acgtgagcca cgaagacccc
120 gaggtccagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa
gacaaagcca 180 cgggaggagc agttcaacag cacgttccgt gtggtcagcg
tcctcaccgt tgtgcaccag 240 gactggctga acggcaagga gtacaagtgc
aaggtctcca acaaaggcct cccagccccc 300 atcgagaaaa ccatctccaa
aaccaaaggg cagccccgag aaccacaggt gtacaccctg 360 cccccatccc
gggaggagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 420
ttctacccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac
480 aagaccacac ctcccatgct ggactccgac ggctccttct tcctctacag
caagctcacc 540 gtggacaaga gcaggtggca gcaggggaac gtcttctcat
gctccgtgat gcatgaggct 600 ctgcacaacc actacacgca gaagagcctc
tccctgtctc cgggtaaa 648 <210> SEQ ID NO 332 <211>
LENGTH: 216 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<223> OTHER INFORMATION: Fragment of immunoglobulin Fc domain
of human IgG2 <400> SEQUENCE: 332 Ala Pro Pro Val Ala Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val
Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 35 40 45 Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55
60 Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln
65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys Gly Gln Pro 100 105 110 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr 115 120 125 Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140 Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 145 150 155 160 Lys Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165 170 175 Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 180 185
190 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
195 200 205 Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> SEQ
ID NO 333 <211> LENGTH: 511 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: IgG2-HC-L10-ShK[1-35, Q16K] fusion polypeptide
<400> SEQUENCE: 333 Met Asp Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val
Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro Gly Ala
Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr
Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50 55 60 Gln Gly
Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr 65 70 75 80
Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 85
90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp
Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Gly Ser Tyr
Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205
Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys 210
215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val
Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala
Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305 310 315 320 Arg
Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly 325 330
335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile
340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro
Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455
460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Ser Cys Ile
465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys Cys
Lys His Ser 485 490 495 Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys Gly Thr Cys 500 505 510 <210> SEQ ID NO 334 <211>
LENGTH: 290 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
VH21SP-IgG2-Fc-L10-ShK(1-35, Q16K) Fusion polypeptide <400>
SEQUENCE: 334 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val
Thr Thr Gly 1 5 10 15 Val His Ser Glu Arg Lys Val Glu Cys Pro Pro
Cys Pro Ala Pro Pro 20 25 30 Val Ala Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser His Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 100 105
110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala
115 120 125 Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 195 200 205 Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 210 215 220 Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230
235 240 Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Arg 245 250 255 Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala
Phe Gln Cys 260 265 270 Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys
Arg Lys Thr Cys Gly 275 280 285 Thr Cys 290 <210> SEQ ID NO
335 <211> LENGTH: 30 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 335
cataagcttc ccaccatgga atggagctgg 30 <210> SEQ ID NO 336
<211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 336 catggatcct
catttacccg gagacaggga g 31 <210> SEQ ID NO 337 <211>
LENGTH: 245 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<223> OTHER INFORMATION: Immunoglobulin Fc domain of human
IgG2 <400> SEQUENCE: 337 Met Glu Trp Ser Trp Val Phe Leu Phe
Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Glu Arg Lys Val
Glu Cys Pro Pro Cys Pro Ala Pro Pro 20 25 30 Val Ala Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser
His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70
75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser 85 90 95 Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln
Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ala 115 120 125 Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 195
200 205 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser 225 230 235 240 Leu Ser Pro Gly Lys 245 <210> SEQ
ID NO 338 <211> LENGTH: 236 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: aKLH 120.6 kappa LC <400> SEQUENCE: 338
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5
10 15 Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser 35 40 45 Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln
Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Arg Leu Ile Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110 His Asn Ser
Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135
140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> SEQ ID NO 339
<211> LENGTH: 467 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: aKLH 120.6 IgG2 HC <400> SEQUENCE: 339 Met Asp
Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15
Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20
25 30 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser
Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln
Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro
Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg
Val Thr Met Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr
Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150
155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr Tyr Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275
280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Phe 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Val His
Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395
400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
405 410 415 Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 <210> SEQ
ID NO 340 <211> LENGTH: 290 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: IgG2 Fc-L10-ShK(1-35) <400> SEQUENCE: 340
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5
10 15 Val His Ser Glu Arg Lys Val Glu Cys Pro Pro Cys Pro Ala Pro
Pro 20 25 30 Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 50 55 60 Ser His Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Phe Arg Val Val
Ser Val Leu Thr Val Val His Gln Asp Trp Leu 100 105 110 Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala 115 120 125 Pro
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro 130 135
140 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Met Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser
Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 245 250 255
Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln Cys 260
265 270 Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys
Gly 275 280 285 Thr Cys 290 <210> SEQ ID NO 341 <211>
LENGTH: 511 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: aKLH
120.6 IgG2-ShK fusion <400> SEQUENCE: 341 Met Asp Met Arg Val
Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly
Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40
45 Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly
50 55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly
Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met
Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser
Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg
Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170
175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln
Thr Tyr Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys
Val Asp Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys
Pro Pro Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295
300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
305 310 315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly
Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415
Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420
425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Arg Ser Cys Ile 465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys
Thr Ala Phe Gln Cys Lys His Ser 485 490 495 Met Lys Tyr Arg Leu Ser
Phe Cys Arg Lys Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID
NO 342 <211> LENGTH: 511 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: aKLH 120.6 IgG2-ShK[1-35, Q16K] fusion
<400> SEQUENCE: 342 Met Asp Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val
Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro Gly Ala
Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr
Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50 55 60 Gln Gly
Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr 65 70 75 80
Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 85
90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp
Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Gly Ser Tyr
Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205
Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys 210
215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val
Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala
Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305 310 315 320 Arg
Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly 325 330
335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile
340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro
Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455
460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Ser Cys Ile
465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys Cys
Lys His Ser 485 490 495 Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys Gly Thr Cys 500 505 510 <210> SEQ ID NO 343 <211>
LENGTH: 471 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: aKLH
120.6 IgG1 HC <400> SEQUENCE: 343 Met Asp Met Arg Val Pro Ala
Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg
Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr
Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50 55
60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr
65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg
Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg
Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185
190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310
315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435
440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> SEQ
ID NO 344 <211> LENGTH: 510 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: aKLH 120.6 IgG1-loop-ShK <400> SEQUENCE:
344 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly
Ala Glu 20 25 30 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys
Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His Met His Trp
Val Arg Gln Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Trp
Ile Asn Pro Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe
Gln Gly Arg Val Thr Met Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr
Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala
Val Tyr Tyr Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125
Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130
135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250
255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Gly Gly 370 375
380 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys
385 390 395 400 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg
Lys Thr Cys 405 410 415 Gly Thr Cys Gly Gly Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val 420 425 430 Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly 435 440 445 Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp 450 455 460 Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 465 470 475 480 Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 485 490 495
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 510
<210> SEQ ID NO 345 <211> LENGTH: 289 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: VH21SP-IgG2-Fc-L10-ShK(1-35, Q16K)
Fusion polypeptide <400> SEQUENCE: 345 Met Glu Trp Ser Trp
Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser
Glu Arg Lys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro 20 25 30 Val
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40
45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
50 55 60 Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser 85 90 95 Thr Phe Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ala 115 120 125 Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170
175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
180 185 190 Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Pro Gly Lys Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Ser 245 250 255 Cys Ile Asp Thr Ile
Pro Lys Ser Arg Cys Thr Ala Phe Gln Cys Lys 260 265 270 His Ser Met
Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys Gly Thr 275 280 285 Cys
<210> SEQ ID NO 346 <211> LENGTH: 289 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: VH21SP-IgG2-Fc-L10-ShK(1-35, Q16K)
Fusion polypeptide <400> SEQUENCE: 346 Met Glu Trp Ser Trp
Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser
Glu Arg Lys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro 20 25 30 Val
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40
45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
50 55 60 Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser 85 90 95 Thr Phe Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ala 115 120 125 Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170
175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
180 185 190 Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Pro Gly Lys Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Ser 245 250 255 Cys Ile Asp Thr Ile
Pro Lys Ser Arg Cys Thr Ala Phe Lys Cys Lys 260 265 270 His Ser Met
Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys Gly Thr 275 280 285 Cys
<210> SEQ ID NO 347 <211> LENGTH: 289 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Fusion polypeptide <400>
SEQUENCE: 347 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val
Thr Thr Gly 1 5 10 15 Val His Ser Glu Arg Lys Val Glu Cys Pro Pro
Cys Pro Ala Pro Pro 20 25 30 Val Ala Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser His Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 100 105
110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala
115 120 125 Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 195 200 205 Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 210 215 220 Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230
235 240 Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Ser 245 250 255 Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe
Lys Cys Lys 260 265 270 His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg
Lys Thr Cys Gly Thr 275 280 285 Cys <210> SEQ ID NO 348
<211> LENGTH: 290 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: IgG2 Fc-L10-ShK(1-35, Q16K) fusion protein <400>
SEQUENCE: 348 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val
Thr Thr Gly 1 5 10 15 Val His Ser Glu Arg Lys Val Glu Cys Pro Pro
Cys Pro Ala Pro Pro 20 25 30 Val Ala Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser His Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 100 105
110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala
115 120 125 Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 195 200 205 Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 210 215 220 Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230
235 240 Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Arg 245 250 255 Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala
Phe Lys Cys 260 265 270 Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys
Arg Lys Thr Cys Gly 275 280 285 Thr Cys 290 <210> SEQ ID NO
349 <211> LENGTH: 24 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Random primer with an extension adapter
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (18)..(23) <223> OTHER INFORMATION: Any
deoxriboneucleotide <400> SEQUENCE: 349 ggccggatag gcctccannn
nnnt 24 <210> SEQ ID NO 350 <211> LENGTH: 45
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Forward primer
<400> SEQUENCE: 350 gtggttgaga ggtgccagat gtgacattgt
gatgactcag tctcc 45 <210> SEQ ID NO 351 <211> LENGTH:
41 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Reverse primer
<400> SEQUENCE: 351 aaccgtttaa acgcggccgc tcaacactct
cccctgttga a 41 <210> SEQ ID NO 352 <211> LENGTH: 43
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Optimized Kozak
seqence <400> SEQUENCE: 352 aagctcgagg tcgactagac caccatggac
atgagggtcc ccg 43 <210> SEQ ID NO 353 <211> LENGTH: 45
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 353 aagctcgagg tcgactagac caccatggac
atgagggtgc ccgct 45 <210> SEQ ID NO 354 <211> LENGTH:
25 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 354 tcatctggat gtcacatctg gcacc 25
<210> SEQ ID NO 355 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 355 ggtgccagat gtgacatcca gatga 25 <210> SEQ ID NO
356 <211> LENGTH: 1398 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: aKLH 120.6-VK1SP-IgG2 Heavy Chain coding
sequence <400> SEQUENCE: 356 atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcc 60 agatgtcagg tgcagctggt
gcagtctggg gctgaggtga agaagcctgg ggcctcagtg 120 aaggtctcct
gcaaggcttc tggatacacc ttcaccggct accacatgca ctgggtgcga 180
caggcccctg gacaagggct tgagtggatg ggatggatca accctaacag tggtggcaca
240 aactatgcac agaagtttca gggcagggtc accatgacca gggacacgtc
catcagcaca 300 gcctacatgg agctgagcag gctgagatct gacgacacgg
ccgtgtatta ctgtgcgaga 360 gatcgtggga gctactactg gttcgacccc
tggggccagg gaaccctggt caccgtctcc 420 tcagcctcca ccaagggccc
atcggtcttc cccctggcgc cctgctccag gagcacctcc 480 gagagcacag
cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 540
tcgtggaact caggcgctct gaccagcggc gtgcacacct tcccagctgt cctacagtcc
600 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcaactt
cggcacccag 660 acctacacct gcaacgtaga tcacaagccc agcaacacca
aggtggacaa gacagttgag 720 cgcaaatgtt gtgtcgagtg cccaccgtgc
ccagcaccac ctgtggcagg accgtcagtc 780 ttcctcttcc ccccaaaacc
caaggacacc ctcatgatct cccggacccc tgaggtcacg 840 tgcgtggtgg
tggacgtgag ccacgaagac cccgaggtcc agttcaactg gtacgtggac 900
ggcgtggagg tgcataatgc caagacaaag ccacgggagg agcagttcaa cagcacgttc
960 cgtgtggtca gcgtcctcac cgttgtgcac caggactggc tgaacggcaa
ggagtacaag 1020 tgcaaggtct ccaacaaagg cctcccagcc cccatcgaga
aaaccatctc caaaaccaaa 1080 gggcagcccc gagaaccaca ggtgtacacc
ctgcccccat cccgggagga gatgaccaag 1140 aaccaggtca gcctgacctg
cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 1200 tgggagagca
atgggcagcc ggagaacaac tacaagacca cacctcccat gctggactcc 1260
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg
1320 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac
gcagaagagc 1380 ctctccctgt ctccgggt 1398 <210> SEQ ID NO 357
<211> LENGTH: 466 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: aKLH 120.6-VK1SP-IgG2 Heavy Chain <400>
SEQUENCE: 357 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu
Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val
Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro Gly Ala Ser Val Lys
Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His
Met His Trp Val Arg Gln Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp
Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala
Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 85 90 95 Ser
Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105
110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe
115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr
Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys 210 215 220 Asn
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu 225 230
235 240 Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
Ala 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Phe 305 310 315 320 Arg Val Val Ser
Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 340 345 350
Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val 355
360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro 405 410 415 Met Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly
465 <210> SEQ ID NO 358 <211> LENGTH: 34 <212>
TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 358 ggatcctcct cctccacccg gagacaggga gagg 34 <210>
SEQ ID NO 359 <211> LENGTH: 34 <212> TYPE: DNA
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 359 tccctgtctc cgggtggagg aggaggatcc ggag 34 <210>
SEQ ID NO 360 <211> LENGTH: 39 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Gly-Gly-ShK-Gly-Gly sequence
<400> SEQUENCE: 360 Gly Gly Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala 1 5 10 15 Phe Gln Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys 20 25 30 Thr Cys Gly Thr Cys Gly
Gly 35 <210> SEQ ID NO 361 <211> LENGTH: 127
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Forward primer
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (6)..(11) <223> OTHER INFORMATION: HINDIII Site
<400> SEQUENCE: 361 tgcagaagct tctagaccac catggaatgg
agctgggtct ttctcttctt cctgtcagta 60 acgactggtg tccactcccg
cagctgcatc gacaccatcc ccaagagccg ctgcaccgcc 120 ttccagt 127
<210> SEQ ID NO 362 <211> LENGTH: 115 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Reverse primer <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (5)..(10)
<223> OTHER INFORMATION: BAMHI Site <400> SEQUENCE: 362
ctccggatcc tcctcctccg caggtgccgc aggtcttgcg gcagaagctc aggcggtact
60 tcatgctgtg cttgcactgg aaggcggtgc agcggctctt ggggatggtg tcgat 115
<210> SEQ ID NO 363 <211> LENGTH: 38 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Forward primer <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(9)
<223> OTHER INFORMATION: BAMHI Site <400> SEQUENCE: 363
gtaggatccg gaggaggagg aagcgacaaa actcacac 38 <210> SEQ ID NO
364 <211> LENGTH: 35 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Reverse primer <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(11) <223>
OTHER INFORMATION: NOTI Site <400> SEQUENCE: 364 cgagcggccg
cttactattt acccggagac aggga 35 <210> SEQ ID NO 365
<211> LENGTH: 879 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: VH21 SP-ShK(1-35)-L10-IgG1 Fc coding sequence
<400> SEQUENCE: 365 atggaatgga gctgggtctt tctcttcttc
ctgtcagtaa cgactggtgt ccactcccgc 60 agctgcatcg acaccatccc
caagagccgc tgcaccgcct tccagtgcaa gcacagcatg 120 aagtaccgcc
tgagcttctg ccgcaagacc tgcggcacct gcggaggagg aggatccgga 180
ggaggaggaa gcgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg
240 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat
ctcccggacc 300 cctgaggtca catgcgtggt ggtggacgtg agccacgaag
accctgaggt caagttcaac 360 tggtacgtgg acggcgtgga ggtgcataat
gccaagacaa agccgcggga ggagcagtac 420 aacagcacgt accgtgtggt
cagcgtcctc accgtcctgc accaggactg gctgaatggc 480 aaggagtaca
agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 540
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat
600 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta
tcccagcgac 660 atcgccgtgg agtgggagag caatgggcag ccggagaaca
actacaagac cacgcctccc 720 gtgctggact ccgacggctc cttcttcctc
tacagcaagc tcaccgtgga caagagcagg 780 tggcagcagg ggaacgtctt
ctcatgctcc gtgatgcatg aggctctgca caaccactac 840 acgcagaaga
gcctctccct gtctccgggt aaatagtaa 879 <210> SEQ ID NO 366
<211> LENGTH: 291 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: VH21 SP-ShK(1-35)-L10-IgG1 Fc <400> SEQUENCE:
366 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15 Val His Ser Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg
Cys Thr 20 25 30 Ala Phe Gln Cys Lys His Ser Met Lys Tyr Arg Leu
Ser Phe Cys Arg 35 40 45 Lys Thr Cys Gly Thr Cys Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 50 55 60 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 65 70 75 80 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 85 90 95 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 100 105 110 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 115 120 125
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 130
135 140 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 145 150 155 160 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 165 170 175 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 180 185 190 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 195 200 205 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 210 215 220 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 225 230 235 240 Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 245 250
255 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
260 265 270 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 275 280 285 Pro Gly Lys 290 <210> SEQ ID NO 367
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 367 cattctagac
caccatggaa tgg 23 <210> SEQ ID NO 368 <211> LENGTH: 29
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 368 cagctgcacc tggcttcctc ctcctccgg 29
<210> SEQ ID NO 369 <211> LENGTH: 192 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: VH21 SP-ShK(1-35, Q16K)-L10 coding
sequence <400> SEQUENCE: 369 atggaatgga gctgggtctt tctcttcttc
ctgtcagtaa cgactggtgt ccactcccgc 60 agctgcatcg acaccatccc
caagagccgc tgcaccgcct tcaagtgcaa gcacagcatg 120 aagtaccgcc
tgagcttctg ccgcaagacc tgcggcacct gcggaggagg aggatccgga 180
ggaggaggaa gc 192 <210> SEQ ID NO 370 <211> LENGTH: 64
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: VH21
SP-ShK(1-35, Q16K)-L10 <400> SEQUENCE: 370 Met Glu Trp Ser
Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His
Ser Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr 20 25 30
Ala Phe Lys Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg 35
40 45 Lys Thr Cys Gly Thr Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 50 55 60 <210> SEQ ID NO 371 <211> LENGTH: 30
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 371 ggaggaggaa gccaggtgca gctggtgcag 30
<210> SEQ ID NO 372 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 372 catgcggccg ctcatttacc c 21 <210> SEQ ID NO 373
<211> LENGTH: 1338 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: aKLH 120.6-HC coding sequence <400>
SEQUENCE: 373 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc
ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggata caccttcacc
ggctaccaca tgcactgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggatgg atcaacccta acagtggtgg cacaaactat 180 gcacagaagt
ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240
atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagagatcgt
300 gggagctact actggttcga cccctggggc cagggaaccc tggtcaccgt
ctcctcagcc 360 tccaccaagg gcccatcggt cttccccctg gcgccctgct
ccaggagcac ctccgagagc 420 acagcggccc tgggctgcct ggtcaaggac
tacttccccg aaccggtgac ggtgtcgtgg 480 aactcaggcg ctctgaccag
cggcgtgcac accttcccag ctgtcctaca gtcctcagga 540 ctctactccc
tcagcagcgt ggtgaccgtg ccctccagca acttcggcac ccagacctac 600
acctgcaacg tagatcacaa gcccagcaac accaaggtgg acaagacagt tgagcgcaaa
660 tgttgtgtcg agtgcccacc gtgcccagca ccacctgtgg caggaccgtc
agtcttcctc 720 ttccccccaa aacccaagga caccctcatg atctcccgga
cccctgaggt cacgtgcgtg 780 gtggtggacg tgagccacga agaccccgag
gtccagttca actggtacgt ggacggcgtg 840 gaggtgcata atgccaagac
aaagccacgg gaggagcagt tcaacagcac gttccgtgtg 900 gtcagcgtcc
tcaccgttgt gcaccaggac tggctgaacg gcaaggagta caagtgcaag 960
gtctccaaca aaggcctccc agcccccatc gagaaaacca tctccaaaac caaagggcag
1020 ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac
caagaaccag 1080 gtcagcctga cctgcctggt caaaggcttc taccccagcg
acatcgccgt ggagtgggag 1140 agcaatgggc agccggagaa caactacaag
accacacctc ccatgctgga ctccgacggc 1200 tccttcttcc tctacagcaa
gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1260 ttctcatgct
ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320
ctgtctccgg gtaaatga 1338 <210> SEQ ID NO 374 <211>
LENGTH: 445 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: aKLH
120.6-HC polypeptide sequence <400> SEQUENCE: 374 Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25
30 His Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln
Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile
Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Ser Tyr Tyr
Trp Phe Asp Pro Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190 Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val
Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Thr Val Glu
Arg Lys Cys Cys Val Glu 210 215 220 Cys Pro Pro Cys Pro Ala Pro Pro
Val Ala Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln 260 265 270 Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280
285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
290 295 300 Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys 325 330 335 Thr Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405
410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445 <210> SEQ ID NO 375 <211> LENGTH: 29
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 375 catctggatg tcgcttcctc ctcctccgg 29
<210> SEQ ID NO 376 <400> SEQUENCE: 376 000 <210>
SEQ ID NO 377 <400> SEQUENCE: 377 000 <210> SEQ ID NO
378 <211> LENGTH: 32 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 378
ggaggaggaa gcgacatcca gatgacccag tc 32 <210> SEQ ID NO 379
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 379 catctcgagc
ggccgctcaa c 21 <210> SEQ ID NO 380 <211> LENGTH: 837
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: VH21
SP-ShK[1-35, Q16K]-L10-aKLH120.6 Light Chain coding sequence
<400> SEQUENCE: 380 atggaatgga gctgggtctt tctcttcttc
ctgtcagtaa cgactggtgt ccactcccgc 60 agctgcatcg acaccatccc
caagagccgc tgcaccgcct tcaagtgcaa gcacagcatg 120 aagtaccgcc
tgagcttctg ccgcaagacc tgcggcacct gcggaggagg aggatccgga 180
ggaggaggaa gcgacatcca gatgacccag tctccatcct ccctgtctgc atctgtagga
240 gacagagtca ccatcacttg ccgggcaagt cagggcatta gaaatgattt
aggctggtat 300 cagcagaaac cagggaaagc ccctaaacgc ctgatctatg
ctgcatccag tttgcaaagt 360 ggggtcccat caaggttcag cggcagtgga
tctgggacag aattcactct cacaatcagc 420 agcctgcagc ctgaagattt
tgcaacttat tactgtctac agcataatag ttacccgctc 480 actttcggcg
gagggaccaa ggtggagatc aaacgaactg tggctgcacc atctgtcttc 540
atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg
600 aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc
cctccaatcg 660 ggtaactccc aggagagtgt cacagagcag gacagcaagg
acagcaccta cagcctcagc 720 agcaccctga cgctgagcaa agcagactac
gagaaacaca aagtctacgc ctgcgaagtc 780 acccatcagg gcctgagctc
gcccgtcaca aagagcttca acaggggaga gtgttga 837 <210> SEQ ID NO
381 <211> LENGTH: 278 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: VH21 SP-ShK[1-35, Q16K]-L10-aKLH120.6 Light
