U.S. patent application number 17/458028 was filed with the patent office on 2022-04-28 for antibody cytokine engrafted compositions and methods of use for immunoregulation.
The applicant listed for this patent is Novarts AG. Invention is credited to Michael DiDonato, Bernhard Hubert Geierstanger, Tobias Junt, Mark Knuth, Shelly Meeusen, Carolina Nicole Simpson, Glen Spraggon, John Trauger.
Application Number | 20220127321 17/458028 |
Document ID | / |
Family ID | 1000006078969 |
Filed Date | 2022-04-28 |
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United States Patent
Application |
20220127321 |
Kind Code |
A1 |
DiDonato; Michael ; et
al. |
April 28, 2022 |
ANTIBODY CYTOKINE ENGRAFTED COMPOSITIONS AND METHODS OF USE FOR
IMMUNOREGULATION
Abstract
The present disclosure provides antibody cytokine engrafted
proteins that bind to and stimulate intracellular signaling through
interleukin 10 receptor. Provided antibody cytokine engrafted
proteins find use in enhancing anti-inflammatory cell responses,
and reducing pro-inflammatory effects in the treatment,
amelioration and prophylaxis of immune related disorders.
Additionally provided are polynucleotides and vectors that encode
antibody cytokine engrafted proteins and host cells capable of
producing antibody cytokine engrafted proteins, as well as methods
of combining antibody cytokine engrafted proteins with other
therapeutics in treating immune related disorder.
Inventors: |
DiDonato; Michael; (San
Diego, CA) ; Geierstanger; Bernhard Hubert; (Solana
Beach, CA) ; Junt; Tobias; (Liestal, CH) ;
Knuth; Mark; (El Cajon, CA) ; Meeusen; Shelly;
(Rancho Santa Fe, CA) ; Simpson; Carolina Nicole;
(Chula Vista, CA) ; Spraggon; Glen; (San Diego,
CA) ; Trauger; John; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novarts AG |
Basel |
|
CH |
|
|
Family ID: |
1000006078969 |
Appl. No.: |
17/458028 |
Filed: |
August 26, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16163857 |
Oct 18, 2018 |
11136366 |
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17458028 |
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15367003 |
Dec 1, 2016 |
10144768 |
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16163857 |
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62263008 |
Dec 4, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/52 20130101;
A61K 45/06 20130101; C07K 2317/565 20130101; C07K 2319/31 20130101;
A61K 39/395 20130101; A61K 38/2066 20130101; C07K 14/5428 20130101;
C07K 2317/56 20130101; C07K 16/00 20130101; C07K 2317/71 20130101;
A61K 2039/505 20130101; C07K 2318/10 20130101; C07K 2317/51
20130101 |
International
Class: |
C07K 14/54 20060101
C07K014/54; A61K 38/20 20060101 A61K038/20; A61K 39/395 20060101
A61K039/395; A61K 45/06 20060101 A61K045/06; C07K 16/00 20060101
C07K016/00 |
Claims
1-36. (canceled)
37. A method of activating monocytes in a patient in need thereof,
comprising administering an antibody cytokine engrafted protein
comprising: (a) a heavy chain variable region (VH), comprising
Complementarity Determining Regions (CDR) HCDR1, HCDR2, HCDR3; and
(b) a light chain variable region (VL), comprising LCDR1, LCDR2,
LCDR3; and (c) an Interleukin 10 (IL10) monomeric molecule
engrafted into a CDR of the VH or the VL.
38. The method of claim 37, wherein monocytes are activated and T
cells or NK cells are not activated.
39. The method of claim 37, wherein administration of the antibody
cytokine engrafted protein reduces TNF.alpha. production.
40-46. (canceled)
47. The method of claim 37, wherein the IL10 monomeric molecule is
engrafted into a heavy chain CDR.
48. The method of claim 47, wherein the heavy chain CDR is HCDR1,
HCDR2 or HCDR3.
49. The method of claim 48, wherein the IL10 monomeric molecule is
engrafted into the HCDR1.
50. The method of claim 37, wherein the IL10 monomeric molecule is
engrafted into a light chain CDR.
51. The method of claim 50, wherein the light chain CDR is LCDR1,
LCDR2 or LCDR3.
52. The method of claim 51, wherein the IL10 monomeric molecule is
engrafted into the LCDR1.