Chain <400> SEQUENCE: 381 Met Glu Trp Ser Trp Val Phe Leu Phe
Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr 20 25 30 Ala Phe Lys Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg 35 40 45 Lys Thr Cys
Gly Thr Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 50 55 60 Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 65 70
75 80 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn
Asp 85 90 95 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Arg Leu Ile 100 105 110 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 115 120 125 Ser Gly Ser Gly Thr Glu Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 130 135 140 Glu Asp Phe Ala Thr Tyr Tyr
Cys Leu Gln His Asn Ser Tyr Pro Leu 145 150 155 160 Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 165 170 175 Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 180 185 190
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 195
200 205 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln 210 215 220 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser 225 230 235 240 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 245 250 255 Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 260 265 270 Phe Asn Arg Gly Glu Cys
275 <210> SEQ ID NO 382 <400> SEQUENCE: 382 000
<210> SEQ ID NO 383 <211> LENGTH: 1038 <212>
TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Coding sequence - IgG2
fragment <400> SEQUENCE: 383 atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtcagg tgcagctggt
ggagtctggg ggaggcgtgg tccagcctgg gaggtccctg 120 agactctcct
gtgcagcgtc tggattcacc ttcagtagct atggcatgca ctgggtccgc 180
caggctccag gcaaggggct ggagtgggtg gcagttatat ggtatgatgg aagtaataaa
240 tactatgcag actccgtgaa gggccgattc actatctcca gagacaattc
caagaacacg 300 ctgtatctgc aaatgaacag cctgagagcc gaggacacgg
ctgtgtatta ctgtgcgagg 360 tataacttca actacggtat ggacgtctgg
ggccaaggga ccacggtcac cgtctctagt 420 gcctccacca agggcccatc
ggtcttcccc ctggcgccct gctccaggag cacctccgag 480 agcacagcgg
ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540
tggaactcag gcgctctgac cagcggcgtg cacaccttcc cagctgtcct acagtcctca
600 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg
cacccagacc 660 tacacctgca acgtagatca caagcccagc aacaccaagg
tggacaagac agttgagcgc 720 aaatgttgtg tcgagtgccc accgtgccca
gcaccacctg tggcaggacc gtcagtcttc 780 ctcttccccc caaaacccaa
ggacaccctc atgatctccc ggacccctga ggtcacgtgc 840 gtggtggtgg
acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 900
gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt
960 gtggtcagcg tcctcaccgt tgtgcaccag gactggctga acggcaagga
gtacaagtgc 1020 aaggtctcca acaaaggc 1038 <210> SEQ ID NO 384
<211> LENGTH: 346 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: IgG2 fragment <400> SEQUENCE: 384 Met Asp Met
Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu
Arg Gly Ala Arg Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly 20 25
30 Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
35 40 45 Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala
Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp
Gly Ser Asn Lys 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys
Ala Arg Tyr Asn Phe Asn Tyr Gly Met Asp 115 120 125 Val Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu 145 150 155
160 Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Asn Phe Gly Thr
Gln Thr Tyr Thr Cys Asn 210 215 220 Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Thr Val Glu Arg 225 230 235 240 Lys Cys Cys Val Glu
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly 245 250 255 Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280
285 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Phe Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Val His Gln Asp
Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly 340 345 <210> SEQ ID NO 385 <211> LENGTH: 37
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Forward primer
<400> SEQUENCE: 385 ttttttttgc gcgctgtgac atccagatga cccagtc
37 <210> SEQ ID NO 386 <400> SEQUENCE: 386 000
<210> SEQ ID NO 387 <211> LENGTH: 510 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: aKLH 120.6 IgG2-ShK[2-35, Q16K]
fusion <400> SEQUENCE: 387 Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys
Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr
Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50 55 60
Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr 65
70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg
Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg
Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185
190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr
Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305 310
315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435
440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser
Cys Ile Asp 465 470 475 480 Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe
Lys Cys Lys His Ser Met 485 490 495 Lys Tyr Arg Leu Ser Phe Cys Arg
Lys Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID NO 388
<211> LENGTH: 684 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS
<222> LOCATION: (1)..(684) <400> SEQUENCE: 388 atg gac
aaa act cac aca tgt cca cct tgt cca gct ccg gaa ctc ctg 48 Met Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15
ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 96
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20
25 30 atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg
agc 144 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser 35 40 45 cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac
ggc gtg gag 192 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu 50 55 60 gtg cat aat gcc aag aca aag ccg cgg gag gag
cag tac aac agc acg 240 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr 65 70 75 80 tac cgt gtg gtc agc gtc ctc acc gtc
ctg cac cag gac tgg ctg aat 288 Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn 85 90 95 ggc aag gag tac aag tgc aag
gtc tcc aac aaa gcc ctc cca gcc ccc 336 Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110 atc gag aaa acc atc
tcc aaa gcc aaa ggg cag ccc cga gaa cca cag 384 Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 gtg tac acc
ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc 432 Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 130 135 140 agc
ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 480 Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150
155 160 gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg
cct 528 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro 165 170 175 ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc
aag ctc acc 576 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr 180 185 190 gtg gac aag agc agg tgg cag cag ggg aac gtc
ttc tca tgc tcc gtg 624 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val 195 200 205 atg cat gag gct ctg cac aac cac tac
acg cag aag agc ctc tcc ctg 672 Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 210 215 220 tct ccg ggt aaa 684 Ser Pro
Gly Lys 225 <210> SEQ ID NO 389 <211> LENGTH: 228
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 389 Met Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 65 70 75 80
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85
90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210
215 220 Ser Pro Gly Lys 225 <210> SEQ ID NO 390 <400>
SEQUENCE: 390 000 <210> SEQ ID NO 391 <211> LENGTH: 744
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Coding sequence
- IgG2 fragment <400> SEQUENCE: 391 atggaatgga gctgggtctt
tctcttcttc ctgtcagtaa cgactggtgt ccactccgag 60 cgcaaagtcg
agtgcccacc gtgcccagca ccacctgtgg caggaccgtc agtcttcctc 120
ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacgtgcgtg
180 gtggtggacg tgagccacga agaccccgag gtccagttca actggtacgt
ggacggcgtg 240 gaggtgcata atgccaagac aaagccacgg gaggagcagt
tcaacagcac gttccgtgtg 300 gtcagcgtcc tcaccgttgt gcaccaggac
tggctgaacg gcaaggagta caagtgcaag 360 gtctccaaca aaggcctccc
agcccccatc gagaaaacca tctccaaaac caaagggcag 420 ccccgagaac
cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag 480
gtcagcctga cctgcctggt caaaggcttc taccccagcg acatcgccgt ggagtgggag
540 agcaatgggc agccggagaa caactacaag accacacctc ccatgctgga
ctccgacggc 600 tccttcttcc tctacagcaa gctcaccgtg gacaagagca
ggtggcagca ggggaacgtc 660 ttctcatgct ccgtgatgca tgaggctctg
cacaaccact acacgcagaa gagcctctcc 720 ctgtctccgg gtaaaggagg agga 744
<210> SEQ ID NO 392 <211> LENGTH: 248 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: IgG2 fragment <400> SEQUENCE:
392 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15 Val His Ser Glu Arg Lys Val Glu Cys Pro Pro Cys Pro Ala
Pro Pro 20 25 30 Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 50 55 60 Ser His Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Phe Arg Val
Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 100 105 110 Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala 115 120 125
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro 130
135 140 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Met Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu 195 200 205 Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu
Ser Pro Gly Lys Gly Gly Gly 245 <210> SEQ ID NO 393
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 393 cattctagac
ccaccatgga catgagggtg 30 <210> SEQ ID NO 394 <211>
LENGTH: 511 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
IgG2-HC-L10-ShK[1-35] fusion polypeptide <400> SEQUENCE: 394
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5
10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala
Glu 20 25 30 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys
Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His Met His Trp Val
Arg Gln Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile
Asn Pro Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln
Gly Arg Val Thr Met Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala
Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val
Tyr Tyr Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp
Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135
140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
145 150 155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser
Asn Phe Gly Thr Gln Thr Tyr Thr Cys 210 215 220 Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu 225 230 235 240 Arg Lys
Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 245 250 255
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260
265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His 275 280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Phe 305 310 315 320 Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385
390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 405 410 415 Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Arg Ser Cys Ile 465 470 475 480 Asp Thr Ile
Pro Lys Ser Arg Cys Thr Ala Phe Gln Cys Lys His Ser 485 490 495 Met
Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys Gly Thr Cys 500 505 510
<210> SEQ ID NO 395 <211> LENGTH: 501 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: truncated IgG2 Fc-L10-ShK(1-35,
Q16K) coding sequence <400> SEQUENCE: 395 ctcccagccc
ccatcgagaa aaccatctcc aaaaccaaag ggcagccccg agaaccacag 60
gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc
120 ctggtcaaag gcttctaccc cagcgacatc gccgtggagt gggagagcaa
tgggcagccg 180 gagaacaact acaagaccac acctcccatg ctggactccg
acggctcctt cttcctctac 240 agcaagctca ccgtggacaa gagcaggtgg
cagcagggga acgtcttctc atgctccgtg 300 atgcatgagg ctctgcacaa
ccactacacg cagaagagcc tctccctgtc tccgggtaaa 360 ggaggaggag
gatccggagg aggaggaagc cgcagctgca tcgacaccat ccccaagagc 420
cgctgcaccg ccttcaagtg caagcacagc atgaagtacc gcctgagctt ctgccgcaag
480 acctgcggca cctgctaatg a 501 <210> SEQ ID NO 396
<211> LENGTH: 165 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: truncated IgG2 Fc-L10-ShK(1-35, Q16K) amino acid
sequence <400> SEQUENCE: 396 Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Thr Lys Gly Gln Pro 1 5 10 15 Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 20 25 30 Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 35 40 45 Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 50 55 60
Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 65
70 75 80 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe 85 90 95 Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys 100 105 110 Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Arg Ser Cys Ile Asp Thr
Ile Pro Lys Ser Arg Cys Thr Ala 130 135 140 Phe Lys Cys Lys His Ser
Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys 145 150 155 160 Thr Cys Gly
Thr Cys 165 <210> SEQ ID NO 397 <211> LENGTH: 129
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Coding sequence
<400> SEQUENCE: 397 ggatccggag gaggaggaag cagctgcatc
gacaccatcc ccaagagccg ctgcaccgcc 60 ttcaagtgca agcacagcat
gaagtaccgc ctgagcttct gccgcaagac ctgcggcacc 120 tgctaatga 129
<210> SEQ ID NO 398 <211> LENGTH: 41 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Polypeptide sequence <400>
SEQUENCE: 398 Gly Ser Gly Gly Gly Gly Ser Ser Cys Ile Asp Thr Ile
Pro Lys Ser 1 5 10 15 Arg Cys Thr Ala Phe Lys Cys Lys His Ser Met
Lys Tyr Arg Leu Ser 20 25 30 Phe Cys Arg Lys Thr Cys Gly Thr Cys 35
40 <210> SEQ ID NO 399 <211> LENGTH: 1407 <212>
TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Coding sequence <400>
SEQUENCE: 399 atggacatga gggtgcccgc tcagctcctg gggctcctgc
tgctgtggct gagaggtgcc 60 agatgtcagg tgcagctggt gcagtctggg
gctgaggtga agaagcctgg ggcctcagtg 120 aaggtctcct gcaaggcttc
tggatacacc ttcaccggct accacatgca ctgggtgcga 180 caggcccctg
gacaagggct tgagtggatg ggatggatca accctaacag tggtggcaca 240
aactatgcac agaagtttca gggcagggtc accatgacca gggacacgtc catcagcaca
300 gcctacatgg agctgagcag gctgagatct gacgacacgg ccgtgtatta
ctgtgcgaga 360 gatcgtggga gctactactg gttcgacccc tggggccagg
gaaccctggt caccgtctcc 420 tcagcctcca ccaagggccc atcggtcttc
cccctggcgc cctgctccag gagcacctcc 480 gagagcacag cggccctggg
ctgcctggtc aaggactact tccccgaacc ggtgacggtg 540 tcgtggaact
caggcgctct gaccagcggc gtgcacacct tcccagctgt cctacagtcc 600
tcaggactct actccctcag cagcgtggtg accgtgccct ccagcaactt cggcacccag
660 acctacacct gcaacgtaga tcacaagccc agcaacacca aggtggacaa
gacagttgag 720 cgcaaatgtt gtgtcgagtg cccaccgtgc ccagcaccac
ctgtggcagg accgtcagtc 780 ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcacg 840 tgcgtggtgg tggacgtgag
ccacgaagac cccgaggtcc agttcaactg gtacgtggac 900 ggcgtggagg
tgcataatgc caagacaaag ccacgggagg agcagttcaa cagcacgttc 960
cgtgtggtca gcgtcctcac cgttgtgcac caggactggc tgaacggcaa ggagtacaag
1020 tgcaaggtct ccaacaaagg cctcccagcc cccatcgaga aaaccatctc
caaaaccaaa 1080 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggagga gatgaccaag 1140 aaccaggtca gcctgacctg cctggtcaaa
ggcttctacc ccagcgacat cgccgtggag 1200 tgggagagca atgggcagcc
ggagaacaac tacaagacca cacctcccat gctggactcc 1260 gacggctcct
tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc
1380 ctctccctgt ctccgggtgg aggagga 1407 <210> SEQ ID NO 400
<211> LENGTH: 469 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Polypeptide sequence <400> SEQUENCE: 400 Met Asp
Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15
Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20
25 30 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser
Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln
Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro
Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg
Val Thr Met Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr
Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150
155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr Tyr Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275
280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Phe 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Val His
Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395
400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
405 410 415 Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Gly Gly Gly 465
<210> SEQ ID NO 401 <211> LENGTH: 510 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: IgG2-HC-L10-ShK[2-35] fusion
polypeptide <400> SEQUENCE: 401 Met Asp Met Arg Val Pro Ala
Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg
Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr
Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50 55
60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr
65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg
Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg
Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185
190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr
Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305 310
315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435
440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser
Cys Ile Asp 465 470 475 480 Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe
Lys Cys Lys His Ser Met 485 490 495 Lys Tyr Arg Leu Ser Phe Cys Arg
Lys Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID NO 402
<211> LENGTH: 291 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: VH21 SP-ShK(1-35, Q16K)-L10-IgG1 Fc <400>
SEQUENCE: 402 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val
Thr Thr Gly 1 5 10 15 Val His Ser Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr 20 25 30 Ala Phe Lys Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg 35 40 45 Lys Thr Cys Gly Thr Cys Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 50 55 60 Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 65 70 75 80 Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 85 90 95 Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 100 105
110 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
115 120 125 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr 130 135 140 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 145 150 155 160 Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 165 170 175 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 180 185 190 Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 195 200 205 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 210 215 220 Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 225 230
235 240 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 245 250 255 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 260 265 270 His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 275 280 285 Pro Gly Lys 290 <210> SEQ ID
NO 403 <211> LENGTH: 509 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: VH21 SP-ShK[1-35, Q16K]-L10-aKLH120.6-HC fusion
polypeptide <400> SEQUENCE: 403 Met Glu Trp Ser Trp Val Phe
Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Arg Ser
Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr 20 25 30 Ala Phe Lys
Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg 35 40 45 Lys
Thr Cys Gly Thr Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 50 55
60 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
65 70 75 80 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Gly Tyr 85 90 95 His Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 100 105 110 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr
Asn Tyr Ala Gln Lys Phe 115 120 125 Gln Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Ile Ser Thr Ala Tyr 130 135 140 Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 145 150 155 160 Ala Arg Asp
Arg Gly Ser Tyr Tyr Trp Phe Asp Pro Trp Gly Gln Gly 165 170 175 Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 180 185
190 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
195 200 205 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp 210 215 220 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu 225 230 235 240 Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 245 250 255 Ser Asn Phe Gly Thr Gln Thr
Tyr Thr Cys Asn Val Asp His Lys Pro 260 265 270 Ser Asn Thr Lys Val
Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu 275 280 285 Cys Pro Pro
Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 290 295 300 Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 305 310
315 320 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Gln 325 330 335 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys 340 345 350 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg
Val Val Ser Val Leu 355 360 365 Thr Val Val His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys 370 375 380 Val Ser Asn Lys Gly Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys 385 390 395 400 Thr Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 405 410 415 Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 420 425 430
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 435
440 445 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp
Gly 450 455 460 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln 465 470 475 480 Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn 485 490 495 His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 500 505 <210> SEQ ID NO 404 <211>
LENGTH: 278 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Fusion
polypeptide <400> SEQUENCE: 404 Met Glu Trp Ser Trp Val Phe
Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Arg Ser
Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr 20 25 30 Ala Phe Lys
Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg 35 40 45 Lys
Thr Cys Gly Thr Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 50 55
60 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
65 70 75 80 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg
Asn Asp 85 90 95 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Arg Leu Ile 100 105 110 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 115 120 125 Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 130 135 140 Glu Asp Phe Ala Thr Tyr
Tyr Cys Leu Gln His Asn Ser Tyr Pro Leu 145 150 155 160 Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 165 170 175 Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 180 185
190 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
195 200 205 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln 210 215 220 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 225 230 235 240 Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 245 250 255 Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 260 265 270 Phe Asn Arg Gly Glu
Cys 275 <210> SEQ ID NO 405 <211> LENGTH: 465
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: anti-DNP 3A4
(W101F) IgG2 Heavy Chain <400> SEQUENCE: 405 Met Asp Met Arg
Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg
Gly Ala Arg Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly 20 25 30
Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35
40 45 Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro
Gly 50 55 60 Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly
Ser Asn Lys 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala
Arg Tyr Asn Phe Asn Tyr Gly Met Asp 115 120 125 Val Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu 145 150 155 160
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165
170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln
Thr Tyr Thr Cys Asn 210 215 220 Val Asp His Lys Pro Ser Asn Thr Lys
Val Asp Lys Thr Val Glu Arg 225 230 235 240 Lys Cys Cys Val Glu Cys
Pro Pro Cys Pro Ala Pro Pro Val Ala Gly 245 250 255 Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290
295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Thr Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met 405 410
415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 450 455 460 Gly 465 <210> SEQ ID NO 406
<211> LENGTH: 510 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: aDNP 3A4 (W101F) IgG2 HC-L10-ShK[1-35, Q16K] fusion
protein <400> SEQUENCE: 406 Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys
Gln Val Gln Leu Val Glu Ser Gly Gly Gly 20 25 30 Val Val Gln Pro
Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr
Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly 50 55 60
Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys 65
70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Asn
Phe Asn Tyr Gly Met Asp 115 120 125 Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu 145 150 155 160 Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185
190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
195 200 205 Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr
Cys Asn 210 215 220 Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
Thr Val Glu Arg 225 230 235 240 Lys Cys Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro Pro Val Ala Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg 305 310
315 320 Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly
Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala
Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met 405 410 415 Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435
440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 450 455 460 Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Ser
Cys Ile Asp 465 470 475 480 Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe
Lys Cys Lys His Ser Met 485 490 495 Lys Tyr Arg Leu Ser Phe Cys Arg
Lys Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID NO 407
<211> LENGTH: 236 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: anti-DNP 3A4 Antibody Light Chain <400>
SEQUENCE: 407 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu
Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser 20 25 30 Val Ser Ala Ser Val Gly Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Ser Arg Arg Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Leu Leu
Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105
110 Ala Asn Ser Phe Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile
115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210>
SEQ ID NO 408 <211> LENGTH: 475 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Polypeptide <400> SEQUENCE:
408 Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser 35 40 45 His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110 Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 130
135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Pro Gly Lys
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 225 230 235 240 Gly
Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro 245 250
255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr 275 280 285 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn 290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335 Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350 Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 355 360 365 Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 370 375
380 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly 435 440 445 Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr 450 455 460 Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 465 470 475 <210> SEQ ID NO 409
<211> LENGTH: 148 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Polypeptide <400> SEQUENCE: 409 Met Lys Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 1 5 10 15 Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 20 25 30
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 35
40 45 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr 50 55 60 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn 65 70 75 80 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro 85 90 95 Ile Glu Lys Thr Ile Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser 100 105 110 Arg Ser Cys Ile Asp Thr Ile
Pro Lys Ser Arg Cys Thr Ala Phe Lys 115 120 125 Cys Lys His Ser Met
Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 130 135 140 Gly Thr Cys
Ala 145 <210> SEQ ID NO 410 <211> LENGTH: 521
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: scFc-Shk
construct <400> SEQUENCE: 410 Met Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 65
70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185
190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 210 215 220 Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly 225 230 235 240 Gly Gly Ser Gly Gly Gly Gly Ser Asp
Lys Thr His Thr Cys Pro Pro 245 250 255 Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310
315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser 340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys 355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp 370 375 380 Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435
440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr 450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly
Gly Gly Ser 465 470 475 480 Gly Gly Gly Gly Ser Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg 485 490 495 Cys Thr Ala Phe Lys Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe 500 505 510 Cys Arg Lys Thr Cys Gly
Thr Cys Ala 515 520 <210> SEQ ID NO 411 <211> LENGTH:
514 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: scFc-Shk
construct <400> SEQUENCE: 411 Met Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 65
70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185
190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 210 215 220 Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly 225 230 235 240 Gly Gly Ser Gly Gly Gly Gly Ser Asp
Lys Thr His Thr Cys Pro Pro 245 250 255 Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310
315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser 340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys 355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp 370 375 380 Glu Leu Gly Gly Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys 385 390 395 400 Thr Ala Phe Gln
Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys 405 410 415 Arg Lys
Thr Cys Gly Thr Cys Gly Gly Thr Lys Asn Gln Val Ser Leu 420 425 430
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 435
440 445 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val 450 455 460 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp 465 470 475 480 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His 485 490 495 Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 500 505 510 Gly Lys <210> SEQ
ID NO 412 <211> LENGTH: 519 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: scFc-Shk construct <400> SEQUENCE: 412 Met
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10
15 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 35 40 45 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Gly Gly Arg Ser Cys 130 135 140
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln Cys Lys His 145
150 155 160 Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys Gly
Thr Cys 165 170 175 Gly Gly Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe 180 185 190 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu 195 200 205 Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe 210 215 220 Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 225 230 235 240 Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 245 250 255 Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser 260 265
270 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