53. The method of claim 37, wherein the antibody cytokine engrafted
protein is capable of less activation of T cells or NK cells when
compared to recombinant human IL10.
54. The method of claim 37, wherein the antibody cytokine engrafted
protein has a longer half-life than rhIL10.
55. The method of claim 37, wherein the IL10 monomeric molecule
consists of SEQ ID NO:209.
56. The method of claim 37, wherein the antibody cytokine engrafted
protein further comprises an IgG class antibody heavy chain.
57. The method of claim 56, wherein the IgG class heavy chain is
selected from IgG1, IgG2, or IgG4.
58. The method of claim 37, wherein the binding specificity of the
CDRs to a target protein is reduced in the presence of the
engrafted IL10 monomeric molecule.
59. The method of claim 37, wherein the binding specificity of the
CDRs to a target protein is retained in the presence of the
engrafted IL10 monomeric molecule.
60. The method of claim 59, wherein the binding specificity of the
CDRs is distinct from the cytokine-receptor binding specificity of
the IL10 monomeric molecule.
61. The method of claim 37, wherein the antibody cytokine engrafted
protein comprises: (i) a heavy chain variable region that comprises
(a) a HCDR1 of SEQ ID NO: 193, (b) a HCDR2 of SEQ ID NO:194, (c) a
HCDR3 of SEQ ID NO:195 and a light chain variable region that
comprises: (d) a LCDR1 of SEQ ID NO:196, (e) a LCDR2 of SEQ ID
NO:197, and (f) a LCDR3 of SEQ ID NO:198; (ii) a heavy chain
variable region that comprises (a) a HCDR1 of SEQ ID NO:97, (b) a
HCDR2 of SEQ ID NO:98, (c) a HCDR3 of SEQ ID NO:99; and a light
chain variable region that comprises: (d) a LCDR1 of SEQ ID NO:100,
(e) a LCDR2 of SEQ ID NO:101, and (f) a LCDR3 of SEQ ID NO:102;
(iii) a heavy chain variable region that comprises (a) a HCDR1 of
SEQ ID NO:1, (b) a HCDR2 of SEQ ID NO:2, (c) a HCDR3 of SEQ ID
NO:3; and a light chain variable region that comprises: (d) a LCDR1
of SEQ ID NO:4, (e) a LCDR2 of SEQ ID NO:5, and (f) a LCDR3 of SEQ
ID NO:6; (iv) a heavy chain variable region that comprises (a) a
HCDR1 of SEQ ID NO:17, (b) a HCDR2 of SEQ ID NO:18, (c) a HCDR3 of
SEQ ID NO:19; and a light chain variable region that comprises: (d)
a LCDR1 of SEQ ID NO:20, (e) a LCDR2 of SEQ ID NO:21, and (f) a
LCDR3 of SEQ ID NO:22; (v) a heavy chain variable region that
comprises (a) a HCDR1 of SEQ ID NO:33, (b) a HCDR2 of SEQ ID NO:34,
(c) a HCDR3 of SEQ ID NO:35; and a light chain variable region that
comprises: (d) a LCDR1 of SEQ ID NO:36, (e) a LCDR2 of SEQ ID
NO:37, and (f) a LCDR3 of SEQ ID NO:38; (vi) a heavy chain variable
region that comprises (a) a HCDR1 of SEQ ID NO:49, (b) a HCDR2 of
SEQ ID NO:50, (c) a HCDR3 of SEQ ID NO:51; and a light chain