275 280 285 Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro 290 295 300 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys 305 310 315 320 Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val 325 330 335 Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp 340 345 350 Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 355 360 365 Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 370 375 380 Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 385 390
395 400 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg 405 410 415 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys 420 425 430 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp 435 440 445 Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys 450 455 460 Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser 465 470 475 480 Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 485 490 495 Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 500 505 510
Leu Ser Leu Ser Pro Gly Lys 515 <210> SEQ ID NO 413
<211> LENGTH: 480 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Polypeptide <400> SEQUENCE: 413 Met Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15 Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35
40 45 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165
170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu 210 215 220 Ser Pro Gly Lys Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly 225 230 235 240 Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr 245 250 255 His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 260 265 270 Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 275 280 285
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 290
295 300 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala 305 310 315 320 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val 325 330 335 Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr 340 345 350 Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr 355 360 365 Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 370 375 380 Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 385 390 395 400 Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 405 410
415 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
420 425 430 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser 435 440 445 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala 450 455 460 Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 465 470 475 480 <210> SEQ ID NO 414
<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 414 aaacctgtgg
cacctgtggc ggtaccaaaa accaggtgtc cctga 45 <210> SEQ ID NO 415
<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 415 tatcgatgca
agaacgaccg cccagttcgt cacgagacgg cggca 45 <210> SEQ ID NO 416
<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 416 aaataccgtc
tcagtttctg tcgtaaaacc tgtggcacct gtggc 45 <210> SEQ ID NO 417
<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 417 cagtacagcg
ggacttaggg attgtatcga tgcaagaacg accgc 45 <210> SEQ ID NO 418
<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 418 caatgcaaac
actcaatgaa ataccgtctc agtttctgtc gtaaa 45 <210> SEQ ID NO 419
<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 419 tgagtgtttg
cattgaaagg cagtacagcg ggacttaggg att 43 <210> SEQ ID NO 420
<211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 420 taatgaattc
gagctccgtc gacaagct 28 <210> SEQ ID NO 421 <211>
LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 421 ccaccggatc caccaccacc tttacccgga
gacagggaga ggct 44 <210> SEQ ID NO 422 <211> LENGTH: 28
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 422 tggtggatcc ggtggtggtg gctccggt 28
<210> SEQ ID NO 423 <211> LENGTH: 40 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 423 gagctcgaat tcattattta cccggagaca gagacaggga 40
<210> SEQ ID NO 424 <211> LENGTH: 36 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 424 ggtggcggtg gctctggtgg tggtggatcc ggtggt 36
<210> SEQ ID NO 425 <211> LENGTH: 39 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 425 agagccaccg ccacctttac ccggagacag ggagaggct 39
<210> SEQ ID NO 426 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 426 ggcggtacca agaaccaggt cagcctga 28 <210> SEQ ID
NO 427 <211> LENGTH: 29 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 427
accgcccagc tcatcacggg atgggggca 29 <210> SEQ ID NO 428
<211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 428 cgtgatgagc
tgggcggtcg ttcttgcatc gatacaatcc ct 42 <210> SEQ ID NO 429
<211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 429 acctggttct
tggtaccgcc acaggtgcca caggttttac ga 42 <210> SEQ ID NO 430
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 430 cactcaatga
aataccgtct cagtttctgt cgt 33 <210> SEQ ID NO 431 <211>
LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 431 gtatttcatt gagtgtttgc attgaaaggc
a 31 <210> SEQ ID NO 432 <211> LENGTH: 28 <212>
TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 432 taatgaattc gagctccgtc gacaagct 28 <210> SEQ ID
NO 433 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 433
cccggagaca gagacaggga 20 <210> SEQ ID NO 434 <211>
LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 434 gtctctgtct ccgggtaaag gcggcggcgg
ca 32 <210> SEQ ID NO 435 <211> LENGTH: 34 <212>
TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 435 agctcgaatt cattaagcac aggtgccaca ggtt 34 <210>
SEQ ID NO 436 <211> LENGTH: 163 <212> TYPE: DNA
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Coding sequence ShK {1-35, Q16K)
with an N-terminal linker <400> SEQUENCE: 436 ggatccggag
gaggaggaag ccgcagctgc atcgacacca tccccaagag ccgctgcacc 60
gccttcaagt gcaagcacag catgaagtac cgcctgagct tctgccgcaa gacctgcggc
120 acctgctaat gagcggccgc tcgaggccgg caaggccgga tcc 163 <210>
SEQ ID NO 437 <211> LENGTH: 42 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK {1-35, Q16K) with an N-terminal
linker <400> SEQUENCE: 437 Gly Ser Gly Gly Gly Gly Ser Arg
Ser Cys Ile Asp Thr Ile Pro Lys 1 5 10 15 Ser Arg Cys Thr Ala Phe
Lys Cys Lys His Ser Met Lys Tyr Arg Leu 20 25 30 Ser Phe Cys Arg
Lys Thr Cys Gly Thr Cys 35 40 <210> SEQ ID NO 438 <211>
LENGTH: 36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
ShK-192 <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION:
L-phosphonophenylalanine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: Between residues 1 and 2 is: {2-[2-Aminoethoxy]ethoxy
}acetic acid <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (22)..(22) <223> OTHER
INFORMATION: Norleucine <400> SEQUENCE: 438 Xaa Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe 1 5 10 15 Gln Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr 20 25 30
Cys Gly Thr Cys 35 <210> SEQ ID NO 439 <211> LENGTH:
281 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: aKLH 120.6
kappa LC-ShK[1-35, Q16K] fusion protein <400> SEQUENCE: 439
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5
10 15 Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser 35 40 45 Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln
Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Arg Leu Ile Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110 His Asn Ser
Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135
140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly 225 230 235 240 Ser Gly
Gly Gly Gly Ser Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser 245 250 255
Arg Cys Thr Ala Phe Lys Cys Lys His Ser Met Lys Tyr Arg Leu Ser 260
265 270 Phe Cys Arg Lys Thr Cys Gly Thr Cys 275 280 <210> SEQ
ID NO 440 <211> LENGTH: 280 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: aKLH 120.6 kappa LC-ShK[2-35, Q16K] fusion
protein <400> SEQUENCE: 440 Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser
Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly
Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60
Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val 65
70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Leu Gln 100 105 110 His Asn Ser Tyr Pro Leu Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185
190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
Gly Gly Gly Gly 225 230 235 240 Ser Gly Gly Gly Gly Ser Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg 245 250 255 Cys Thr Ala Phe Lys Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe 260 265 270 Cys Arg Lys Thr Cys
Gly Thr Cys 275 280 <210> SEQ ID NO 441 <211> LENGTH:
34 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 441 aacaggggag agtgtggagg aggaggatcc ggag 34
<210> SEQ ID NO 442 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 442 catgcggccg ctcattagca gg 22 <210> SEQ ID NO 443
<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Forward primer sequence <400> SEQUENCE: 443
ggacactgac atggactgaa ggagta 26 <210> SEQ ID NO 444
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Reverse primer sequence <400> SEQUENCE: 444
ctcctgggag ttacccgatt g 21 <210> SEQ ID NO 445 <211>
LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Reverse primer sequence <400> SEQUENCE: 445 gatgggccct
tggtggaggc tgaggagacg gtgaccgtgg 40 <210> SEQ ID NO 446
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 446 aagctcgagg
tcgactagac caccatggac atgagg 36 <210> SEQ ID NO 447
<211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 447 aaccgtttaa
acgcggccgc tcaacactct cccctgttga a 41 <210> SEQ ID NO 448
<211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 448 aagctcgagg
tcgactagac caccatggaa ttgggactga g 41 <210> SEQ ID NO 449
<211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 449 aaccgtttaa
acgcggccgc tcatttaccc ggagacaggg a 41 <210> SEQ ID NO 450
<211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 450 ttttttttgc
gcgctgtgac atccagatga cccagtc 37 <210> SEQ ID NO 451
<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 451 aaaaaacgta
cgtttgatat ccactttggt cc 32 <210> SEQ ID NO 452 <211>
LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 452 ctgtgtatta ctgtgcgagg tataacttca
actacggtat ggacgtctgg 50 <210> SEQ ID NO 453 <211>
LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 453 ccagacgtcc ataccgtagt tgaagttata
cctcgcacag taatacacag 50 <210> SEQ ID NO 454 <211>
LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 454 ctgtgtatta ctgtgcgagg tataactaca
actacggtat ggacgtctgg 50 <210> SEQ ID NO 455 <211>
LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 455 ccagacgtcc ataccgtagt tgtagttata
cctcgcacag taatacacag 50 <210> SEQ ID NO 456 <211>
LENGTH: 59 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 456 aagctcgagg tcgactagac caccatggac
atgagggtgc ccgctcagct cctggggct 59 <210> SEQ ID NO 457
<211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 457 aaccgtttaa
acgcggccgc tcatttaccc ggagacaggg a 41 <210> SEQ ID NO 458
<211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 458 ggacaagaca
gttgagcgca aatcttctgt cgagtgccca ccgtgcccag 50 <210> SEQ ID
NO 459 <211> LENGTH: 50 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 459
ctgggcacgg tgggcactcg acagaagatt tgcgctcaac tgtcttgtcc 50
<210> SEQ ID NO 460 <211> LENGTH: 59 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 460 aagctcgagg tcgactagac caccatggac atgagggtgc
ccgctcagct cctggggct 59 <210> SEQ ID NO 461 <211>
LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 461 aaccgtttaa acgcggccgc tcatttaccc
ggagacaggg a 41 <210> SEQ ID NO 462 <211> LENGTH: 511
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: aKLH 120.6
IgG2-ShK[1-35, R1A, I4A, Q16K] fusion protein <400> SEQUENCE:
462 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly
Ala Glu 20 25 30 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys
Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His Met His Trp
Val Arg Gln Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Trp
Ile Asn Pro Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe
Gln Gly Arg Val Thr Met Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr
Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala
Val Tyr Tyr Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125
Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130
135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser 145 150 155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser
Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys 210 215 220 Asn Val Asp His
Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu 225 230 235 240 Arg
Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 245 250
255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 275 280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Phe 305 310 315 320 Arg Val Val Ser Val Leu Thr
Val Val His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr
Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375
380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 405 410 415 Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Ala Ser Cys Ala 465 470 475 480 Asp Thr
Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys Cys Lys His Ser 485 490 495
Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys Gly Thr Cys 500 505
510 <210> SEQ ID NO 463 <211> LENGTH: 511 <212>
TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: aKLH 120.6 IgG2-ShK[1-35,
R1A, Q16K, K30E] fusion protein <400> SEQUENCE: 463 Met Asp
Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15
Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20
25 30 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser
Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln
Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro
Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg
Val Thr Met Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr
Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150
155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr Tyr Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275
280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Phe 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Val His
Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395
400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
405 410 415 Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Ala Ser Cys Ile 465 470 475 480 Asp Thr Ile Pro Lys
Ser Arg Cys Thr Ala Phe Lys Cys Lys His Ser 485 490 495 Met Lys Tyr
Arg Leu Ser Phe Cys Arg Glu Thr Cys Gly Thr Cys 500 505 510
<210> SEQ ID NO 464 <211> LENGTH: 511 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: aKLH 120.6 IgG2-ShK[1-35, R1H, I4A,
Q16K] fusion protein <400> SEQUENCE: 464 Met Asp Met Arg Val
Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly
Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40
45 Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly
50 55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly
Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met
Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser
Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg
Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170
175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln
Thr Tyr Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys
Val Asp Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys
Pro Pro Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295
300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
305 310 315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly
Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415
Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420
425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser His Ser Cys Ala 465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys
Thr Ala Phe Lys Cys Lys His Ser 485 490 495 Met Lys Tyr Arg Leu Ser
Phe Cys Arg Lys Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID
NO 465 <211> LENGTH: 511 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: aKLH 120.6 IgG2-ShK[1-35, R1H, Q16K, K30E]
fusion protein <400> SEQUENCE: 465 Met Asp Met Arg Val Pro
Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala
Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys
Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45
Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50
55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly
Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr
Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg
Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp
Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180
185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser 195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
Tyr Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro
Pro Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305
310 315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu
Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln
Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425
430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
His Ser Cys Ile 465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys Cys Lys His Ser 485 490 495 Met Lys Tyr Arg Leu Ser Phe
Cys Arg Glu Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID NO
466 <211> LENGTH: 511 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: aKLH 120.6 HC (IgG2)-ShK[1-35, R1K, 14A, Q16K]
fusion protein <400> SEQUENCE: 466 Met Asp Met Arg Val Pro
Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala
Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys
Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45
Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50
55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly
Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr
Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg
Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp
Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180
185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser 195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
Tyr Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro
Pro Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305
310 315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu
Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln
Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425
430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Lys Ser Cys Ala 465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys Cys Lys His Ser 485 490 495 Met Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID NO
467 <211> LENGTH: 511 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: aKLH 120.6 IgG2-ShK[1-35, R1K, Q16K, K30E]
fusion protein <400> SEQUENCE: 467 Met Asp Met Arg Val Pro
Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala
Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys
Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45
Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50
55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly
Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr
Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg
Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp
Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180
185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser 195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
Tyr Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro
Pro Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305
310 315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu
Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln
Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425
430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Lys Ser Cys Ile 465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys Cys Lys His Ser 485 490 495 Met Lys Tyr Arg Leu Ser Phe
Cys Arg Glu Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID NO
468 <211> LENGTH: 37 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 468
aggaggagga agcgccagct gcgccgacac catcccc 37 <210> SEQ ID NO
469 <211> LENGTH: 37 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 469
ggggatggtg tcggcgcagc tggcgcttcc tcctcct 37 <210> SEQ ID NO
470 <211> LENGTH: 30 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 470
gaggaggagg aagcgccagc tgcatcgaca 30 <210> SEQ ID NO 471
<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 471 gagcttctgc
cgcgagacct gcggcac 27 <210> SEQ ID NO 472 <211> LENGTH:
27 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 472 cgatgcagct ggcgcttcct cctcctc 27
<210> SEQ ID NO 473 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 473 gtgccgcagg tctcgcggca gaagctc 27 <210> SEQ ID
NO 474 <211> LENGTH: 36 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 474
ggaggaggaa gccacagctg cgccgacacc atcccc 36 <210> SEQ ID NO
475 <211> LENGTH: 36 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 475
ggggatggtg tcggcgcagc tgtggcttcc tcctcc 36 <210> SEQ ID NO
476 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 476
ggaggaggaa gccacagctg catcgac 27 <210> SEQ ID NO 477
<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 477 gtcgatgcag
ctgtggcttc ctcctcc 27 <210> SEQ ID NO 478 <211> LENGTH:
45 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 478 ccggaggagg aggaagcaag agctgcgccg
acaccatccc caaga 45 <210> SEQ ID NO 479 <211> LENGTH:
45 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 479 tcttggggat ggtgtcggcg cagctcttgc
ttcctcctcc tccgg 45 <210> SEQ ID NO 480 <211> LENGTH:
35 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 480 cggaggagga ggaagcaaga gctgcatcga cacca 35
<210> SEQ ID NO 481 <211> LENGTH: 33 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 481 tggtgtcgat gcagctcttg cttcctcctc ctc 33 <210>
SEQ ID NO 482 <211> LENGTH: 30 <212> TYPE: DNA
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 482 cattctagaa ccaccatgga catgagggtg 30
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 482
<210> SEQ ID NO 1 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: Stichodactyla helianthus <400>
SEQUENCE: 1 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala
Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys
Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 2
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
Radianthus magnifica <400> SEQUENCE: 2 Arg Thr Cys Lys Asp
Leu Ile Pro Val Ser Glu Cys Thr Asp Ile Arg 1 5 10 15 Cys Arg Thr
Ser Met Lys Tyr Arg Leu Asn Leu Cys Arg Lys Thr Cys 20 25 30 Gly
Ser Cys 35 <210> SEQ ID NO 3 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: Anemonia erythraea
<400> SEQUENCE: 3 Arg Ala Cys Lys Asp Tyr Leu Pro Lys Ser Glu
Cys Thr Gln Phe Arg 1 5 10 15 Cys Arg Thr Ser Met Lys Tyr Lys Tyr
Thr Asn Cys Lys Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ
ID NO 4 <211> LENGTH: 38 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Toxin peptide analog <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION:
Carboxy-terminal residue is optionally amidated <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(2) <223> OTHER INFORMATION: Xaa 1 and Xaa 2 are absent,
or Xaa 1 is absent and Xaa 2 is Glu, Ser, Ala, or Thr, or Xaa 1 is
Arg or Ala and Xaa 2 is Glu, Ser, Ala, or Thr <220> FEATURE:
<221> NAME/KEY: DISULFID <222> LOCATION: (3)..(35)
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa 4 is an
alkyl, basic, or acidic amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (6)..(6)
<223> OTHER INFORMATION: Xaa 6 is Thr, Tyr, Ala, or Leu
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (7)..(7) <223> OTHER INFORMATION: Xaa 7 is Leu,
Ile, Ala, or Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (8)..(8) <223> OTHER
INFORMATION: Xaa 8 is Pro, Ala, Arg, Lys, 1-Nal, or Glu <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(9)..(9) <223> OTHER INFORMATION: Xaa 9 is Lys, Ala, Val or
or an acidic amino acid residue <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (10)..(10) <223>
OTHER INFORMATION: Xaa 10 is Ser, Glu, Arg, or Ala <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(11)..(11) <223> OTHER INFORMATION: Xaa 11 is Arg, Glu, or
Ala <220> FEATURE: <221> NAME/KEY: DISULFID <222>
LOCATION: (12)..(28) <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (13)..(13) <223> OTHER
INFORMATION: Xaa 13 is Thr, Ala, Arg, Lys, 1-Nal, or Glu
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (14)..(14) <223> OTHER INFORMATION: Xaa 14 is Gln,
Ala or an acidic amino acid reside <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (15)..(15) <223>
OTHER INFORMATION: Xaa 15 is an alkyl or aromatic amino acid
residue <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (16)..(16) <223> OTHER INFORMATION: Xaa
16 is a basic, alkyl, or aromatic amino acid residue other than
Ala, Gln, Glu or Arg <220> FEATURE: <221> NAME/KEY:
DISULFID <222> LOCATION: (17)..(32) <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (18)..(18)
<223> OTHER INFORMATION: Xaa 18 is an Ala or an acidic or
basic amino acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: Xaa 19 is Thr, Ala or a basic amino acid residue
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (20)..(20) <223> OTHER INFORMATION: Xaa 20 is Ser,
Ala or a basic amino acid residue <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Xaa 21 is an alkyl or aromatic amino acid
residue, other than Ala or Met <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (22)..(22) <223>
OTHER INFORMATION: Xaa 22 is Lys or Ala <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (23)..(23)
<223> OTHER INFORMATION: Xaa 23 is Tyr or Ala <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(24)..(24) <223> OTHER INFORMATION: Xaa 24 is Arg, Lys, or
Ala <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (25)..(25) <223> OTHER INFORMATION: Xaa
25 is Tyr, Leu, or Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (26)..(26) <223> OTHER
INFORMATION: Xaa 26 is Ser, Thr, Asn, Ala, or an aromatic amino
acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (27)..(27) <223> OTHER
INFORMATION: Xaa 27 is Leu, Ala, Asn, or an aromatic amino acid
residue <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (29)..(29) <223> OTHER INFORMATION: Xaa
29 is 1-Nal, 2-Nal, Ala, or an basic amino acid residue <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(30)..(30) <223> OTHER INFORMATION: Xaa 30 is Ala or an
acidic or basic amino acid residue <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (31)..(31) <223>
OTHER INFORMATION: Xaa 31 is Thr, Ala or an aromatic amino acid
residue <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (33)..(33) <223> OTHER INFORMATION: Xaa
33 is Gly, Ala, Arg, Lys, 1-Nal, or Glu <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (34)..(34)
<223> OTHER INFORMATION: Xaa 34 is Thr, Ser, Ala, Lys, or an
aromatic amino acid residue <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (36)..(38) <223>
OTHER INFORMATION: Xaa in positions 36-38 are each independently
absent are independently a neutral, basic, acidic, or N-alkylated
amino acid residue <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (38)..(38) <223> OTHER
INFORMATION: Carboxy-terminal residue is optionally amidated
<400> SEQUENCE: 4 Xaa Xaa Cys Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa 1 5 10 15 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Cys 20 25 30 Xaa Xaa Cys Xaa Xaa Xaa 35
<210> SEQ ID NO 5 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: Actinia equina <400> SEQUENCE: 5
Gly Cys Lys Asp Asn Phe Ser Ala Asn Thr Cys Lys His Val Lys Ala 1 5
10 15 Asn Asn Asn Cys Gly Ser Gln Lys Tyr Ala Thr Asn Cys Ala Lys
Thr
20 25 30 Cys Gly Lys Cys 35 <210> SEQ ID NO 6 <211>
LENGTH: 36 <212> TYPE: PRT <213> ORGANISM: Anemonia
sulcata <400> SEQUENCE: 6 Ala Cys Lys Asp Asn Phe Ala Ala Ala
Thr Cys Lys His Val Lys Glu 1 5 10 15 Asn Lys Asn Cys Gly Ser Gln
Lys Tyr Ala Thr Asn Cys Ala Lys Thr 20 25 30 Cys Gly Lys Cys 35
<210> SEQ ID NO 7 <211> LENGTH: 37 <212> TYPE:
PRT <213> ORGANISM: Bunodosoma granulifera <400>
SEQUENCE: 7 Val Cys Arg Asp Trp Phe Lys Glu Thr Ala Cys Arg His Ala
Lys Ser 1 5 10 15 Leu Gly Asn Cys Arg Thr Ser Gln Lys Tyr Arg Ala
Asn Cys Ala Lys 20 25 30 Thr Cys Glu Leu Cys 35 <210> SEQ ID
NO 8 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: Stichodactyla helianthus <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: Pegylated
<400> SEQUENCE: 8 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg
Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu
Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ
ID NO 9 <211> LENGTH: 38 <212> TYPE: PRT <213>
ORGANISM: Orthochirus scrobiculosus <400> SEQUENCE: 9 Gly Val
Ile Ile Asn Val Lys Cys Lys Ile Ser Arg Gln Cys Leu Glu 1 5 10 15
Pro Cys Lys Lys Ala Gly Met Arg Phe Gly Lys Cys Met Asn Gly Lys 20
25 30 Cys Ala Cys Thr Pro Lys 35 <210> SEQ ID NO 10
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: HmK peptide <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norvaline <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 10 Arg Thr Cys Lys Asp
Leu Ile Pro Val Ser Glu Cys Thr Asp Ile Lys 1 5 10 15 Cys Arg Thr
Ser Xaa Lys Tyr Arg Leu Asn Leu Cys Arg Lys Thr Cys 20 25 30 Gly
Ser Cys 35 <210> SEQ ID NO 11 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (16)..(16) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 11 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Xaa 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 12 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (26)..(26) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 12 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Xaa Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 13 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK[1-35, Q16K]
polypeptide <400> SEQUENCE: 13 Arg Ser Cys Ile Asp Thr Ile
Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met
Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 14 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination toxin peptide analog
(C-Terminal Amide) <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 14 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 15 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 15 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His
Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 16 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK PEGylated
Toxin Peptide <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <400> SEQUENCE: 16 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 17 <211> LENGTH: 36
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK reference
peptide <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: L-phosphonotyrosine <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(2) <223> OTHER INFORMATION: Between residues 1 and 2
is: {2-[2-Aminoethoxy]ethoxy }acetic acid <400> SEQUENCE: 17
Xaa Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe 1 5
10 15 Gln Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr 20 25 30 Cys Gly Thr Cys 35 <210> SEQ ID NO 18
<211> LENGTH: 36 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK reference peptide <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(2) <223>
OTHER INFORMATION: Between residues 1 and 2 is:
{2-[2-Aminoethoxy]ethoxy }acetic acid <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: L-phosphonotyrosine <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(22)..(22) <223> OTHER INFORMATION: Norleucine <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(36)..(36) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 18 Xaa Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys
Thr Ala Phe 1 5 10 15 Gln Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser
Phe Cys Arg Lys Thr 20 25 30 Cys Gly Thr Cys 35 <210> SEQ ID
NO 19 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: ShK peptide <400> SEQUENCE: 19 Arg Arg Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys
Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 20 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 20 Arg Ser Cys Arg Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 21 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 21
Arg Ser Cys Ile Arg Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 22 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 22 Arg Ser Cys Ile Asp Arg Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 23 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 23
Arg Ser Cys Ile Asp Thr Arg Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 24 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 24 Arg Ser Cys Ile Asp Thr Ile Arg
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 25 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 25
Arg Ser Cys Ile Asp Thr Ile Pro Arg Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 26 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 26 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Arg Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 27 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 27
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Arg Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 28 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 28 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Arg Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 29 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 29 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Arg Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 30 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 30
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Arg 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 31 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 31 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Arg His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 32 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 32
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys Arg Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 33 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 33 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Arg Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 34 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 34
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Arg Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 35 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 35 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Arg
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 36 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 36
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Arg Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 37 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 37 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Arg Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 38 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 38
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Arg Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 39 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 39 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Arg Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 40 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 40
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Arg Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 41 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 41 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Arg Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 42 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
42
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Arg Thr Cys 35 <210> SEQ ID NO 43 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 43 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Arg Cys 35
<210> SEQ ID NO 44 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 44
Glu Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 45 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 45 Arg Glu Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 46 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 46
Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 47 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 47 Arg Ser Cys Ile Glu Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 48 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 48
Arg Ser Cys Ile Asp Glu Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 49 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 49 Arg Ser Cys Ile Asp Thr Glu Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 50 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 50
Arg Ser Cys Ile Asp Thr Ile Glu Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 51 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 51 Arg Ser Cys Ile Asp Thr Ile Pro
Glu Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 52 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 52
Arg Ser Cys Ile Asp Thr Ile Pro Lys Glu Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 53 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 53 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Glu Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 54 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 54
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Glu Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 55 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptdie <400> SEQUENCE: 55 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Glu Phe Gln 1 5 10 15
Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20
25 30 Gly Thr Cys 35 <210> SEQ ID NO 56 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 56 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Glu Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 57 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 57
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Glu 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 58 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 58 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Glu His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 59 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 59
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys Glu Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 60 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 60 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Glu Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 61 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Shk Peptide <400> SEQUENCE: 61
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Glu Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 62 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 62 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Glu
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 63 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 63
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Glu Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 64 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 64 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Glu Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 65 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 65
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Glu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 66 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 66 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Glu Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 67 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 67
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Glu Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 68 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <400> SEQUENCE: 68 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Glu Lys Thr Cys 20 25 30 Gly Thr Cys
35 <210> SEQ ID NO 69 <211> LENGTH: 35 <212>
TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: ShK peptide <400>
SEQUENCE: 69 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe
Cys Arg Glu Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
70 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: ShK peptide <400> SEQUENCE: 70 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys
Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Glu Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 71 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 71 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Glu Thr Cys 35 <210>
SEQ ID NO 72 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE: 72
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Glu Cys 35 <210> SEQ ID NO 73 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 73 Xaa Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 74 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (2)..(2) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 74 Arg Xaa Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 75 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (4)..(4) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 75 Arg Ser Cys Xaa Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 76 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (5)..(5) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 76 Arg Ser Cys Ile Xaa
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 77 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (6)..(6) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 77 Arg Ser Cys Ile Asp
Xaa Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 78 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (7)..(7) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 78 Arg Ser Cys Ile Asp
Thr Xaa Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 79 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (8)..(8) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 79 Arg Ser Cys Ile Asp
Thr Ile Xaa Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 80 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (9)..(9) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine
<400> SEQUENCE: 80 Arg Ser Cys Ile Asp Thr Ile Pro Xaa Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 81 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (10)..(10)
<223> OTHER INFORMATION: 3-(1-naphthyl)alanine <400>
SEQUENCE: 81 Arg Ser Cys Ile Asp Thr Ile Pro Lys Xaa Arg Cys Thr
Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
82 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: ShK peptide <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (11)..(11) <223>
OTHER INFORMATION: 3-(1-naphthyl)alanine <400> SEQUENCE: 82
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Xaa Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 83 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (13)..(13) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 83 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Xaa Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 84 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (14)..(14) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 84 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Xaa Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 85 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (15)..(15) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 85 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Xaa Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 86 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Linker sequence
<400> SEQUENCE: 86 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210>
SEQ ID NO 87 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (18)..(18)
<223> OTHER INFORMATION: 3-(1-naphthyl)alanine <400>
SEQUENCE: 87 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Gln 1 5 10 15 Cys Xaa His Ser Met Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
88 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: ShK peptide <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (19)..(19) <223>
OTHER INFORMATION: 3-(1-naphthyl)alanine <400> SEQUENCE: 88
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys Xaa Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 89 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (20)..(20) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 89 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Xaa Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 90 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptdie
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 90 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 91 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (22)..(22) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 91 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15
Cys Lys His Ser Met Xaa Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20
25 30 Gly Thr Cys 35 <210> SEQ ID NO 92 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (23)..(23) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 92 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Xaa Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 93 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (24)..(24) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 93 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Xaa Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 94 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (25)..(25) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 94 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Xaa Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 95 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (27)..(27) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 95 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Xaa Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 96 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (29)..(29) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 96 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Xaa Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 97 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (30)..(30) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 97 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Xaa Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 98 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShKpeptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (31)..(31) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 98 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Xaa Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 99 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (33)..(33) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 99 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Xaa
Thr Cys 35 <210> SEQ ID NO 100 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (34)..(34) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 100 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Xaa Cys 35 <210> SEQ ID NO 101 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 101 Ala Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 102 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
102 Arg Ala Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 103 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
103 Arg Ser Cys Ala Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 104
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 104 Arg Ser Cys Ile
Ala Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 105 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 105 Arg Ser Cys Ile Asp Ala Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 106 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
106 Arg Ser Cys Ile Asp Thr Ala Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 107
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 107 Arg Ser Cys Ile
Asp Thr Ile Ala Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 108 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 108 Arg Ser Cys Ile Asp Thr Ile Pro Ala Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 109 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
109 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ala Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 110
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 110 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Ala Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 111 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Shk peptide
<400> SEQUENCE: 111 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Ala Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 112 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
112 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 113
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Shk peptide <400> SEQUENCE: 113 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Ala 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 114 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 114 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Ala His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 115 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
115 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys Ala Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 116
<211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
116 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ala Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 117
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 117 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Ala Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 118 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 118 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Ala Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 119 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
119 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Ala Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 120
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 120 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Ala Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 121 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 121 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Ala Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 122 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
122 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ala Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 123
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 123 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Ala Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 124 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 124 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Ala Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 125 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
125 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Ala
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 126
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 126 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Ala Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 127 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 127 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Ala Thr Cys 35 <210>
SEQ ID NO 128 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
128 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Ala Cys 35 <210> SEQ ID NO 129
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide
<400> SEQUENCE: 129 Lys Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 130 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
130 Arg Lys Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 131
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 131 Arg Ser Cys Lys
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 132 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 132 Arg Ser Cys Ile Lys Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 133 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
133 Arg Ser Cys Ile Asp Lys Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 134
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 134 Arg Ser Cys Ile
Asp Thr Lys Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 135 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 135 Arg Ser Cys Ile Asp Thr Ile Lys Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 136 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
136 Arg Ser Cys Ile Asp Thr Ile Pro Lys Lys Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 137
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 137 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Lys Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 138 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 138 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Lys Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 139 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptdie <400> SEQUENCE:
139 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Lys Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 140
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 140 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Lys Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 141 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 141 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys Lys Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 142 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
142 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15
Cys Lys His Lys Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20
25 30 Gly Thr Cys 35 <210> SEQ ID NO 143 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 143 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Lys Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 144 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
144 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Lys Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 145
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 145 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Lys Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 146 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 146 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Lys Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 147 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
147 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Lys Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 148
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 148 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Lys Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 149 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 149 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Lys Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 150 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <400> SEQUENCE:
150 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Lys Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 151
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK peptide <400> SEQUENCE: 151 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Lys Thr Cys 35 <210> SEQ ID NO 152 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<400> SEQUENCE: 152 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Lys Cys 35 <210>
SEQ ID NO 153 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Shk peptide <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (16)..(16)
<223> OTHER INFORMATION: Ornithine <400> SEQUENCE: 153
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Xaa 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 154 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (16)..(16) <223> OTHER INFORMATION:
alpha, gamma-diaminobutyric acid <400> SEQUENCE: 154 Arg Ser
Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Xaa 1 5 10 15
Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20
25 30 Gly Thr Cys 35 <210> SEQ ID NO 155 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (16)..(16)
<223> OTHER INFORMATION: 3-amino-6-hydroxy-2-piperidone
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norleucine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 155 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Xaa 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 156 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK peptide <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Norleucine <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 156
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Asn 1 5
10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 157 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
peptide <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 157 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe His 1 5 10 15 Cys Lys His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 158 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK PEGylated toxin peptide analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: PEGylated
<400> SEQUENCE: 158 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 159 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK PEGylated toxin peptide analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: PEGylated
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (16)..(16) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 159 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Xaa 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 160 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK PEGylated
toxin peptide analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (27)..(27) <223> OTHER
INFORMATION: 3-(1-naphthyl)alanine <400> SEQUENCE: 160 Arg
Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10
15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Xaa Cys Arg Lys Thr Cys
20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 161 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: ShK
PEGylated toxin peptide analog <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1) <223>
OTHER INFORMATION: PEGylated <400> SEQUENCE: 161 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Ala Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 162 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (27)..(27) <223> OTHER
INFORMATION: 3-(1-naphthyl)alanine <400> SEQUENCE: 162 Arg
Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10
15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Xaa Cys Arg Lys Thr Cys
20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 163 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
PEGylated Toxin Peptide Analog <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1) <223>
OTHER INFORMATION: PEGylated <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (35)..(35) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 163 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 164 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: PEGylated Toxin
Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: PEGylated <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION
<400> SEQUENCE: 164 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 165 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: PEGylated Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: PEGylated
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norvaline
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 165 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 166 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: PEGylated Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: PEGylated
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 166 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Gln Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 167 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: PEGylated Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: PEGylated
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norleucine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 167 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Ala Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 168 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: PEGylated Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: PEGylated
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norleucine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 168 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Glu His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 169 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: PEGylated Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: PEGylated
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norleucine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 169 Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 170 <211> LENGTH: 36 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: PEGylated Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: PEGylated
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norleucine
<400> SEQUENCE: 170 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Ala 35
<210> SEQ ID NO 171 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: PEGylated Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: PEGylated
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norvaline
<400> SEQUENCE: 171 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Ala 35
<210> SEQ ID NO 172 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: PEGylated Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: PEGylated
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norvaline
<400> SEQUENCE: 172 Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Ala 35
<210> SEQ ID NO 173 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: PEGylated Toxin Peptide Analog
<220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: PEGylated <400> SEQUENCE: 173
Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Gln Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys Ala 35 <210> SEQ ID NO 174
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: PEGylated Toxin Peptide Analog <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (16)..(16)
<223> OTHER INFORMATION: PEGylated <400> SEQUENCE: 174