variable region that comprises: (d) a LCDR1 of SEQ ID NO:52, (e) a
LCDR2 of SEQ ID NO:53, and (f) a LCDR3 of SEQ ID NO:54; (vii) a
heavy chain variable region that comprises (a) a HCDR1 of SEQ ID
NO:65, (b) a HCDR2 of SEQ ID NO:66, (c) a HCDR3 of SEQ ID NO:67;
and a light chain variable region that comprises: (d) a LCDR1 of
SEQ ID NO:68, (e) a LCDR2 of SEQ ID NO:69, and (f) a LCDR3 of SEQ
ID NO:70; (viii) a heavy chain variable region that comprises (a) a
HCDR1 of SEQ ID NO:81, (b) a HCDR2 of SEQ ID NO:82, (c) a HCDR3 of
SEQ ID NO:83; and a light chain variable region that comprises: (d)
a LCDR1 of SEQ ID NO:84, (e) a LCDR2 of SEQ ID NO:85, and (f) a
LCDR3 of SEQ ID NO:86; (ix) a heavy chain variable region that
comprises (a) a HCDR1 of SEQ ID NO:113, (b) a HCDR2 of SEQ ID
NO:114, (c) a HCDR3 of SEQ ID NO:115; and a light chain variable
region that comprises: (d) a LCDR1 of SEQ ID NO:116, (e) a LCDR2 of
SEQ ID NO:117, and (f) a LCDR3 of SEQ ID NO:118; (x) a heavy chain
variable region that comprises (a) a HCDR1 of SEQ ID NO:129, (b) a
HCDR2 of SEQ ID NO:130, (c) a HCDR3 of SEQ ID NO:131; and a light
chain variable region that comprises: (d) a LCDR1 of SEQ ID NO:132,
(e) a LCDR2 of SEQ ID NO:133, and (f) a LCDR3 of SEQ ID NO:134;
(xi) a heavy chain variable region that comprises (a) a HCDR1 of
SEQ ID NO:145, (b) a HCDR2 of SEQ ID NO:146, (c) a HCDR3 of SEQ ID
NO:147; and a light chain variable region that comprises: (d) a
LCDR1 of SEQ ID NO:148, (e) a LCDR2 of SEQ ID NO:149, and (f) a
LCDR3 of SEQ ID NO:150; (xii) a heavy chain variable region that
comprises (a) a HCDR1 of SEQ ID NO:161, (b) a HCDR2 of SEQ ID
NO:162, (c) a HCDR3 of SEQ ID NO:163; and a light chain variable
region that comprises: (d) a LCDR1 of SEQ ID NO:164, (e) a LCDR2 of
SEQ ID NO:165, and (f) a LCDR3 of SEQ ID NO:166; (xiii) a heavy
chain variable region that comprises (a) a HCDR1 of SEQ ID NO:177,
(b) a HCDR2 of SEQ ID NO:178, (c) a HCDR3 of SEQ ID NO:179; and a
light chain variable region that comprises: (d) a LCDR1 of SEQ ID
NO:180, (e) a LCDR2 of SEQ ID NO:181, and (f) a LCDR3 of SEQ ID
NO:182; (xiv) a heavy chain variable region that comprises (a) a
HCDR1 of SEQ ID NO:210, (b) a HCDR2 of SEQ ID NO:211, (c) a HCDR3
of SEQ ID NO:212; and a light chain variable region that comprises:
(d) a LCDR1 of SEQ ID NO:213, (e) a LCDR2 of SEQ ID NO:214, and (f)
a LCDR3 of SEQ ID NO:215; or (xv) a heavy chain variable region
that comprises (a) a HCDR1 of SEQ ID NO:226, (b) a HCDR2 of SEQ ID
NO:227, (c) a HCDR3 of SEQ ID NO:228; and a light chain variable
region that comprises: (d) a LCDR1 of SEQ ID NO:229, (e) a LCDR2 of
SEQ ID NO:230, and (f) a LCDR3 of SEQ ID NO:231.