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 175 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination toxin peptide analog (free acid) <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Norleucine <400> SEQUENCE: 175
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5
10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 176 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination toxin peptide analog (free acid) <400> SEQUENCE:
176 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Lys
1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 177
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Combination toxin peptide analog (free acid)
<400> SEQUENCE: 177 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Arg His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 178 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination toxin peptide analog
(free acid) <400> SEQUENCE: 178 Arg Ser Cys Ile Asp Thr Ile
Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Ala
Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 179 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination toxin peptide analog
(free acid) <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <400> SEQUENCE: 179 Arg Ser Cys Ile Asp Thr Ile
Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa
Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 180 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination toxin peptide analog
(free acid) <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (27)..(27) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <400> SEQUENCE: 180 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Xaa Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 181 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
toxin peptide analog (free acid) <400> SEQUENCE: 181 Arg Ser
Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15
Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Lys Lys Thr Cys 20
25 30 Gly Thr Cys 35 <210> SEQ ID NO 182 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
toxin peptide analog (free acid) <400> SEQUENCE: 182 Arg Ser
Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15
Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Arg Thr Cys 20
25 30 Gly Thr Cys 35 <210> SEQ ID NO 183 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
toxin peptide analog (free acid) <400> SEQUENCE: 183 Arg Ser
Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15
Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Glu Thr Cys 20
25 30 Gly Thr Cys 35 <210> SEQ ID NO 184 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
toxin peptide analog (free acid) <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (27)..(27) <223>
OTHER INFORMATION: 3-(1-naphthyl)alanine <400> SEQUENCE: 184
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Xaa Cys Lys Arg Thr
Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 185 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
toxin peptide analog (free acid) <400> SEQUENCE: 185 Arg Ser
Cys Glu Asp Thr Ile Pro Lys Arg Arg Cys Thr Ala Ala Lys 1 5 10 15
Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Arg Thr Cys 20
25 30 Gly Thr Cys 35 <210> SEQ ID NO 186 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 186
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 187 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(27)..(27) <223> OTHER INFORMATION: 3-(1-naphthyl)alanine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 187 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Xaa Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 188 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide} <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 188 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 189 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 189
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Arg Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 190 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide} <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 190 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe
Cys Arg Glu Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
191 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (26)..(26) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 191 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Xaa Phe Cys Lys Arg Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 192 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 192
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Tyr Phe Cys Lys Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 193 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(26)..(26) <223> OTHER INFORMATION: 3-(2-naphthyl)alanine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 193 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Xaa Phe Cys Lys Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 194 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 194
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Glu Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 195 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 195 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Glu His Ser Met Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
196 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 196 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Arg Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 197 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (26)..(26) <223> OTHER
INFORMATION: 3-(1-naphthyl)alanine <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (35)..(35) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 197 Arg Ser Cys
Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Xaa Phe Cys Lys Arg Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 198 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 198
Arg Ser Cys Glu Asp Thr Ile Pro Glu Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Arg His Ser Met Lys Tyr Arg Leu Ser Phe Cys Lys Arg Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 199 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 199 Arg Ser Cys Glu Asp Thr Ile Pro Lys Glu Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Arg His Ser Met Lys Tyr Arg Leu Ser Phe
Cys Lys Arg Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
200 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 200 Arg Ser Cys Glu Asp Thr Ile Pro
Glu Glu Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Arg His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Lys Arg Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 201 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 201 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Lys Arg Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 202 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 202
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Trp Cys Lys Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 203 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Norleucine <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 203 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe
Cys Lys Arg Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 204 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Norleucine <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 204
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Glu His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 205 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Norleucine <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 205 Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
206 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 206 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Ala Lys 1 5 10 15 Cys Lys His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 207 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (27)..(27) <223> OTHER
INFORMATION: 3-(2-naphthyl)alanine <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (35)..(35) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 207 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Met Lys Tyr Arg Leu Ser Xaa Cys Lys Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 208 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 208
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Arg His Ser Met Lys Tyr Arg Leu Ser Phe Cys Lys Arg Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 209 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 209 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Phe Phe
Cys Lys Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
210 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 210 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Lys Ala Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 211 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 211 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Arg
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Lys Arg Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 212 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 212
Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Arg His Ser Met Lys Tyr Arg Leu Ser Phe Cys Lys Arg Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 213 <211>
LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 213
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Lys Arg Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 214 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Norleucine <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 214 Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Ala Lys 1 5 10 15 Cys Glu His Ser Xaa Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
215 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 215 Arg Ser Cys Glu Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Glu His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 216 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 216 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 217 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Norleucine <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 217
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Lys 1 5
10 15 Cys Glu His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 218 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Norleucine <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 218 Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe
Cys Arg Arg Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
219 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 219 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Glu His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Arg Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 220 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 220 Arg Ser Cys Glu
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Glu
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Arg Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 221 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Norleucine <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 221
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Ala Lys 1 5
10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Arg Thr
Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 222 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Norleucine <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 222
Arg Ser Cys Ile Asp Thr Ile Pro Glu Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 223 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Norleucine <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 223 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Ala Lys 1 5 10 15 Cys Glu His Ser Xaa Lys Tyr Arg Leu Ser Phe
Cys Arg Arg Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
224 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 224 Arg Ser Cys Glu Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Ala Lys 1 5 10 15 Cys Lys His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Arg Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 225 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 225 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Glu Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 226 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (26)..(26)
<223> OTHER INFORMATION: 3-(1-naphthyl)alanine <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 226 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Xaa Phe
Cys Lys Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
227 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norleucine <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (26)..(26) <223> OTHER INFORMATION:
3-(1-naphthyl)alanine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 227 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Xaa Phe Cys Lys Arg Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 228 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 228
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Tyr Cys Lys Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 229 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 229 Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Thr Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
230 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 230 Arg Ser Cys Glu Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Ser Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys
20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 231 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Combination Toxin Peptide Analog {C-Terminal Amide } <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Norleucine <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(35)..(35) <223> OTHER INFORMATION: AMIDATION <400>
SEQUENCE: 231 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe
Cys Lys Ala Thr Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO
232 <211> LENGTH: 35 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Combination Toxin Peptide Analog {C-Terminal
Amide } <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norvaline <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 232 Arg Ser Cys Glu Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 233 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Combination Toxin Peptide Analog
{C-Terminal Amide } <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Aminobutyric acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (35)..(35) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 233 Arg Ser Cys
Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 234 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Combination
Toxin Peptide Analog {C-Terminal Amide } <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 234
Arg Ser Cys Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Gln Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 235 <211>
LENGTH: 36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Toxin
Peptide Analog with C-Terminal extension <400> SEQUENCE: 235
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys Ala 35 <210> SEQ ID NO 236
<211> LENGTH: 36 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Toxin Peptide Analog with C-Terminal extension
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norleucine
<400> SEQUENCE: 236 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Ala 35
<210> SEQ ID NO 237 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension <400> SEQUENCE: 237 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Asn Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Ala 35
<210> SEQ ID NO 238 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norvaline <400> SEQUENCE: 238 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Ala 35
<210> SEQ ID NO 239 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Norvaline <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (36)..(36) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 239 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Ala 35
<210> SEQ ID NO 240 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension <400> SEQUENCE: 240 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Gln Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys
Ala
35 <210> SEQ ID NO 241 <211> LENGTH: 36 <212>
TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Toxin Peptide Analog with
C-Terminal extension <400> SEQUENCE: 241 Arg Ser Cys Glu Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His
Ser Gln Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys Ala 35 <210> SEQ ID NO 242 <211> LENGTH: 36
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norvaline <400> SEQUENCE: 242 Arg Ser Cys
Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys Ala 35 <210> SEQ ID NO 243 <211> LENGTH:
36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norleucine <400> SEQUENCE: 243 Arg Ser Cys
Glu Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys Ala 35 <210> SEQ ID NO 244 <211> LENGTH:
36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norleucine <400> SEQUENCE: 244 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys Glu 35 <210> SEQ ID NO 245 <211> LENGTH:
36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norleucine <400> SEQUENCE: 245 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys Tyr 35 <210> SEQ ID NO 246 <211> LENGTH:
36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norleucine <400> SEQUENCE: 246 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys Val 35 <210> SEQ ID NO 247 <211> LENGTH:
36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norvaline <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (36)..(36) <223>
OTHER INFORMATION: Beta Ala <400> SEQUENCE: 247 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys Xaa 35 <210> SEQ ID NO 248 <211> LENGTH:
36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension {and C-Terminal Amide }
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norvaline
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (36)..(36) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 248 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Asp 35
<210> SEQ ID NO 249 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension {and C-Terminal Amide } <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norvaline <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (36)..(36) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 249 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys Glu 35 <210> SEQ ID NO 250 <211> LENGTH:
36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension {and C-Terminal Amide }
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norleucine
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (36)..(36) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 250 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15
Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20
25 30 Gly Thr Cys Asp 35 <210> SEQ ID NO 251 <211>
LENGTH: 36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Toxin
Peptide Analog with C-Terminal extension {and C-Terminal Amide }
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norvaline
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (36)..(36) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 251 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Ser 35
<210> SEQ ID NO 252 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension {and C-Terminal Amide } <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norvaline <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (36)..(36) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 252 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys Thr 35 <210> SEQ ID NO 253 <211> LENGTH:
36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Toxin Peptide
Analog with C-Terminal extension {and C-Terminal Amide }
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norvaline
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (36)..(36) <223> OTHER INFORMATION: Aminoisobutyric
acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (36)..(36) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 253 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys Xaa 35
<210> SEQ ID NO 254 <211> LENGTH: 36 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Toxin Peptide Analog with C-Terminal
extension {and C-Terminal Amide } <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21) <223>
OTHER INFORMATION: Norvaline <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (36)..(36) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 254 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys Ala 35 <210> SEQ ID NO 255 <211> LENGTH:
35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Norleucine <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 255 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys
His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 256 <400> SEQUENCE: 256
000 <210> SEQ ID NO 257 <211> LENGTH: 35 <212>
TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Position 21 Toxin Peptide
Analog <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION:
Methinine oxide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (35)..(35) <223> OTHER INFORMATION:
AMIDATION <400> SEQUENCE: 257 Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Xaa Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35
<210> SEQ ID NO 258 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 258 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Ser Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 259 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 259 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Thr Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 260 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Norvaline
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (35)..(35) <223> OTHER INFORMATION: AMIDATION
<400> SEQUENCE: 260
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 261 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Position 21 Toxin Peptide Analog <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (35)..(35) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 261 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Gln Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 262 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 262 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser His Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 263 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 263 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Tyr Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 264 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 264 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Asn Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 265 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 265 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Val Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 266 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 266 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Leu Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 267 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 267 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Trp Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 268 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Aminobutyric acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (35)..(35) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 268 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 269 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Cyclohexylalanine <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (35)..(35) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 269 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 270 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Cyclohexylglycine <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (35)..(35) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 270 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35
<210> SEQ ID NO 271 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Position 21 Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION:
4-hydroxyproline (or hydroxyproline) <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (35)..(35)
<223> OTHER INFORMATION: AMIDATION <400> SEQUENCE: 271
Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5
10 15 Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys 20 25 30 Gly Thr Cys 35 <210> SEQ ID NO 272 <211>
LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Position 21 Toxin Peptide Analog <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (35)..(35) <223>
OTHER INFORMATION: AMIDATION <400> SEQUENCE: 272 Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys
Lys His Ser Phe Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25
30 Gly Thr Cys 35 <210> SEQ ID NO 273 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 273 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Ile Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 274 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Position 21
Toxin Peptide Analog <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: AMIDATION <400> SEQUENCE: 274 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys
His Ser Asp Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30
Gly Thr Cys 35 <210> SEQ ID NO 275 <211> LENGTH: 83
<212> TYPE: PRT <213> ORGANISM: Anemonia erythraea
<220> FEATURE: <221> NAME/KEY: SIGNAL <222>
LOCATION: (1)..(83) <223> OTHER INFORMATION: Putative signal
sequence <400> SEQUENCE: 275 Met Lys Gly Gln Met Ile Ile Cys
Leu Val Leu Ile Ala Leu Cys Met 1 5 10 15 Ser Val Val Val Met Ala
Gln Asn Leu Arg Ala Glu Glu Leu Glu Lys 20 25 30 Ala Asn Pro Lys
Asp Glu Arg Val Arg Ser Phe Glu Arg Asn Gln Lys 35 40 45 Arg Ala
Cys Lys Asp Tyr Leu Pro Lys Ser Glu Cys Thr Gln Phe Arg 50 55 60
Cys Arg Thr Ser Met Lys Tyr Lys Tyr Thr Asn Cys Lys Lys Thr Cys 65
70 75 80 Gly Thr Cys <210> SEQ ID NO 276 <211> LENGTH:
74 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Hmk peptide
sequence <400> SEQUENCE: 276 Met Lys Ser Gln Met Ile Ala Ala
Val Leu Leu Ile Ala Phe Cys Leu 1 5 10 15 Cys Val Val Val Thr Ala
Arg Met Glu Leu Gln Asp Val Glu Asp Met 20 25 30 Glu Asn Gly Phe
Gln Lys Arg Arg Thr Cys Lys Asp Leu Ile Pro Val 35 40 45 Ser Glu
Cys Thr Asp Ile Arg Cys Arg Thr Ser Met Lys Tyr Arg Leu 50 55 60
Asn Leu Cys Arg Lys Thr Cys Gly Ser Cys 65 70 <210> SEQ ID NO
277 <211> LENGTH: 738 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (1)..(738) <400> SEQUENCE: 277 atg
gaa tgg agc tgg gtc ttt ctc ttc ttc ctg tca gta acg act ggt 48 Met
Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10
15 gtc cac tcc gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa
96 Val His Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
20 25 30 ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc
aag gac 144 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp 35 40 45 acc ctc atg atc tcc cgg acc cct gag gtc aca tgc
gtg gtg gtg gac 192 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp 50 55 60 gtg agc cac gaa gac cct gag gtc aag ttc
aac tgg tac gtg gac ggc 240 Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly 65 70 75 80 gtg gag gtg cat aat gcc aag aca
aag ccg cgg gag gag cag tac aac 288 Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95 agc acg tac cgt gtg gtc
agc gtc ctc acc gtc ctg cac cag gac tgg 336 Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp 100 105 110 ctg aat ggc aag
gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 384 Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125 gcc ccc
atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa 432 Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140
cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac 480
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 145
150 155 160 cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc
gac atc 528 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile 165 170 175 gcc gtg gag tgg gag agc aat ggg cag ccg gag aac
aac tac aag acc 576 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr 180 185 190 acg cct ccc gtg ctg gac tcc gac ggc tcc
ttc ttc ctc tac agc aag 624 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys 195 200 205 ctc acc gtg gac aag agc agg tgg
cag cag ggg aac gtc ttc tca tgc 672 Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys 210 215 220 tcc gtg atg cat gag gct
ctg cac aac cac tac acg cag aag agc ctc 720 Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu 225 230 235 240 tcc ctg tct
ccg ggt aaa 738 Ser Leu Ser Pro Gly Lys 245 <210> SEQ ID NO
278 <211> LENGTH: 246 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 278 Met Glu Trp Ser
Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35
40 45 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp 50 55 60 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly 65 70 75 80 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn 85 90 95
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 100
105 110 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro 115 120 125 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu 130 135 140 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn 145 150 155 160 Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175 Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190 Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205 Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 225
230 235 240 Ser Leu Ser Pro Gly Lys 245 <210> SEQ ID NO 279
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Linker sequence <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(2) <223>
OTHER INFORMATION: Xaa in positions 1-2 are each independently any
amino acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: Xaa in positions 4-5 are each independently any amino
acid residue <400> SEQUENCE: 279 Xaa Xaa Asn Xaa Xaa Gly 1 5
<210> SEQ ID NO 280 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptidyl linker <400>
SEQUENCE: 280 Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> SEQ
ID NO 281 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Peptidyl linker <400> SEQUENCE: 281 Gly
Gly Gly Gly Gly 1 5 <210> SEQ ID NO 282 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptidyl linker
<400> SEQUENCE: 282 Gly Gly Gly Gly Gly Gly Gly 1 5
<210> SEQ ID NO 283 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptidyl linker <400>
SEQUENCE: 283 Gly Gly Gly Gly Ser 1 5 <210> SEQ ID NO 284
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Peptidyl linker <400> SEQUENCE: 284 Gly Gly Gly
Gly Gly Lys 1 5 <210> SEQ ID NO 285 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptidyl linker
<400> SEQUENCE: 285 Gly Gly Gly Gly Gly Lys Arg 1 5
<210> SEQ ID NO 286 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptidyl linker <400>
SEQUENCE: 286 Gly Gly Gly Lys Gly Gly Gly Gly 1 5 <210> SEQ
ID NO 287 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Peptidyl linker <400> SEQUENCE: 287 Gly
Gly Gly Asn Gly Ser Gly Gly 1 5 <210> SEQ ID NO 288
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Peptidyl linker <400> SEQUENCE: 288 Gly Gly Gly
Cys Gly Gly Gly Gly 1 5 <210> SEQ ID NO 289 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Peptidyl linker <400> SEQUENCE: 289 Gly Pro Asn Gly Gly 1 5
<210> SEQ ID NO 290 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptidyl linker <400>
SEQUENCE: 290 Gly Gly Gly Lys Gly Gly Gly Gly 1 5 <210> SEQ
ID NO 291 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Peptidyl linker <400> SEQUENCE: 291 Gly
Gly Gly Gly Ser 1 5 <210> SEQ ID NO 292 <211> LENGTH:
10 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptidyl linker
<400> SEQUENCE: 292 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1
5 10 <210> SEQ ID NO 293 <211> LENGTH: 25 <212>
TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Peptidyl linker <400>
SEQUENCE: 293 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25
<210> SEQ ID NO 294 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptide linker <400> SEQUENCE:
294 Gly Gly Glu Gly Gly Gly
1 5 <210> SEQ ID NO 295 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Peptide linker <400>
SEQUENCE: 295 Gly Gly Glu Glu Glu Gly Gly Gly 1 5 <210> SEQ
ID NO 296 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Peptide linker <400> SEQUENCE: 296 Gly Glu
Glu Glu Gly 1 5 <210> SEQ ID NO 297 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptide linker
<400> SEQUENCE: 297 Gly Glu Glu Glu 1 <210> SEQ ID NO
298 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Peptide linker <400> SEQUENCE: 298 Gly Gly
Asp Gly Gly Gly 1 5 <210> SEQ ID NO 299 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptide linker
<400> SEQUENCE: 299 Gly Gly Asp Asp Asp Gly Gly 1 5
<210> SEQ ID NO 300 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptide linker <400> SEQUENCE:
300 Gly Asp Asp Asp Gly 1 5 <210> SEQ ID NO 301 <211>
LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Peptide linker <400> SEQUENCE: 301 Gly Asp Asp Asp 1
<210> SEQ ID NO 302 <211> LENGTH: 21 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptide linker <400> SEQUENCE:
302 Gly Gly Gly Gly Ser Asp Asp Ser Asp Glu Gly Ser Asp Gly Glu Asp
1 5 10 15 Gly Gly Gly Gly Ser 20 <210> SEQ ID NO 303
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Peptide linker <400> SEQUENCE: 303 Trp Glu Trp
Glu Trp 1 5 <210> SEQ ID NO 304 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptide linker
<400> SEQUENCE: 304 Phe Glu Phe Glu Phe 1 5 <210> SEQ
ID NO 305 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Peptide linker <400> SEQUENCE: 305 Glu Glu
Glu Trp Trp Trp 1 5 <210> SEQ ID NO 306 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptide linker
<400> SEQUENCE: 306 Glu Glu Glu Phe Phe Phe 1 5 <210>
SEQ ID NO 307 <211> LENGTH: 7 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptide linker <400> SEQUENCE:
307 Trp Trp Glu Glu Glu Trp Trp 1 5 <210> SEQ ID NO 308
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Peptide linker <400> SEQUENCE: 308 Phe Phe Glu
Glu Glu Phe Phe 1 5 <210> SEQ ID NO 309 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Peptide linker
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(2) <223> OTHER INFORMATION: Xaa in positions
1-2 are each independently any amino acid residue <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(4)..(5) <223> OTHER INFORMATION: Xaa in positions 4-5 are
each independently any amino acid residue <400> SEQUENCE: 309
Xaa Xaa Tyr Xaa Xaa Gly 1 5 <210> SEQ ID NO 310 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Peptide linker <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(2) <223> OTHER
INFORMATION: Xaa in positions 1-2 are each independently any amino
acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (4)..(5) <223> OTHER
INFORMATION: Xaa in positions 4-5 are each independently any amino
acid residue <400> SEQUENCE: 310 Xaa Xaa Ser Xaa Xaa Gly 1 5
<210> SEQ ID NO 311 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Peptide linker <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(2)
<223> OTHER INFORMATION: Xaa in positions 1-2 are each
independently any amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (4)..(5)
<223> OTHER INFORMATION: Xaa in positions 4-5 are each
independently any amino acid residue <400> SEQUENCE: 311 Xaa
Xaa Thr Xaa Xaa Gly 1 5 <210> SEQ ID NO 312 <211>
LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Flexible peptide linker <400> SEQUENCE: 312 Gly Ser Gly Ser
Ala Thr Gly Gly Ser Gly Ser Thr Ala Ser Ser Gly 1 5 10 15 Ser Gly
Ser Ala Thr His 20 <210> SEQ ID NO 313 <211> LENGTH: 22
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Flexible
peptide linker <400> SEQUENCE: 313 His Gly Ser Gly Ser Ala
Thr Gly Gly Ser Gly Ser Thr Ala Ser Ser 1 5 10 15 Gly Ser Gly Ser
Ala Thr 20 <210> SEQ ID NO 314 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Flexible
peptide linker <400> SEQUENCE: 314 Ala Glu Ala Ala Ala Lys
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 Ala Gly Gly
<210> SEQ ID NO 315 <211> LENGTH: 35 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Reference peptide <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(1) <223> OTHER INFORMATION: Nalpha branched PEGylated
<400> SEQUENCE: 315 Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His Ser Met Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly Thr Cys 35 <210>
SEQ ID NO 316 <211> LENGTH: 36 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: PEGylated Toxin Peptide Analog
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: 20kDa
PEG-[Lys16]Shk-Ala <400> SEQUENCE: 316 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 1 5 10 15 Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys Ala 35 <210> SEQ ID NO 317 <211> LENGTH: 35
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: ShK peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (22)..(22) <223> OTHER INFORMATION:
Diaminopropionic acid <400> SEQUENCE: 317 Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln 1 5 10 15 Cys Lys His
Ser Met Xaa Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 20 25 30 Gly
Thr Cys 35 <210> SEQ ID NO 318 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: modifed
VH21signal peptide <400> SEQUENCE: 318 Met Glu Trp Ser Trp
Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser
<210> SEQ ID NO 319 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 319 catgaattcc ccaccatgga atggagctgg 30 <210> SEQ
ID NO 320 <211> LENGTH: 38 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 320
cacggtgggc actcgacttt gcgctcggag tggacacc 38 <210> SEQ ID NO
321 <211> LENGTH: 132 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: ShK[2-35] with N-terminal linker extension
<400> SEQUENCE: 321 ggaggaggag gatccggagg aggaggaagc
agctgcatcg acaccatccc caagagccgc 60 tgcaccgcct tccagtgcaa
gcacagcatg aagtaccgcc tgagcttctg ccgcaagacc 120 tgcggcacct gc 132
<210> SEQ ID NO 322 <211> LENGTH: 44 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK[2-35] with N-terminal linker
extension <400> SEQUENCE: 322 Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Ser Cys Ile Asp Thr Ile 1 5 10 15 Pro Lys Ser Arg Cys Thr
Ala Phe Gln Cys Lys His Ser Met Lys Tyr 20 25 30 Arg Leu Ser Phe
Cys Arg Lys Thr Cys Gly Thr Cys 35 40 <210> SEQ ID NO 323
<211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 323 gtccactccg
agcgcaaagt cgagtgccca ccgtgcc 37 <210> SEQ ID NO 324
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 324 tcctcctcct
ttacccggag acagggagag 30 <210> SEQ ID NO 325 <211>
LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 325 gctgcaccgc cttcaagtgc aagcacagc
29 <210> SEQ ID NO 326 <211> LENGTH: 29 <212>
TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 326 gctgtgcttg cacttgaagg cggtgcagc 29 <210> SEQ ID
NO 327
<211> LENGTH: 132 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: ShK[2-35, K16] with N-terminal linker extension
<400> SEQUENCE: 327 ggaggaggag gatccggagg aggaggaagc
agctgcatcg acaccatccc caagagccgc 60 tgcaccgcct tcaagtgcaa
gcacagcatg aagtaccgcc tgagcttctg ccgcaagacc 120 tgcggcacct gc 132
<210> SEQ ID NO 328 <211> LENGTH: 44 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK[2-35, Q16K] with N-terminal
linker extension <400> SEQUENCE: 328 Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Ser Cys Ile Asp Thr Ile 1 5 10 15 Pro Lys Ser Arg
Cys Thr Ala Phe Lys Cys Lys His Ser Met Lys Tyr 20 25 30 Arg Leu
Ser Phe Cys Arg Lys Thr Cys Gly Thr Cys 35 40 <210> SEQ ID NO
329 <211> LENGTH: 28 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 329
ccgggtaaag gaggaggagg atccggag 28 <210> SEQ ID NO 330
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 330 catgcggccg
ctcattagca ggtg 24 <210> SEQ ID NO 331 <211> LENGTH:
648 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: Coding sequence of fragment of immunoglobulin Fc
domain of human IgG2 <400> SEQUENCE: 331 gcaccacctg
tggcaggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 60
atgatctccc ggacccctga ggtcacgtgc gtggtggtgg acgtgagcca cgaagacccc
120 gaggtccagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa
gacaaagcca 180 cgggaggagc agttcaacag cacgttccgt gtggtcagcg
tcctcaccgt tgtgcaccag 240 gactggctga acggcaagga gtacaagtgc
aaggtctcca acaaaggcct cccagccccc 300 atcgagaaaa ccatctccaa
aaccaaaggg cagccccgag aaccacaggt gtacaccctg 360 cccccatccc
gggaggagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 420
ttctacccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac
480 aagaccacac ctcccatgct ggactccgac ggctccttct tcctctacag
caagctcacc 540 gtggacaaga gcaggtggca gcaggggaac gtcttctcat
gctccgtgat gcatgaggct 600 ctgcacaacc actacacgca gaagagcctc
tccctgtctc cgggtaaa 648 <210> SEQ ID NO 332 <211>
LENGTH: 216 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<223> OTHER INFORMATION: Fragment of immunoglobulin Fc domain
of human IgG2 <400> SEQUENCE: 332 Ala Pro Pro Val Ala Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val
Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 35 40 45 Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55
60 Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln
65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys Gly Gln Pro 100 105 110 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr 115 120 125 Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140 Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 145 150 155 160 Lys Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165 170 175 Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 180 185
190 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
195 200 205 Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> SEQ
ID NO 333 <211> LENGTH: 511 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: IgG2-HC-L10-ShK[1-35, Q16K] fusion polypeptide
<400> SEQUENCE: 333 Met Asp Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val
Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro Gly Ala
Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr
Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50 55 60 Gln Gly
Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr 65 70 75 80
Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 85
90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp
Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Gly Ser Tyr
Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205
Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys 210
215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val
Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala
Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305 310 315 320 Arg
Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly 325 330
335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile
340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro
Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455
460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Ser Cys Ile
465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys Cys
Lys His Ser
485 490 495 Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys Gly Thr
Cys 500 505 510 <210> SEQ ID NO 334 <211> LENGTH: 290
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION:
VH21SP-IgG2-Fc-L10-ShK(1-35, Q16K) Fusion polypeptide <400>
SEQUENCE: 334 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val
Thr Thr Gly 1 5 10 15 Val His Ser Glu Arg Lys Val Glu Cys Pro Pro
Cys Pro Ala Pro Pro 20 25 30 Val Ala Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser His Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 100 105
110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala
115 120 125 Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 195 200 205 Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 210 215 220 Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230
235 240 Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Arg 245 250 255 Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala
Phe Gln Cys 260 265 270 Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys
Arg Lys Thr Cys Gly 275 280 285 Thr Cys 290 <210> SEQ ID NO
335 <211> LENGTH: 30 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 335
cataagcttc ccaccatgga atggagctgg 30 <210> SEQ ID NO 336
<211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 336 catggatcct
catttacccg gagacaggga g 31 <210> SEQ ID NO 337 <211>
LENGTH: 245 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<223> OTHER INFORMATION: Immunoglobulin Fc domain of human
IgG2 <400> SEQUENCE: 337 Met Glu Trp Ser Trp Val Phe Leu Phe
Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Glu Arg Lys Val
Glu Cys Pro Pro Cys Pro Ala Pro Pro 20 25 30 Val Ala Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser
His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70
75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser 85 90 95 Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln
Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ala 115 120 125 Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 195
200 205 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser 225 230 235 240 Leu Ser Pro Gly Lys 245 <210> SEQ
ID NO 338 <211> LENGTH: 236 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: aKLH 120.6 kappa LC <400> SEQUENCE: 338
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5
10 15 Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser 35 40 45 Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln
Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Arg Leu Ile Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110 His Asn Ser
Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135
140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> SEQ ID NO 339
<211> LENGTH: 467 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: aKLH 120.6 IgG2 HC <400> SEQUENCE: 339 Met Asp
Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15
Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20
25 30 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser
Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln
Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro
Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg
Val Thr Met Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr
Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150
155 160
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165
170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr
Gln Thr Tyr Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290
295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Phe 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp
Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys
Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410
415 Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 <210> SEQ ID NO
340 <211> LENGTH: 290 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: IgG2 Fc-L10-ShK(1-35) <400> SEQUENCE: 340
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5
10 15 Val His Ser Glu Arg Lys Val Glu Cys Pro Pro Cys Pro Ala Pro
Pro 20 25 30 Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 50 55 60 Ser His Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Phe Arg Val Val
Ser Val Leu Thr Val Val His Gln Asp Trp Leu 100 105 110 Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala 115 120 125 Pro
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro 130 135
140 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Met Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser
Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 245 250 255
Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln Cys 260
265 270 Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys
Gly 275 280 285 Thr Cys 290 <210> SEQ ID NO 341 <211>
LENGTH: 511 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: aKLH
120.6 IgG2-ShK fusion <400> SEQUENCE: 341 Met Asp Met Arg Val
Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly
Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40
45 Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly
50 55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly
Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met
Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser
Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg
Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170
175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln
Thr Tyr Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys
Val Asp Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys
Pro Pro Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295
300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
305 310 315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly
Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415
Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420
425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Arg Ser Cys Ile 465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys
Thr Ala Phe Gln Cys Lys His Ser 485 490 495 Met Lys Tyr Arg Leu Ser
Phe Cys Arg Lys Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID
NO 342 <211> LENGTH: 511 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: aKLH 120.6 IgG2-ShK[1-35, Q16K] fusion
<400> SEQUENCE: 342 Met Asp Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val
Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro Gly Ala
Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45
Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50
55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly
Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr
Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg
Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp
Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180
185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser 195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
Tyr Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro
Pro Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305
310 315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu
Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln
Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425
430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Arg Ser Cys Ile 465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys Thr
Ala Phe Lys Cys Lys His Ser 485 490 495 Met Lys Tyr Arg Leu Ser Phe
Cys Arg Lys Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID NO
343 <211> LENGTH: 471 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: aKLH 120.6 IgG1 HC <400> SEQUENCE: 343 Met
Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10
15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu
20 25 30 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala
Ser Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg
Gln Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn
Pro Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly
Arg Val Thr Met Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr
Tyr Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145
150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265
270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 370 375 380 Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390
395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys
465 470 <210> SEQ ID NO 344 <211> LENGTH: 510
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: aKLH 120.6
IgG1-loop-ShK <400> SEQUENCE: 344 Met Asp Met Arg Val Pro Ala
Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg
Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr
Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50 55
60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr
65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg
Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg
Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185
190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290
295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Gly Gly 370 375 380 Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys 385 390 395 400 Cys
Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys 405 410
415 Gly Thr Cys Gly Gly Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
420 425 430 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly 435 440 445 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp 450 455 460 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp 465 470 475 480 Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His 485 490 495 Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 510 <210> SEQ ID
NO 345 <211> LENGTH: 289 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: VH21SP-IgG2-Fc-L10-ShK(1-35, Q16K) Fusion
polypeptide <400> SEQUENCE: 345 Met Glu Trp Ser Trp Val Phe
Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Glu Arg
Lys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro 20 25 30 Val Ala Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55
60 Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser 85 90 95 Thr Phe Arg Val Val Ser Val Leu Thr Val Val His
Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ala 115 120 125 Pro Ile Glu Lys Thr Ile Ser Lys
Thr Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185
190 Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 225 230 235 240 Leu Ser Pro Gly Lys Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Ser 245 250 255 Cys Ile Asp Thr Ile Pro Lys
Ser Arg Cys Thr Ala Phe Gln Cys Lys 260 265 270 His Ser Met Lys Tyr
Arg Leu Ser Phe Cys Arg Lys Thr Cys Gly Thr 275 280 285 Cys
<210> SEQ ID NO 346 <211> LENGTH: 289 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: VH21SP-IgG2-Fc-L10-ShK(1-35, Q16K)
Fusion polypeptide <400> SEQUENCE: 346 Met Glu Trp Ser Trp
Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser
Glu Arg Lys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro 20 25 30 Val
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40
45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
50 55 60 Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser 85 90 95 Thr Phe Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ala 115 120 125 Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170
175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
180 185 190 Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Pro Gly Lys Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Ser 245 250 255 Cys Ile Asp Thr Ile
Pro Lys Ser Arg Cys Thr Ala Phe Lys Cys Lys 260 265 270 His Ser Met
Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys Gly Thr 275 280 285 Cys
<210> SEQ ID NO 347 <211> LENGTH: 289 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Fusion polypeptide <400>
SEQUENCE: 347 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val
Thr Thr Gly 1 5 10 15 Val His Ser Glu Arg Lys Val Glu Cys Pro Pro
Cys Pro Ala Pro Pro 20 25 30 Val Ala Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser His Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 100 105
110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala
115 120 125 Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 195 200 205 Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 210 215 220 Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230
235 240 Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Ser 245 250 255 Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe
Lys Cys Lys 260 265 270 His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg
Lys Thr Cys Gly Thr 275 280 285 Cys <210> SEQ ID NO 348
<211> LENGTH: 290 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: IgG2 Fc-L10-ShK(1-35, Q16K) fusion protein <400>
SEQUENCE: 348 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val
Thr Thr Gly
1 5 10 15 Val His Ser Glu Arg Lys Val Glu Cys Pro Pro Cys Pro Ala
Pro Pro 20 25 30 Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 50 55 60 Ser His Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Phe Arg Val
Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 100 105 110 Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala 115 120 125
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro 130
135 140 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Met Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu 195 200 205 Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu
Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 245 250
255 Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys Cys
260 265 270 Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr
Cys Gly 275 280 285 Thr Cys 290 <210> SEQ ID NO 349
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Random primer with an extension adapter <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(18)..(23) <223> OTHER INFORMATION: Any deoxriboneucleotide
<400> SEQUENCE: 349 ggccggatag gcctccannn nnnt 24 <210>
SEQ ID NO 350 <211> LENGTH: 45 <212> TYPE: DNA
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Forward primer <400> SEQUENCE:
350 gtggttgaga ggtgccagat gtgacattgt gatgactcag tctcc 45
<210> SEQ ID NO 351 <211> LENGTH: 41 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Reverse primer <400> SEQUENCE:
351 aaccgtttaa acgcggccgc tcaacactct cccctgttga a 41 <210>
SEQ ID NO 352 <211> LENGTH: 43 <212> TYPE: DNA
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Optimized Kozak seqence <400>
SEQUENCE: 352 aagctcgagg tcgactagac caccatggac atgagggtcc ccg 43
<210> SEQ ID NO 353 <211> LENGTH: 45 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 353 aagctcgagg tcgactagac caccatggac atgagggtgc ccgct 45
<210> SEQ ID NO 354 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 354 tcatctggat gtcacatctg gcacc 25 <210> SEQ ID NO
355 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 355
ggtgccagat gtgacatcca gatga 25 <210> SEQ ID NO 356
<211> LENGTH: 1398 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: aKLH 120.6-VK1SP-IgG2 Heavy Chain coding
sequence <400> SEQUENCE: 356 atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcc 60 agatgtcagg tgcagctggt
gcagtctggg gctgaggtga agaagcctgg ggcctcagtg 120 aaggtctcct
gcaaggcttc tggatacacc ttcaccggct accacatgca ctgggtgcga 180
caggcccctg gacaagggct tgagtggatg ggatggatca accctaacag tggtggcaca
240 aactatgcac agaagtttca gggcagggtc accatgacca gggacacgtc
catcagcaca 300 gcctacatgg agctgagcag gctgagatct gacgacacgg
ccgtgtatta ctgtgcgaga 360 gatcgtggga gctactactg gttcgacccc
tggggccagg gaaccctggt caccgtctcc 420 tcagcctcca ccaagggccc
atcggtcttc cccctggcgc cctgctccag gagcacctcc 480 gagagcacag
cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 540
tcgtggaact caggcgctct gaccagcggc gtgcacacct tcccagctgt cctacagtcc
600 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcaactt
cggcacccag 660 acctacacct gcaacgtaga tcacaagccc agcaacacca
aggtggacaa gacagttgag 720 cgcaaatgtt gtgtcgagtg cccaccgtgc
ccagcaccac ctgtggcagg accgtcagtc 780 ttcctcttcc ccccaaaacc
caaggacacc ctcatgatct cccggacccc tgaggtcacg 840 tgcgtggtgg
tggacgtgag ccacgaagac cccgaggtcc agttcaactg gtacgtggac 900
ggcgtggagg tgcataatgc caagacaaag ccacgggagg agcagttcaa cagcacgttc
960 cgtgtggtca gcgtcctcac cgttgtgcac caggactggc tgaacggcaa
ggagtacaag 1020 tgcaaggtct ccaacaaagg cctcccagcc cccatcgaga
aaaccatctc caaaaccaaa 1080 gggcagcccc gagaaccaca ggtgtacacc
ctgcccccat cccgggagga gatgaccaag 1140 aaccaggtca gcctgacctg
cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 1200 tgggagagca
atgggcagcc ggagaacaac tacaagacca cacctcccat gctggactcc 1260
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg
1320 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac
gcagaagagc 1380 ctctccctgt ctccgggt 1398 <210> SEQ ID NO 357
<211> LENGTH: 466 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: aKLH 120.6-VK1SP-IgG2 Heavy Chain <400>
SEQUENCE: 357 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu
Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val
Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro Gly Ala Ser Val Lys
Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His
Met His Trp Val Arg Gln Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp
Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala
Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 85 90 95 Ser
Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105
110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe
115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205
Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys 210
215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val
Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala
Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305 310 315 320 Arg
Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly 325 330
335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile
340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro
Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455
460 Pro Gly 465 <210> SEQ ID NO 358 <211> LENGTH: 34
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 358 ggatcctcct cctccacccg gagacaggga gagg 34
<210> SEQ ID NO 359 <211> LENGTH: 34 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 359 tccctgtctc cgggtggagg aggaggatcc ggag 34 <210>
SEQ ID NO 360 <211> LENGTH: 39 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Gly-Gly-ShK-Gly-Gly sequence
<400> SEQUENCE: 360 Gly Gly Arg Ser Cys Ile Asp Thr Ile Pro
Lys Ser Arg Cys Thr Ala 1 5 10 15 Phe Gln Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys Arg Lys 20 25 30 Thr Cys Gly Thr Cys Gly
Gly 35 <210> SEQ ID NO 361 <211> LENGTH: 127
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Forward primer
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (6)..(11) <223> OTHER INFORMATION: HINDIII Site
<400> SEQUENCE: 361 tgcagaagct tctagaccac catggaatgg
agctgggtct ttctcttctt cctgtcagta 60 acgactggtg tccactcccg
cagctgcatc gacaccatcc ccaagagccg ctgcaccgcc 120 ttccagt 127
<210> SEQ ID NO 362 <211> LENGTH: 115 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Reverse primer <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (5)..(10)
<223> OTHER INFORMATION: BAMHI Site <400> SEQUENCE: 362
ctccggatcc tcctcctccg caggtgccgc aggtcttgcg gcagaagctc aggcggtact
60 tcatgctgtg cttgcactgg aaggcggtgc agcggctctt ggggatggtg tcgat 115