62. The method of claim 37, wherein the antibody cytokine engrafted
protein comprises: (i) a heavy chain variable region (VH) that
comprises SEQ ID NO:205, and a light chain variable region (VL)
that comprises SEQ ID NO:206; (ii) a heavy chain variable region
(VH) that comprises SEQ ID NO: 109, and a light chain variable
region (VL) that comprises SEQ ID NO: 110; (iii) a heavy chain
variable region (VH) that comprises SEQ ID NO:13, and a light chain
variable region (VL) that comprises SEQ ID NO:14; (iv) a heavy
chain variable region (VH) that comprises SEQ ID NO:29, and a light
chain variable region (VL) that comprises SEQ ID NO:30; (v) a heavy
chain variable region (VH) that comprises SEQ ID NO:45, and a light
chain variable region (VL) that comprises SEQ ID NO:46; (vi) a
heavy chain variable region (VH) that comprises SEQ ID NO:61, and a
light chain variable region (VL) that comprises SEQ ID NO:62; (vii)
a heavy chain variable region (VH) that comprises SEQ ID NO:77, and
a light chain variable region (VL) that comprises SEQ ID NO:78;
(viii) a heavy chain variable region (VH) that comprises SEQ ID
NO:93, and a light chain variable region (VL) that comprises SEQ ID
NO:94; (ix) a heavy chain variable region (VH) that comprises SEQ
ID NO:125, and a light chain variable region (VL) that comprises
SEQ ID NO:126; (x) a heavy chain variable region (VH) that
comprises SEQ ID NO:141, and a light chain variable region (VL)
that comprises SEQ ID NO:142; (xi) a heavy chain variable region
(VH) that comprises SEQ ID NO:157, and a light chain variable
region (VL) that comprises SEQ ID NO:158; (xii) a heavy chain
variable region (VH) that comprises SEQ ID NO:173, and a light
chain variable region (VL) that comprises SEQ ID NO:174; (xiii) a
heavy chain variable region (VH) that comprises SEQ ID NO:189, and
a light chain variable region (VL) that comprises SEQ ID NO:190;
(xiv) a heavy chain variable region (VH) that comprises SEQ ID
NO:222, and a light chain variable region (VL) that comprises SEQ
ID NO:223; or (xv) a heavy chain variable region (VH) that
comprises SEQ ID NO:238, and a light chain variable region (VL)
that comprises SEQ ID NO:239.
Description
FIELD
[0001] The present disclosure relates to antibody-cytokine
engrafted compositions that bind to interleukin-ten receptor
(IL10R), and stimulate signaling through this receptor.
BACKGROUND
[0002] Interleukin 10 (IL10) was identified as a cytokine synthesis
inhibition factor, exerting effects on Thl helper cells and antigen
presenting cells (APC's). IL10, a 161 amino acid protein that
exists as a domain-swapped non-covalent homodimer, binds to the
high affinity IL-10 receptor I (IL-10RI) with a stoichiometry of
one homodimer to four IL-10RI subunits that recruits IL-10R2 and
activates signal transduction through JAKI/TYK2 pathway. IL10
inhibits monocyte and macrophage synthesis of pro-inflammatory
cytokines, e.g., IL-1, IL-6, IL-8, IL-12, TNF-alpha, GM-CSF, and
reactive oxygen and nitrogen intermediates, and has shown strong
efficacy in numerous preclinical models of autoimmunity. IL10
efficacy is thought to result from its ability to inhibit
activation and effector function of monocytes, macrophages, and T
cells. However, IL10 is a pleiotropic immunoregulatory cytokine
with a very broad spectrum of biological activities. IL10 has also
been shown to promote growth and differentiation of B cells, NK
cells, cytotoxic and helper T cells, mast cells, granulocytes,
dendritic cells, keratinocytes, and even endothelial cells,
indicating it also possesses pro-inflammatory functions (Moore et
al., Annu Rev Immunol. 2001;19:683-765).
[0003] IL10 as a therapeutic for the treatment of autoimmune and
inflammatory conditions, specifically inflammatory bowel disease
(IBD), is supported in animal model systems as well as genetics.
For example, knockout of IL10 in mice and mice with a defect in the
IL-10R develop a spontaneous onset of colitis. In humans, 9-16% of
Crohn's Disease patients have a NOD2 mutation that is associated
with defective or reduced IL10 production, and 20% of ulcerative
colitis (UC) patients carry a disease-associated IL10 SNP in the
3'UTR. Lastly, certain mutations in IL-10R1 in humans cause a rare,
severe form of Crohn's Disease (Glocker at al., N Engl J Med. 2009;
361(21):2033-45).
[0004] Despite the strong genetic linkage data, recombinant human
IL10 demonstrated tolerability and safety in healthy volunteers and
specific patient populations up to 25 .mu.g/kg, and mild effects in
a subset of recipients up to 100 .mu.g/kg; however only mild
clinical efficacy was found for the specific patient populations
and clinical development was discontinued for lack of efficacy.
Reviews of the studies have been undertaken and several
possibilities for the results have been identified, for example,
limited efficacy is due to poor exposure of the target organ
(colon) due to short half-life of IL10; the pro-inflammatory
effects of IL10 that manifested at high doses and counterbalanced
the anti-inflammatory efficacy of low dose IL10. (Lindsay and
Hodgson, Aliment Pharmacol Ther. 2001; 15(11) 1709-1716) Consistent
with this possibility, high doses of IL10 were associated with
elevated systemic IFN.gamma., granzyme and neopterin levels, which
are associated with increased inflammation (Tilg et al., Gut 2002;
50(2)191-5).