<210> SEQ ID NO 363 <211> LENGTH: 38 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Forward primer <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (4)..(9)
<223> OTHER INFORMATION: BAMHI Site <400> SEQUENCE: 363
gtaggatccg gaggaggagg aagcgacaaa actcacac 38 <210> SEQ ID NO
364 <211> LENGTH: 35 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Reverse primer <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (4)..(11) <223>
OTHER INFORMATION: NOTI Site <400> SEQUENCE: 364 cgagcggccg
cttactattt acccggagac aggga 35 <210> SEQ ID NO 365
<211> LENGTH: 879 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: VH21 SP-ShK(1-35)-L10-IgG1 Fc coding sequence
<400> SEQUENCE: 365 atggaatgga gctgggtctt tctcttcttc
ctgtcagtaa cgactggtgt ccactcccgc 60 agctgcatcg acaccatccc
caagagccgc tgcaccgcct tccagtgcaa gcacagcatg 120 aagtaccgcc
tgagcttctg ccgcaagacc tgcggcacct gcggaggagg aggatccgga 180
ggaggaggaa gcgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg
240 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat
ctcccggacc 300 cctgaggtca catgcgtggt ggtggacgtg agccacgaag
accctgaggt caagttcaac 360 tggtacgtgg acggcgtgga ggtgcataat
gccaagacaa agccgcggga ggagcagtac 420 aacagcacgt accgtgtggt
cagcgtcctc accgtcctgc accaggactg gctgaatggc 480 aaggagtaca
agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 540
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat
600 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta
tcccagcgac 660 atcgccgtgg agtgggagag caatgggcag ccggagaaca
actacaagac cacgcctccc 720 gtgctggact ccgacggctc cttcttcctc
tacagcaagc tcaccgtgga caagagcagg 780 tggcagcagg ggaacgtctt
ctcatgctcc gtgatgcatg aggctctgca caaccactac 840 acgcagaaga
gcctctccct gtctccgggt aaatagtaa 879 <210> SEQ ID NO 366
<211> LENGTH: 291 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: VH21 SP-ShK(1-35)-L10-IgG1 Fc <400> SEQUENCE:
366 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15 Val His Ser Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg
Cys Thr 20 25 30 Ala Phe Gln Cys Lys His Ser Met Lys Tyr Arg Leu
Ser Phe Cys Arg 35 40 45 Lys Thr Cys Gly Thr Cys Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 50 55 60 Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 65 70 75 80 Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 85 90 95 Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 100 105 110 Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 115 120 125
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 130
135 140 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 145 150 155 160 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 165 170 175 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 180 185 190
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 195
200 205 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 210 215 220 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 225 230 235 240 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 245 250 255 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 260 265 270 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 275 280 285 Pro Gly Lys 290
<210> SEQ ID NO 367 <211> LENGTH: 23 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 367 cattctagac caccatggaa tgg 23 <210> SEQ ID NO
368 <211> LENGTH: 29 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 368
cagctgcacc tggcttcctc ctcctccgg 29 <210> SEQ ID NO 369
<211> LENGTH: 192 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: VH21 SP-ShK(1-35, Q16K)-L10 coding sequence
<400> SEQUENCE: 369 atggaatgga gctgggtctt tctcttcttc
ctgtcagtaa cgactggtgt ccactcccgc 60 agctgcatcg acaccatccc
caagagccgc tgcaccgcct tcaagtgcaa gcacagcatg 120 aagtaccgcc
tgagcttctg ccgcaagacc tgcggcacct gcggaggagg aggatccgga 180
ggaggaggaa gc 192 <210> SEQ ID NO 370 <211> LENGTH: 64
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: VH21
SP-ShK(1-35, Q16K)-L10 <400> SEQUENCE: 370 Met Glu Trp Ser
Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His
Ser Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr 20 25 30
Ala Phe Lys Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg 35
40 45 Lys Thr Cys Gly Thr Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 50 55 60 <210> SEQ ID NO 371 <211> LENGTH: 30
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 371 ggaggaggaa gccaggtgca gctggtgcag 30
<210> SEQ ID NO 372 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 372 catgcggccg ctcatttacc c 21 <210> SEQ ID NO 373
<211> LENGTH: 1338 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: aKLH 120.6-HC coding sequence <400>
SEQUENCE: 373 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc
ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggata caccttcacc
ggctaccaca tgcactgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggatgg atcaacccta acagtggtgg cacaaactat 180 gcacagaagt
ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240
atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagagatcgt
300 gggagctact actggttcga cccctggggc cagggaaccc tggtcaccgt
ctcctcagcc 360 tccaccaagg gcccatcggt cttccccctg gcgccctgct
ccaggagcac ctccgagagc 420 acagcggccc tgggctgcct ggtcaaggac
tacttccccg aaccggtgac ggtgtcgtgg 480 aactcaggcg ctctgaccag
cggcgtgcac accttcccag ctgtcctaca gtcctcagga 540 ctctactccc
tcagcagcgt ggtgaccgtg ccctccagca acttcggcac ccagacctac 600
acctgcaacg tagatcacaa gcccagcaac accaaggtgg acaagacagt tgagcgcaaa
660 tgttgtgtcg agtgcccacc gtgcccagca ccacctgtgg caggaccgtc
agtcttcctc 720 ttccccccaa aacccaagga caccctcatg atctcccgga
cccctgaggt cacgtgcgtg 780 gtggtggacg tgagccacga agaccccgag
gtccagttca actggtacgt ggacggcgtg 840 gaggtgcata atgccaagac
aaagccacgg gaggagcagt tcaacagcac gttccgtgtg 900 gtcagcgtcc
tcaccgttgt gcaccaggac tggctgaacg gcaaggagta caagtgcaag 960
gtctccaaca aaggcctccc agcccccatc gagaaaacca tctccaaaac caaagggcag
1020 ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac
caagaaccag 1080 gtcagcctga cctgcctggt caaaggcttc taccccagcg
acatcgccgt ggagtgggag 1140 agcaatgggc agccggagaa caactacaag
accacacctc ccatgctgga ctccgacggc 1200 tccttcttcc tctacagcaa
gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1260 ttctcatgct
ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320
ctgtctccgg gtaaatga 1338 <210> SEQ ID NO 374 <211>
LENGTH: 445 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: aKLH
120.6-HC polypeptide sequence <400> SEQUENCE: 374 Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25
30 His Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln
Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile
Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Ser Tyr Tyr
Trp Phe Asp Pro Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190 Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val
Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Thr Val Glu
Arg Lys Cys Cys Val Glu 210 215 220 Cys Pro Pro Cys Pro Ala Pro Pro
Val Ala Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln 260 265 270 Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280
285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
290 295 300 Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys 325 330 335 Thr Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395
400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405
410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445 <210> SEQ ID NO 375 <211> LENGTH: 29
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 375 catctggatg tcgcttcctc ctcctccgg 29
<210> SEQ ID NO 376 <400> SEQUENCE: 376 000 <210>
SEQ ID NO 377 <400> SEQUENCE: 377 000 <210> SEQ ID NO
378 <211> LENGTH: 32 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 378
ggaggaggaa gcgacatcca gatgacccag tc 32 <210> SEQ ID NO 379
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 379 catctcgagc
ggccgctcaa c 21 <210> SEQ ID NO 380 <211> LENGTH: 837
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: VH21
SP-ShK[1-35, Q16K]-L10-aKLH120.6 Light Chain coding sequence
<400> SEQUENCE: 380 atggaatgga gctgggtctt tctcttcttc
ctgtcagtaa cgactggtgt ccactcccgc 60 agctgcatcg acaccatccc
caagagccgc tgcaccgcct tcaagtgcaa gcacagcatg 120 aagtaccgcc
tgagcttctg ccgcaagacc tgcggcacct gcggaggagg aggatccgga 180
ggaggaggaa gcgacatcca gatgacccag tctccatcct ccctgtctgc atctgtagga
240 gacagagtca ccatcacttg ccgggcaagt cagggcatta gaaatgattt
aggctggtat 300 cagcagaaac cagggaaagc ccctaaacgc ctgatctatg
ctgcatccag tttgcaaagt 360 ggggtcccat caaggttcag cggcagtgga
tctgggacag aattcactct cacaatcagc 420 agcctgcagc ctgaagattt
tgcaacttat tactgtctac agcataatag ttacccgctc 480 actttcggcg
gagggaccaa ggtggagatc aaacgaactg tggctgcacc atctgtcttc 540
atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg
600 aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc
cctccaatcg 660 ggtaactccc aggagagtgt cacagagcag gacagcaagg
acagcaccta cagcctcagc 720 agcaccctga cgctgagcaa agcagactac
gagaaacaca aagtctacgc ctgcgaagtc 780 acccatcagg gcctgagctc
gcccgtcaca aagagcttca acaggggaga gtgttga 837 <210> SEQ ID NO
381 <211> LENGTH: 278 <212> TYPE: PRT <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: VH21 SP-ShK[1-35, Q16K]-L10-aKLH120.6 Light
Chain <400> SEQUENCE: 381 Met Glu Trp Ser Trp Val Phe Leu Phe
Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Arg Ser Cys Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr 20 25 30 Ala Phe Lys Cys Lys
His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg 35 40 45 Lys Thr Cys
Gly Thr Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 50 55 60 Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 65 70
75 80 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn
Asp 85 90 95 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Arg Leu Ile 100 105 110 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 115 120 125 Ser Gly Ser Gly Thr Glu Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 130 135 140 Glu Asp Phe Ala Thr Tyr Tyr
Cys Leu Gln His Asn Ser Tyr Pro Leu 145 150 155 160 Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 165 170 175 Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 180 185 190
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 195
200 205 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln 210 215 220 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser 225 230 235 240 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 245 250 255 Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 260 265 270 Phe Asn Arg Gly Glu Cys
275 <210> SEQ ID NO 382 <400> SEQUENCE: 382 000
<210> SEQ ID NO 383 <211> LENGTH: 1038 <212>
TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Coding sequence - IgG2
fragment <400> SEQUENCE: 383 atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtcagg tgcagctggt
ggagtctggg ggaggcgtgg tccagcctgg gaggtccctg 120 agactctcct
gtgcagcgtc tggattcacc ttcagtagct atggcatgca ctgggtccgc 180
caggctccag gcaaggggct ggagtgggtg gcagttatat ggtatgatgg aagtaataaa
240 tactatgcag actccgtgaa gggccgattc actatctcca gagacaattc
caagaacacg 300 ctgtatctgc aaatgaacag cctgagagcc gaggacacgg
ctgtgtatta ctgtgcgagg 360 tataacttca actacggtat ggacgtctgg
ggccaaggga ccacggtcac cgtctctagt 420 gcctccacca agggcccatc
ggtcttcccc ctggcgccct gctccaggag cacctccgag 480 agcacagcgg
ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540
tggaactcag gcgctctgac cagcggcgtg cacaccttcc cagctgtcct acagtcctca
600 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg
cacccagacc 660 tacacctgca acgtagatca caagcccagc aacaccaagg
tggacaagac agttgagcgc 720 aaatgttgtg tcgagtgccc accgtgccca
gcaccacctg tggcaggacc gtcagtcttc 780 ctcttccccc caaaacccaa
ggacaccctc atgatctccc ggacccctga ggtcacgtgc 840 gtggtggtgg
acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 900
gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt
960 gtggtcagcg tcctcaccgt tgtgcaccag gactggctga acggcaagga
gtacaagtgc 1020 aaggtctcca acaaaggc 1038 <210> SEQ ID NO 384
<211> LENGTH: 346 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: IgG2 fragment <400> SEQUENCE: 384 Met Asp Met
Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu
Arg Gly Ala Arg Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly 20 25
30 Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
35 40 45 Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala
Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp
Gly Ser Asn Lys 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys
Ala Arg Tyr Asn Phe Asn Tyr Gly Met Asp
115 120 125 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser
Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu 145 150 155 160 Ser Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val
Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn 210 215 220 Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg 225 230
235 240 Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg 305 310 315 320 Val Val Ser Val
Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly 340 345 <210> SEQ ID NO 385
<211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Forward primer <400> SEQUENCE: 385 ttttttttgc
gcgctgtgac atccagatga cccagtc 37 <210> SEQ ID NO 386
<400> SEQUENCE: 386 000 <210> SEQ ID NO 387 <211>
LENGTH: 510 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: aKLH
120.6 IgG2-ShK[2-35, Q16K] fusion <400> SEQUENCE: 387 Met Asp
Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15
Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20
25 30 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser
Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln
Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro
Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg
Val Thr Met Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr
Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150
155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr Tyr Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275
280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Phe 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Val His
Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395
400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
405 410 415 Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Ser Cys Ile Asp 465 470 475 480 Thr Ile Pro Lys Ser
Arg Cys Thr Ala Phe Lys Cys Lys His Ser Met 485 490 495 Lys Tyr Arg
Leu Ser Phe Cys Arg Lys Thr Cys Gly Thr Cys 500 505 510 <210>
SEQ ID NO 388 <211> LENGTH: 684 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (1)..(684) <400>
SEQUENCE: 388 atg gac aaa act cac aca tgt cca cct tgt cca gct ccg
gaa ctc ctg 48 Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu 1 5 10 15 ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa
ccc aag gac acc ctc 96 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 20 25 30 atg atc tcc cgg acc cct gag gtc aca
tgc gtg gtg gtg gac gtg agc 144 Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 35 40 45 cac gaa gac cct gag gtc aag
ttc aac tgg tac gtg gac ggc gtg gag 192 His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 gtg cat aat gcc aag
aca aag ccg cgg gag gag cag tac aac agc acg 240 Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 65 70 75 80 tac cgt gtg
gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat 288 Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95 ggc
aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 336 Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105
110 atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag
384 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
115 120 125 gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac
cag gtc 432 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val 130 135 140 agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc
gac atc gcc gtg 480 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 145 150 155 160 gag tgg gag agc aat ggg cag ccg gag
aac aac tac aag acc acg cct 528 Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro 165 170 175 ccc gtg ctg gac tcc gac ggc
tcc ttc ttc ctc tac agc aag ctc acc 576 Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 gtg gac aag agc agg
tgg cag cag ggg aac gtc ttc tca tgc tcc gtg 624 Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205 atg cat gag
gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg 672 Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 tct
ccg ggt aaa 684 Ser Pro Gly Lys 225 <210> SEQ ID NO 389
<211> LENGTH: 228 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 389 Met Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20
25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser 35 40 45 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 130 135 140 Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150
155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Pro Gly Lys 225
<210> SEQ ID NO 390 <400> SEQUENCE: 390 000 <210>
SEQ ID NO 391 <211> LENGTH: 744 <212> TYPE: DNA
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Coding sequence - IgG2 fragment
<400> SEQUENCE: 391 atggaatgga gctgggtctt tctcttcttc
ctgtcagtaa cgactggtgt ccactccgag 60 cgcaaagtcg agtgcccacc
gtgcccagca ccacctgtgg caggaccgtc agtcttcctc 120 ttccccccaa
aacccaagga caccctcatg atctcccgga cccctgaggt cacgtgcgtg 180
gtggtggacg tgagccacga agaccccgag gtccagttca actggtacgt ggacggcgtg
240 gaggtgcata atgccaagac aaagccacgg gaggagcagt tcaacagcac
gttccgtgtg 300 gtcagcgtcc tcaccgttgt gcaccaggac tggctgaacg
gcaaggagta caagtgcaag 360 gtctccaaca aaggcctccc agcccccatc
gagaaaacca tctccaaaac caaagggcag 420 ccccgagaac cacaggtgta
caccctgccc ccatcccggg aggagatgac caagaaccag 480 gtcagcctga
cctgcctggt caaaggcttc taccccagcg acatcgccgt ggagtgggag 540
agcaatgggc agccggagaa caactacaag accacacctc ccatgctgga ctccgacggc
600 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca
ggggaacgtc 660 ttctcatgct ccgtgatgca tgaggctctg cacaaccact
acacgcagaa gagcctctcc 720 ctgtctccgg gtaaaggagg agga 744
<210> SEQ ID NO 392 <211> LENGTH: 248 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: IgG2 fragment <400> SEQUENCE:
392 Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15 Val His Ser Glu Arg Lys Val Glu Cys Pro Pro Cys Pro Ala
Pro Pro 20 25 30 Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val 50 55 60 Ser His Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Phe Arg Val
Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 100 105 110 Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala 115 120 125
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro 130
135 140 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Met Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu 195 200 205 Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu
Ser Pro Gly Lys Gly Gly Gly 245 <210> SEQ ID NO 393
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 393 cattctagac
ccaccatgga catgagggtg 30 <210> SEQ ID NO 394 <211>
LENGTH: 511 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
IgG2-HC-L10-ShK[1-35] fusion polypeptide <400> SEQUENCE: 394
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5
10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala
Glu 20 25 30 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys
Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His Met His Trp Val
Arg Gln Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile
Asn Pro Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln
Gly Arg Val Thr Met Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala
Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val
Tyr Tyr Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp
Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135
140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
145 150 155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser
Asn Phe Gly Thr Gln Thr Tyr Thr Cys 210 215 220 Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu 225 230 235 240 Arg Lys
Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 245 250 255
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260
265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His 275 280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Phe 305 310 315 320 Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385
390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 405 410 415 Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460
Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Ser Cys Ile 465
470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln Cys Lys
His Ser 485 490 495 Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys
Gly Thr Cys 500 505 510 <210> SEQ ID NO 395 <211>
LENGTH: 501 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
truncated IgG2 Fc-L10-ShK(1-35, Q16K) coding sequence <400>
SEQUENCE: 395 ctcccagccc ccatcgagaa aaccatctcc aaaaccaaag
ggcagccccg agaaccacag 60 gtgtacaccc tgcccccatc ccgggaggag
atgaccaaga accaggtcag cctgacctgc 120 ctggtcaaag gcttctaccc
cagcgacatc gccgtggagt gggagagcaa tgggcagccg 180 gagaacaact
acaagaccac acctcccatg ctggactccg acggctcctt cttcctctac 240
agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg
300 atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc
tccgggtaaa 360 ggaggaggag gatccggagg aggaggaagc cgcagctgca
tcgacaccat ccccaagagc 420 cgctgcaccg ccttcaagtg caagcacagc
atgaagtacc gcctgagctt ctgccgcaag 480 acctgcggca cctgctaatg a 501
<210> SEQ ID NO 396 <211> LENGTH: 165 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: truncated IgG2 Fc-L10-ShK(1-35,
Q16K) amino acid sequence <400> SEQUENCE: 396 Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro 1 5 10 15 Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 20 25 30
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 35
40 45 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr 50 55 60 Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr 65 70 75 80 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe 85 90 95 Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys 100 105 110 Ser Leu Ser Leu Ser Pro Gly
Lys Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Arg Ser Cys
Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala 130 135 140 Phe Lys Cys
Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys 145 150 155 160
Thr Cys Gly Thr Cys 165 <210> SEQ ID NO 397 <211>
LENGTH: 129 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Coding
sequence <400> SEQUENCE: 397 ggatccggag gaggaggaag cagctgcatc
gacaccatcc ccaagagccg ctgcaccgcc 60 ttcaagtgca agcacagcat
gaagtaccgc ctgagcttct gccgcaagac ctgcggcacc 120 tgctaatga 129
<210> SEQ ID NO 398 <211> LENGTH: 41 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Polypeptide sequence <400>
SEQUENCE: 398 Gly Ser Gly Gly Gly Gly Ser Ser Cys Ile Asp Thr Ile
Pro Lys Ser 1 5 10 15 Arg Cys Thr Ala Phe Lys Cys Lys His Ser Met
Lys Tyr Arg Leu Ser 20 25 30 Phe Cys Arg Lys Thr Cys Gly Thr Cys 35
40 <210> SEQ ID NO 399 <211> LENGTH: 1407 <212>
TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Coding sequence <400>
SEQUENCE: 399 atggacatga gggtgcccgc tcagctcctg gggctcctgc
tgctgtggct gagaggtgcc 60 agatgtcagg tgcagctggt gcagtctggg
gctgaggtga agaagcctgg ggcctcagtg 120 aaggtctcct gcaaggcttc
tggatacacc ttcaccggct accacatgca ctgggtgcga 180 caggcccctg
gacaagggct tgagtggatg ggatggatca accctaacag tggtggcaca 240
aactatgcac agaagtttca gggcagggtc accatgacca gggacacgtc catcagcaca
300 gcctacatgg agctgagcag gctgagatct gacgacacgg ccgtgtatta
ctgtgcgaga 360 gatcgtggga gctactactg gttcgacccc tggggccagg
gaaccctggt caccgtctcc 420 tcagcctcca ccaagggccc atcggtcttc
cccctggcgc cctgctccag gagcacctcc 480 gagagcacag cggccctggg
ctgcctggtc aaggactact tccccgaacc ggtgacggtg 540 tcgtggaact
caggcgctct gaccagcggc gtgcacacct tcccagctgt cctacagtcc 600
tcaggactct actccctcag cagcgtggtg accgtgccct ccagcaactt cggcacccag
660 acctacacct gcaacgtaga tcacaagccc agcaacacca aggtggacaa
gacagttgag 720 cgcaaatgtt gtgtcgagtg cccaccgtgc ccagcaccac
ctgtggcagg accgtcagtc 780 ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcacg 840 tgcgtggtgg tggacgtgag
ccacgaagac cccgaggtcc agttcaactg gtacgtggac 900 ggcgtggagg
tgcataatgc caagacaaag ccacgggagg agcagttcaa cagcacgttc 960
cgtgtggtca gcgtcctcac cgttgtgcac caggactggc tgaacggcaa ggagtacaag
1020 tgcaaggtct ccaacaaagg cctcccagcc cccatcgaga aaaccatctc
caaaaccaaa 1080 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggagga gatgaccaag 1140 aaccaggtca gcctgacctg cctggtcaaa
ggcttctacc ccagcgacat cgccgtggag 1200 tgggagagca atgggcagcc
ggagaacaac tacaagacca cacctcccat gctggactcc 1260 gacggctcct
tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc
1380 ctctccctgt ctccgggtgg aggagga 1407 <210> SEQ ID NO 400
<211> LENGTH: 469 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Polypeptide sequence <400> SEQUENCE: 400 Met Asp
Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15
Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20
25 30 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser
Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His Met His Trp Val Arg Gln
Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro
Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg
Val Thr Met Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr
Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150
155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr Tyr Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275
280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Phe 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Val His
Gln Asp Trp Leu Asn Gly 325 330 335
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 340
345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln
Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460
Pro Gly Gly Gly Gly 465 <210> SEQ ID NO 401 <211>
LENGTH: 510 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
IgG2-HC-L10-ShK[2-35] fusion polypeptide <400> SEQUENCE: 401
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5
10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala
Glu 20 25 30 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys
Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His Met His Trp Val
Arg Gln Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Trp Ile
Asn Pro Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln
Gly Arg Val Thr Met Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr Ala
Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala Val
Tyr Tyr Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp
Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135
140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
145 150 155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser
Asn Phe Gly Thr Gln Thr Tyr Thr Cys 210 215 220 Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu 225 230 235 240 Arg Lys
Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 245 250 255
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260
265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His 275 280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Phe 305 310 315 320 Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385
390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 405 410 415 Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Ser Cys Ile Asp 465 470 475 480 Thr Ile Pro
Lys Ser Arg Cys Thr Ala Phe Lys Cys Lys His Ser Met 485 490 495 Lys
Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys Gly Thr Cys 500 505 510
<210> SEQ ID NO 402 <211> LENGTH: 291 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: VH21 SP-ShK(1-35, Q16K)-L10-IgG1 Fc
<400> SEQUENCE: 402 Met Glu Trp Ser Trp Val Phe Leu Phe Phe
Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Arg Ser Cys Ile Asp
Thr Ile Pro Lys Ser Arg Cys Thr 20 25 30 Ala Phe Lys Cys Lys His
Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg 35 40 45 Lys Thr Cys Gly
Thr Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 50 55 60 Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 65 70 75 80
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 85
90 95 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His 100 105 110 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 115 120 125 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr 130 135 140 Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 145 150 155 160 Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 165 170 175 Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 180 185 190 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 195 200 205
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 210
215 220 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 225 230 235 240 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 245 250 255 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 260 265 270 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 275 280 285 Pro Gly Lys 290
<210> SEQ ID NO 403 <211> LENGTH: 509 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: VH21 SP-ShK[1-35,
Q16K]-L10-aKLH120.6-HC fusion polypeptide <400> SEQUENCE: 403
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5
10 15 Val His Ser Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys
Thr 20 25 30 Ala Phe Lys Cys Lys His Ser Met Lys Tyr Arg Leu Ser
Phe Cys Arg 35 40 45 Lys Thr Cys Gly Thr Cys Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser 50 55 60 Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala 65 70 75 80 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 85 90 95 His Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 100 105 110 Gly Trp Ile
Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 115 120 125 Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 130 135
140 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
145 150 155 160 Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe Asp Pro Trp
Gly Gln Gly 165 170 175 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe 180 185 190 Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala Ala Leu 195 200 205
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 210
215 220 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 225 230 235 240 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser 245 250 255 Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys
Asn Val Asp His Lys Pro 260 265 270 Ser Asn Thr Lys Val Asp Lys Thr
Val Glu Arg Lys Cys Cys Val Glu 275 280 285 Cys Pro Pro Cys Pro Ala
Pro Pro Val Ala Gly Pro Ser Val Phe Leu 290 295 300 Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 305 310 315 320 Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln 325 330
335 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
340 345 350 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser
Val Leu 355 360 365 Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys 370 375 380 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys 385 390 395 400 Thr Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser 405 410 415 Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 420 425 430 Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 435 440 445 Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 450 455
460 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
465 470 475 480 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn 485 490 495 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 500 505 <210> SEQ ID NO 404 <211> LENGTH: 278
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Fusion
polypeptide <400> SEQUENCE: 404 Met Glu Trp Ser Trp Val Phe
Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Arg Ser
Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr 20 25 30 Ala Phe Lys
Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg 35 40 45 Lys
Thr Cys Gly Thr Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 50 55
60 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
65 70 75 80 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg
Asn Asp 85 90 95 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Arg Leu Ile 100 105 110 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 115 120 125 Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 130 135 140 Glu Asp Phe Ala Thr Tyr
Tyr Cys Leu Gln His Asn Ser Tyr Pro Leu 145 150 155 160 Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 165 170 175 Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 180 185
190 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
195 200 205 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln 210 215 220 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 225 230 235 240 Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 245 250 255 Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 260 265 270 Phe Asn Arg Gly Glu
Cys 275 <210> SEQ ID NO 405 <211> LENGTH: 465
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: anti-DNP 3A4
(W101F) IgG2 Heavy Chain <400> SEQUENCE: 405 Met Asp Met Arg
Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg
Gly Ala Arg Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly 20 25 30
Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35
40 45 Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro
Gly 50 55 60 Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly
Ser Asn Lys 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala
Arg Tyr Asn Phe Asn Tyr Gly Met Asp 115 120 125 Val Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu 145 150 155 160
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165
170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln
Thr Tyr Thr Cys Asn 210 215 220 Val Asp His Lys Pro Ser Asn Thr Lys
Val Asp Lys Thr Val Glu Arg 225 230 235 240 Lys Cys Cys Val Glu Cys
Pro Pro Cys Pro Ala Pro Pro Val Ala Gly 245 250 255 Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290
295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Thr Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met 405 410
415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 450 455 460 Gly 465 <210> SEQ ID NO 406
<211> LENGTH: 510 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: aDNP 3A4 (W101F) IgG2 HC-L10-ShK[1-35, Q16K] fusion
protein <400> SEQUENCE: 406 Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys
Gln Val Gln Leu Val Glu Ser Gly Gly Gly 20 25 30 Val Val Gln Pro
Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr
Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly 50 55 60
Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys 65
70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn 85 90 95
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100
105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Asn Phe Asn Tyr Gly Met
Asp 115 120 125 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala
Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu 145 150 155 160 Ser Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr
Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn 210 215 220
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg 225
230 235 240 Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
Ala Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg 305 310 315 320 Val Val Ser
Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu 340 345
350 Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Met 405 410 415 Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Ser Cys Ile Asp 465 470
475 480 Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys Cys Lys His Ser
Met 485 490 495 Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys Gly Thr
Cys 500 505 510 <210> SEQ ID NO 407 <211> LENGTH: 236
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: anti-DNP 3A4
Antibody Light Chain <400> SEQUENCE: 407 Met Asp Met Arg Val
Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly
Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Val
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40
45 Gln Gly Ile Ser Arg Arg Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60 Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser
Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln 100 105 110 Ala Asn Ser Phe Pro Phe Thr Phe
Gly Pro Gly Thr Lys Val Asp Ile 115 120 125 Lys Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170
175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 225 230 235 <210> SEQ ID NO 408 <211> LENGTH:
475 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Polypeptide
<400> SEQUENCE: 408 Met Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 65 70 75 80
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85
90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210
215 220 Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly 225 230 235 240 Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His
Thr Cys Pro Pro 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330
335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys 355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp 370 375 380 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440 445 Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455
460 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475
<210> SEQ ID NO 409 <211> LENGTH: 148 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Polypeptide <400> SEQUENCE:
409 Met Lys Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
1 5 10 15 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 20 25 30 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 35 40 45
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 50
55 60 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn 65 70 75 80 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro 85 90 95 Ile Glu Lys Thr Ile Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser 100 105 110 Arg Ser Cys Ile Asp Thr Ile Pro Lys
Ser Arg Cys Thr Ala Phe Lys 115 120 125 Cys Lys His Ser Met Lys Tyr
Arg Leu Ser Phe Cys Arg Lys Thr Cys 130 135 140 Gly Thr Cys Ala 145
<210> SEQ ID NO 410 <211> LENGTH: 521 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: scFc-Shk construct <400>
SEQUENCE: 410 Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 65 70 75 80 Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95 Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105
110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser
Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 225 230
235 240 Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro
Pro 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335 Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 355
360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp 370 375 380 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440 445 Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460 Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser 465 470 475
480 Gly Gly Gly Gly Ser Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg
485 490 495 Cys Thr Ala Phe Lys Cys Lys His Ser Met Lys Tyr Arg Leu
Ser Phe 500 505 510 Cys Arg Lys Thr Cys Gly Thr Cys Ala 515 520
<210> SEQ ID NO 411 <211> LENGTH: 514 <212> TYPE:
PRT <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: scFc-Shk construct <400>
SEQUENCE: 411 Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 65 70 75 80 Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95 Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105
110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser
Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 225 230
235 240 Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro
Pro 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn 290 295 300 Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335 Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 355
360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp 370 375 380 Glu Leu Gly Gly Arg Ser Cys Ile Asp Thr Ile Pro Lys
Ser Arg Cys 385 390 395 400 Thr Ala Phe Gln Cys Lys His Ser Met Lys
Tyr Arg Leu Ser Phe Cys 405 410 415 Arg Lys Thr Cys Gly Thr Cys Gly
Gly Thr Lys Asn Gln Val Ser Leu 420 425 430 Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 435 440 445 Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 450 455 460 Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 465 470 475
480 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
485 490 495 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro 500 505 510 Gly Lys <210> SEQ ID NO 412 <211>
LENGTH: 519 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
scFc-Shk construct <400> SEQUENCE: 412 Met Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20
25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser 35 40 45 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Gly Gly Arg Ser Cys 130 135 140 Ile
Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Gln Cys Lys His 145 150
155 160 Ser Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys Thr Cys Gly Thr
Cys 165 170 175 Gly Gly Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe 180 185 190 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu 195 200 205 Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe 210 215 220 Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly 225 230 235 240 Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 245 250 255 Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser 260 265 270
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 275
280 285 Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro 290 295 300 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys 305 310 315 320 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val 325 330 335 Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp 340 345 350 Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 355 360 365 Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 370 375 380 Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 385 390 395
400 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
405 410 415 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys 420 425 430 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 435 440 445 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys 450 455 460 Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser 465 470 475 480 Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 485 490 495 Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 500 505 510 Leu
Ser Leu Ser Pro Gly Lys 515 <210> SEQ ID NO 413 <211>
LENGTH: 480 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION:
Polypeptide <400> SEQUENCE: 413 Met Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55
60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185
190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 210 215 220 Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly 225 230 235 240 Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Asp Lys Thr 245 250 255 His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser 260 265 270 Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 275 280 285 Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 290 295 300 Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 305 310
315 320 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val 325 330 335 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr 340 345 350 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr 355 360 365 Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu 370 375 380 Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys 385 390 395 400 Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 405 410 415 Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 420 425 430
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 435
440 445 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala 450 455 460 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 465 470 475 480 <210> SEQ ID NO 414 <211>
LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 414 aaacctgtgg cacctgtggc ggtaccaaaa
accaggtgtc cctga 45 <210> SEQ ID NO 415 <211> LENGTH:
45 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 415 tatcgatgca agaacgaccg cccagttcgt
cacgagacgg cggca 45 <210> SEQ ID NO 416 <211> LENGTH:
45 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 416 aaataccgtc tcagtttctg tcgtaaaacc
tgtggcacct gtggc 45 <210> SEQ ID NO 417 <211> LENGTH:
45 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 417 cagtacagcg ggacttaggg attgtatcga
tgcaagaacg accgc 45 <210> SEQ ID NO 418 <211> LENGTH:
45 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer
sequence
<400> SEQUENCE: 418 caatgcaaac actcaatgaa ataccgtctc
agtttctgtc gtaaa 45 <210> SEQ ID NO 419 <211> LENGTH:
43 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 419 tgagtgtttg cattgaaagg cagtacagcg
ggacttaggg att 43 <210> SEQ ID NO 420 <211> LENGTH: 28
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 420 taatgaattc gagctccgtc gacaagct 28
<210> SEQ ID NO 421 <211> LENGTH: 44 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 421 ccaccggatc caccaccacc tttacccgga gacagggaga ggct 44
<210> SEQ ID NO 422 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 422 tggtggatcc ggtggtggtg gctccggt 28 <210> SEQ ID
NO 423 <211> LENGTH: 40 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 423
gagctcgaat tcattattta cccggagaca gagacaggga 40 <210> SEQ ID
NO 424 <211> LENGTH: 36 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 424
ggtggcggtg gctctggtgg tggtggatcc ggtggt 36 <210> SEQ ID NO
425 <211> LENGTH: 39 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 425
agagccaccg ccacctttac ccggagacag ggagaggct 39 <210> SEQ ID NO
426 <211> LENGTH: 28 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 426
ggcggtacca agaaccaggt cagcctga 28 <210> SEQ ID NO 427
<211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 427 accgcccagc
tcatcacggg atgggggca 29 <210> SEQ ID NO 428 <211>
LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 428 cgtgatgagc tgggcggtcg ttcttgcatc
gatacaatcc ct 42 <210> SEQ ID NO 429 <211> LENGTH: 42
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 429 acctggttct tggtaccgcc acaggtgcca
caggttttac ga 42 <210> SEQ ID NO 430 <211> LENGTH: 33
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 430 cactcaatga aataccgtct cagtttctgt cgt 33
<210> SEQ ID NO 431 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 431 gtatttcatt gagtgtttgc attgaaaggc a 31 <210> SEQ
ID NO 432 <211> LENGTH: 28 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 432
taatgaattc gagctccgtc gacaagct 28 <210> SEQ ID NO 433
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 433 cccggagaca
gagacaggga 20 <210> SEQ ID NO 434 <211> LENGTH: 32
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 434 gtctctgtct ccgggtaaag gcggcggcgg ca 32
<210> SEQ ID NO 435 <211> LENGTH: 34 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 435 agctcgaatt cattaagcac aggtgccaca ggtt 34 <210>
SEQ ID NO 436 <211> LENGTH: 163 <212> TYPE: DNA
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Coding sequence ShK {1-35, Q16K)
with an N-terminal linker <400> SEQUENCE: 436 ggatccggag
gaggaggaag ccgcagctgc atcgacacca tccccaagag ccgctgcacc 60
gccttcaagt gcaagcacag catgaagtac cgcctgagct tctgccgcaa gacctgcggc
120 acctgctaat gagcggccgc tcgaggccgg caaggccgga tcc 163 <210>
SEQ ID NO 437 <211> LENGTH: 42 <212> TYPE: PRT
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK {1-35, Q16K) with an N-terminal
linker <400> SEQUENCE: 437 Gly Ser Gly Gly Gly Gly Ser Arg
Ser Cys Ile Asp Thr Ile Pro Lys 1 5 10 15 Ser Arg Cys Thr Ala Phe
Lys Cys Lys His Ser Met Lys Tyr Arg Leu 20 25 30 Ser Phe Cys Arg
Lys Thr Cys Gly Thr Cys 35 40 <210> SEQ ID NO 438 <211>
LENGTH: 36 <212> TYPE: PRT <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: ShK-192 <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: L-phosphonophenylalanine <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(1)..(2) <223> OTHER INFORMATION: Between residues 1 and 2
is: {2-[2-Aminoethoxy]ethoxy }acetic acid <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (22)..(22)
<223> OTHER INFORMATION: Norleucine <400> SEQUENCE: 438
Xaa Arg Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe 1 5
10 15 Gln Cys Lys His Ser Xaa Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr 20 25 30 Cys Gly Thr Cys 35 <210> SEQ ID NO 439
<211> LENGTH: 281 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: aKLH 120.6 kappa LC-ShK[1-35, Q16K] fusion protein
<400> SEQUENCE: 439 Met Asp Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Arg
Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro
Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val 65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85
90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu
Gln 100 105 110 His Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210
215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly
Gly 225 230 235 240 Ser Gly Gly Gly Gly Ser Arg Ser Cys Ile Asp Thr
Ile Pro Lys Ser 245 250 255 Arg Cys Thr Ala Phe Lys Cys Lys His Ser
Met Lys Tyr Arg Leu Ser 260 265 270 Phe Cys Arg Lys Thr Cys Gly Thr
Cys 275 280 <210> SEQ ID NO 440 <211> LENGTH: 280
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: aKLH 120.6
kappa LC-ShK[2-35, Q16K] fusion protein <400> SEQUENCE: 440
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5
10 15 Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser 35 40 45 Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln
Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Arg Leu Ile Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110 His Asn Ser
Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135
140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly 225 230 235 240 Ser Gly
Gly Gly Gly Ser Ser Cys Ile Asp Thr Ile Pro Lys Ser Arg 245 250 255
Cys Thr Ala Phe Lys Cys Lys His Ser Met Lys Tyr Arg Leu Ser Phe 260
265 270 Cys Arg Lys Thr Cys Gly Thr Cys 275 280 <210> SEQ ID
NO 441 <211> LENGTH: 34 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 441
aacaggggag agtgtggagg aggaggatcc ggag 34 <210> SEQ ID NO 442
<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 442 catgcggccg
ctcattagca gg 22 <210> SEQ ID NO 443 <211> LENGTH: 26
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Forward primer
sequence <400> SEQUENCE: 443 ggacactgac atggactgaa ggagta 26
<210> SEQ ID NO 444 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Reverse primer sequence <400>
SEQUENCE: 444 ctcctgggag ttacccgatt g 21 <210> SEQ ID NO 445
<211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Reverse primer sequence <400> SEQUENCE: 445
gatgggccct tggtggaggc tgaggagacg gtgaccgtgg 40 <210> SEQ ID
NO 446 <211> LENGTH: 36 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 446
aagctcgagg tcgactagac caccatggac atgagg 36 <210> SEQ ID NO
447 <211> LENGTH: 41 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 447
aaccgtttaa acgcggccgc tcaacactct cccctgttga a 41 <210> SEQ ID
NO 448 <211> LENGTH: 41 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 448
aagctcgagg tcgactagac caccatggaa ttgggactga g 41
<210> SEQ ID NO 449 <211> LENGTH: 41 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 449 aaccgtttaa acgcggccgc tcatttaccc ggagacaggg a 41
<210> SEQ ID NO 450 <211> LENGTH: 37 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 450 ttttttttgc gcgctgtgac atccagatga cccagtc 37
<210> SEQ ID NO 451 <211> LENGTH: 32 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 451 aaaaaacgta cgtttgatat ccactttggt cc 32 <210>
SEQ ID NO 452 <211> LENGTH: 50 <212> TYPE: DNA
<213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 452 ctgtgtatta ctgtgcgagg tataacttca actacggtat
ggacgtctgg 50 <210> SEQ ID NO 453 <211> LENGTH: 50
<212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 453 ccagacgtcc ataccgtagt tgaagttata
cctcgcacag taatacacag 50 <210> SEQ ID NO 454 <211>
LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 454 ctgtgtatta ctgtgcgagg tataactaca
actacggtat ggacgtctgg 50 <210> SEQ ID NO 455 <211>
LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 455 ccagacgtcc ataccgtagt tgtagttata
cctcgcacag taatacacag 50 <210> SEQ ID NO 456 <211>
LENGTH: 59 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 456 aagctcgagg tcgactagac caccatggac
atgagggtgc ccgctcagct cctggggct 59 <210> SEQ ID NO 457
<211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 457 aaccgtttaa
acgcggccgc tcatttaccc ggagacaggg a 41 <210> SEQ ID NO 458
<211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 458 ggacaagaca
gttgagcgca aatcttctgt cgagtgccca ccgtgcccag 50 <210> SEQ ID
NO 459 <211> LENGTH: 50 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 459
ctgggcacgg tgggcactcg acagaagatt tgcgctcaac tgtcttgtcc 50
<210> SEQ ID NO 460 <211> LENGTH: 59 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 460 aagctcgagg tcgactagac caccatggac atgagggtgc
ccgctcagct cctggggct 59 <210> SEQ ID NO 461 <211>
LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 461 aaccgtttaa acgcggccgc tcatttaccc
ggagacaggg a 41 <210> SEQ ID NO 462 <211> LENGTH: 511
<212> TYPE: PRT <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: aKLH 120.6
IgG2-ShK[1-35, R1A, I4A, Q16K] fusion protein <400> SEQUENCE:
462 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly
Ala Glu 20 25 30 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys
Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Gly Tyr His Met His Trp
Val Arg Gln Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Trp
Ile Asn Pro Asn Ser Gly Gly Thr 65 70 75 80 Asn Tyr Ala Gln Lys Phe
Gln Gly Arg Val Thr Met Thr Arg Asp Thr 85 90 95 Ser Ile Ser Thr
Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 100 105 110 Thr Ala
Val Tyr Tyr Cys Ala Arg Asp Arg Gly Ser Tyr Tyr Trp Phe 115 120 125
Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130
135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser 145 150 155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser
Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys 210 215 220 Asn Val Asp His
Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu 225 230 235 240 Arg
Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 245 250
255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 275 280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Phe 305 310 315 320 Arg Val Val Ser Val Leu Thr
Val Val His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr
Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375
380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 405 410 415 Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 435 440 445
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450
455 460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Cys
Ala 465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys
Cys Lys His Ser 485 490 495 Met Lys Tyr Arg Leu Ser Phe Cys Arg Lys
Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID NO 463
<211> LENGTH: 511 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: aKLH 120.6 IgG2-ShK[1-35, R1A, Q16K, K30E] fusion
protein <400> SEQUENCE: 463 Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys
Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr
Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50 55 60
Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr 65
70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg
Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg
Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185
190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr
Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305 310
315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435
440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
Ser Cys Ile 465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala
Phe Lys Cys Lys His Ser 485 490 495 Met Lys Tyr Arg Leu Ser Phe Cys
Arg Glu Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID NO 464
<211> LENGTH: 511 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: aKLH 120.6 IgG2-ShK[1-35, R1H, I4A, Q16K] fusion
protein <400> SEQUENCE: 464 Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys
Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr
Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50 55 60
Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr 65
70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg
Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg
Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185
190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr
Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305 310
315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435
440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His
Ser Cys Ala 465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala
Phe Lys Cys Lys His Ser 485 490 495 Met Lys Tyr Arg Leu Ser Phe Cys
Arg Lys Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID NO 465
<211> LENGTH: 511 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: aKLH 120.6 IgG2-ShK[1-35, R1H, Q16K, K30E] fusion
protein <400> SEQUENCE: 465 Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys
Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr
Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50 55 60
Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr
65 70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg
Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg
Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185
190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr
Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305 310
315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435
440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His
Ser Cys Ile 465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala
Phe Lys Cys Lys His Ser 485 490 495 Met Lys Tyr Arg Leu Ser Phe Cys
Arg Glu Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID NO 466
<211> LENGTH: 511 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: aKLH 120.6 HC (IgG2)-ShK[1-35, R1K, 14A, Q16K] fusion
protein <400> SEQUENCE: 466 Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys
Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr
Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50 55 60
Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr 65
70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg
Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg
Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185
190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr
Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305 310
315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435
440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 450 455 460 Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys
Ser Cys Ala 465 470 475 480 Asp Thr Ile Pro Lys Ser Arg Cys Thr Ala
Phe Lys Cys Lys His Ser 485 490 495 Met Lys Tyr Arg Leu Ser Phe Cys
Arg Lys Thr Cys Gly Thr Cys 500 505 510 <210> SEQ ID NO 467
<211> LENGTH: 511 <212> TYPE: PRT <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: aKLH 120.6 IgG2-ShK[1-35, R1K, Q16K, K30E] fusion
protein <400> SEQUENCE: 467 Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys
Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr
Phe Thr Gly Tyr His Met His Trp Val Arg Gln Ala Pro Gly 50 55 60
Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr 65
70 75 80 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg
Asp Thr 85 90 95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg
Gly Ser Tyr Tyr Trp Phe 115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185
190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
195 200 205 Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr
Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro Pro Val Ala 245 250 255 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met
260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 275 280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Phe 305 310 315 320 Arg Val Val Ser Val Leu Thr
Val Val His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr
Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375
380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 405 410 415 Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Lys Ser Cys Ile 465 470 475 480 Asp Thr
Ile Pro Lys Ser Arg Cys Thr Ala Phe Lys Cys Lys His Ser 485 490 495
Met Lys Tyr Arg Leu Ser Phe Cys Arg Glu Thr Cys Gly Thr Cys 500 505
510 <210> SEQ ID NO 468 <211> LENGTH: 37 <212>
TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220>
FEATURE: <223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 468 aggaggagga agcgccagct gcgccgacac catcccc 37
<210> SEQ ID NO 469 <211> LENGTH: 37 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 469 ggggatggtg tcggcgcagc tggcgcttcc tcctcct 37
<210> SEQ ID NO 470 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 470 gaggaggagg aagcgccagc tgcatcgaca 30 <210> SEQ
ID NO 471 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 471
gagcttctgc cgcgagacct gcggcac 27 <210> SEQ ID NO 472
<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 472 cgatgcagct
ggcgcttcct cctcctc 27 <210> SEQ ID NO 473 <211> LENGTH:
27 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE
<220> FEATURE: <223> OTHER INFORMATION: Primer sequence
<400> SEQUENCE: 473 gtgccgcagg tctcgcggca gaagctc 27
<210> SEQ ID NO 474 <211> LENGTH: 36 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 474 ggaggaggaa gccacagctg cgccgacacc atcccc 36
<210> SEQ ID NO 475 <211> LENGTH: 36 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 475 ggggatggtg tcggcgcagc tgtggcttcc tcctcc 36
<210> SEQ ID NO 476 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 476 ggaggaggaa gccacagctg catcgac 27 <210> SEQ ID
NO 477 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: ARTIFICIAL SEQUENCE <220> FEATURE: <223>
OTHER INFORMATION: Primer sequence <400> SEQUENCE: 477
gtcgatgcag ctgtggcttc ctcctcc 27 <210> SEQ ID NO 478
<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 478 ccggaggagg
aggaagcaag agctgcgccg acaccatccc caaga 45 <210> SEQ ID NO 479
<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 479 tcttggggat
ggtgtcggcg cagctcttgc ttcctcctcc tccgg 45 <210> SEQ ID NO 480
<211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 480 cggaggagga
ggaagcaaga gctgcatcga cacca 35 <210> SEQ ID NO 481
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
ARTIFICIAL SEQUENCE <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 481 tggtgtcgat
gcagctcttg cttcctcctc ctc 33 <210> SEQ ID NO 482 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ARTIFICIAL
SEQUENCE <220> FEATURE: <223> OTHER INFORMATION: Primer
sequence <400> SEQUENCE: 482 cattctagaa ccaccatgga catgagggtg
30
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