[0005] Antibody cytokine engrafted proteins have been developed to
generate a more effective IL10 therapeutic that confers an improved
profile that address the clinical failings of recombinant human
IL10. This improved therapeutic profile includes strong
anti-inflammatory IL10 potency with a marked reduction in
pro-inflammatory activity, extended half-life, ease of
administration and stability. Thus, the disclosure provides
improved IL10 antibody cytokine engrafted proteins and methods of
treating immune related disorders.
SUMMARY
[0006] The disclosure provides for antibody-cytokine engrafted
proteins having preferred therapeutic profiles over recombinant
IL10 and IL10 constructs known and used in the clinic. In
particular, provided are antibody cytokine engrafted proteins that
maintain the desired anti-inflammatory activity of native or
recombinant human IL10; however, a marked reduction in
pro-inflammatory activity is retained. Additionally, the antibody
cytokine engrafted proteins convey improved half-life, stability
and ease of administration over recombinant human IL10 protein
(rhIL10). The preferred properties of these compositions result in
preferable therapeutic compositions over those previously used in
the clinic or described in the literature. The present disclosure
thus provides antibody cytokine engrafted proteins that bind to and
promote signalling through IL10 receptors and stimulate certain
cell types. Provided are antibody cytokine engrafted proteins
comprising: (i) an immunoglobulin heavy chain sequence comprising a
heavy chain variable region (VH) and a heavy chain constant region
comprising CH1, CH2 and CH3 regions, and (ii) an immunoglobulin
light chain sequence comprising a light chain variable region (VL),
and a light chain constant region (CL); wherein a monomeric IL10
molecule is engrafted into a complementarity determining region
(CDR) of the VH or the VL.
[0007] In some embodiments, the antibody cytokine engrafted protein
includes a variable heavy chain comprising SEQ ID NO: 13 and a
variable light chain comprising SEQ ID NO: 14. In some embodiments,
the antibody cytokine engrafted protein includes a variable heavy
chain comprising SEQ ID NO: 29 and a variable light chain
comprising SEQ ID NO: 30. In some embodiments, the antibody
cytokine engrafted protein includes a variable heavy chain
comprising SEQ ID NO: 45 and a variable light chain comprising SEQ
ID NO: 46. In some embodiments, the antibody cytokine engrafted
protein includes a variable heavy chain comprising SEQ ID NO: 61
and a variable light chain comprising SEQ ID NO: 62. In some
embodiments, the antibody cytokine engrafted protein includes a
variable heavy chain comprising SEQ ID NO: 77 and a variable light
chain comprising SEQ ID NO: 78. In some embodiments, the antibody
cytokine engrafted protein a variable heavy chain comprising SEQ ID
NO: 93 and a variable light chain comprising SEQ ID NO: 94. In some
embodiments, the antibody cytokine engrafted protein includes a
variable heavy chain comprising SEQ ID NO: 109 and a variable light
chain comprising SEQ ID NO: 110. In some embodiments, the antibody
cytokine engrafted protein includes a variable heavy chain
comprising SEQ ID NO: 125 and a variable light chain comprising SEQ
ID NO: 126. In some embodiments, the antibody cytokine engrafted
protein includes a variable heavy chain comprising SEQ ID NO: 141
and a variable light chain comprising SEQ ID NO: 142. In some
embodiments, the antibody cytokine engrafted protein includes a
variable heavy chain comprising SEQ ID NO: 157 and a variable light
chain comprising SEQ ID NO: 158. In some embodiments, the antibody
cytokine engrafted protein includes a variable heavy chain
comprising SEQ ID NO: 173 and a variable light chain comprising SEQ
ID NO: 174. In some embodiments, the antibody cytokine engrafted
protein includes a variable heavy chain comprising SEQ ID NO: 189
and a variable light chain comprising SEQ ID NO: 190. In some
embodiments, the antibody cytokine engrafted protein includes a
variable heavy chain comprising SEQ ID NO: 205 and a variable light
chain comprising SEQ ID NO: 206. In some embodiments, the antibody
cytokine engrafted protein includes a variable heavy chain
comprising SEQ ID NO: 222 and a variable light chain comprising SEQ
ID NO: 223. In some embodiments, the antibody cytokine engrafted
protein includes a variable heavy chain comprising SEQ ID NO: 238
and a variable light chain comprising SEQ ID NO: 239.
[0008] In certain embodiments, the antibody cytokine engrafted
protein comprises an IgG class antibody Fc region. In particular
embodiments, the immunoglobulin is selected from IgG1, IgG2, or
IgG4 subclass Fc region. In some embodiments, the antibody cytokine
engrafted protein optionally contains at least one modification
that modulates (i.e., increases or decreases) binding of the
antibody or antibody fragment to an Fc receptor. The immunoglobulin
heavy chain of the antibody cytokine engrafted protein optionally
comprises a modification conferring modified effector function. In
particular embodiments the immunoglobulin heavy chain comprises a
mutation conferring reduced effector function selected from any of
D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234A/L235A,
P329A/L234A/L235A, and P329G/L234A/L235A.
[0009] In a related aspect, the disclosure further provides
polynucleotides encoding at least a heavy chain and/or a light
chain protein of an antibody cytokine engrafted protein as
described herein. In another related aspect, the disclosure further
provides host cells suitable for the production of an antibody
cytokine engrafted protein as described herein. In particular
embodiments, host cells comprise nucleic acids encoding a heavy
chain and/or light chain polypeptide of the antibody cytokine
engrafted protein. In still another aspect, methods for producing
antibody cytokine engrafted proteins of the disclosure are
provided, comprising culturing provided host cells as described
herein under conditions suitable for expression, formation, and
secretion of the antibody cytokine engrafted protein and recovering
the antibody cytokine engrafted protein from the culture. In a
further aspect, the disclosure further provides kits comprising an
antibody cytokine engrafted protein, as described herein.
[0010] In another related aspect, the disclosure further provides
compositions comprising an antibody cytokine engrafted protein, as
described herein, and a pharmaceutically acceptable carrier. In
some embodiments, the disclosure provides pharmaceutical
compositions comprising an antibody cytokine engrafted protein of
the disclosure for administering to an individual.
[0011] In another aspect, the disclosure provides methods of
treating an immune related disorder in an individual in need
thereof, comprising administering to the individual a
therapeutically effective amount of an antibody cytokine engrafted
protein of the disclosure, as described herein. In a further
aspect, the disclosure provides an antibody cytokine engrafted
protein for use in treatment or prophylaxis of an immune related
disorder in an individual.
[0012] In some embodiments, the patient has an immune related
disorder, for example, inflammatory bowel disease, Crohn's disease,
ulcerative colitis, rheumatoid arthritis, psoriasis, type I
diabetes, acute pancreatitis, uveitis, Sjogren's disease, Behcet's
disease, sarcoidosis, and graft versus host disease (GVHD). In
particular embodiments the inflammatory bowel disease is Crohn's
disease or ulcerative colitis.
[0013] In some embodiments the disclosure provides an antibody
cytokine engrafted protein comprising: [0014] (a) a heavy chain
variable region (VH), comprising Complementarity Determining
Regions (CDR) HCDR1, HCDR2, HCDR3; and [0015] (b) a light chain
variable region (VL), comprising LCDR1, LCDR2, LCDR3; and [0016]
(c) an Interleukin 10 (IL10) monomeric molecule engrafted into a
CDR of the VH or the VL.
[0017] The antibody cytokine engrafted protein wherein the IL10
monomeric molecule is engrafted into a heavy chain CDR.
[0018] The antibody cytokine engrafted protein wherein the heavy
chain CDR is selected from HCDR1, HCDR2 or HCDR3.
[0019] The antibody cytokine engrafted protein wherein the IL10
monomeric molecule is engrafted into HCDR1.
[0020] The antibody cytokine engrafted protein wherein the IL10
monomeric molecule is engrafted into a light chain CDR.
[0021] The antibody cytokine engrafted protein wherein the light
chain CDR is selected from LCDR1, LCDR2 or LCDR3.
[0022] The antibody cytokine engrafted protein wherein the IL10
monomeric molecule is engrafted into a LCDR1.
[0023] The antibody cytokine engrafted protein wherein the antibody
cytokine engrafted protein has less activation of T cells or NK
cells when compared to IL10.
[0024] The antibody cytokine engrafted protein wherein the antibody
cytokine engrafted protein has a longer half-life than rhIL10.
[0025] The antibody cytokine engrafted protein wherein the IL10
monomeric molecule consists of SEQ ID NO:209.
[0026] The antibody cytokine engrafted protein further comprising
an IgG class antibody heavy chain.
[0027] The antibody cytokine engrafted protein wherein the IgG
class heavy chain is selected from IgG1, IgG2, or IgG4.
[0028] The antibody cytokine engrafted protein wherein the binding
specificity of the CDRs to a target protein is reduced by the
presence of the engrafted IL10 monomeric molecule.
[0029] The antibody cytokine engrafted protein wherein the binding
specificity of the CDRs to a target protein is retained in the
presence of the engrafted IL10 monomeric molecule.
[0030] The antibody cytokine engrafted protein wherein the binding
specificity of the CDRs is to a target protein distinct from the
cytokine-receptor binding specificity of the IL10 monomeric
molecule.
[0031] The antibody cytokine engrafted protein wherein the binding
specificity of the CDRs is to a non-human target.
[0032] The antibody cytokine engrafted protein wherein the antibody
cytokine engrafted protein is humanized or human.
[0033] An antibody cytokine engrafted protein comprising: [0034]
(i) a heavy chain variable region that comprises (a) a HCDR1 of SEQ
ID NO: 193, (b) a HCDR2 of SEQ ID NO:194, (c) a HCDR3 of SEQ ID
NO:195 and a light chain variable region that comprises: (d) a
LCDR1 of SEQ ID NO:196, (e) a LCDR2 of SEQ ID NO:197, and (f) a
LCDR3 of SEQ ID NO:198; [0035] (ii) a heavy chain variable region
that comprises (a) a HCDR1 of SEQ ID NO:97, (b) a HCDR2 of SEQ ID
NO:98, (c) a HCDR3 of SEQ ID NO:99; and a light chain variable
region that comprises: (d) a LCDR1 of SEQ ID NO:100, (e) a LCDR2 of
SEQ ID NO:101, and (f) a LCDR3 of SEQ ID NO:102; [0036] (iii) a
heavy chain variable region that comprises (a) a HCDR1 of SEQ ID
NO:1, (b) a HCDR2 of SEQ ID NO:2, (c) a HCDR3 of SEQ ID NO:3; and a
light chain variable region that comprises: (d) a LCDR1 of SEQ ID
NO:4, (e) a LCDR2 of SEQ ID NO:5, and (f) a LCDR3 of SEQ ID NO:6;
[0037] (iv) a heavy chain variable region that comprises (a) a
HCDR1 of SEQ ID NO:17, (b) a HCDR2 of SEQ ID NO:18, (c) a HCDR3 of
SEQ ID NO:19; and a light chain variable region that comprises: (d)
a LCDR1 of SEQ ID NO:20, (e) a LCDR2 of SEQ ID NO:21, and (f) a
LCDR3 of SEQ ID NO:22; [0038] (v) a heavy chain variable region
that comprises (a) a HCDR1 of SEQ ID NO:33, (b) a HCDR2 of SEQ ID
NO:34, (c) a HCDR3 of SEQ ID NO:35; and a light chain variable
region that comprises: (d) a LCDR1 of SEQ ID NO:36, (e) a LCDR2 of
SEQ ID NO:37, and (f) a LCDR3 of SEQ ID NO:38; [0039] (vi) a heavy
chain variable region that comprises (a) a HCDR1 of SEQ ID NO:49,
(b) a HCDR2 of SEQ ID NO:50, (c) a HCDR3 of SEQ ID NO:51; and a
light chain variable region that comprises: (d) a LCDR1 of SEQ ID
NO:52, (e) a LCDR2 of SEQ ID NO:53, and (f) a LCDR3 of SEQ ID
NO:54; [0040] (vii) a heavy chain variable region that comprises
(a) a HCDR1 of SEQ ID NO:65, (b) a HCDR2 of SEQ ID NO:66, (c) a
HCDR3 of SEQ ID NO:67; and a light chain variable region that
comprises: (d) a LCDR1 of SEQ ID NO:68, (e) a LCDR2 of SEQ ID
NO:69, and (f) a LCDR3 of SEQ ID NO:70; [0041] (viii) a heavy chain
variable region that comprises (a) a HCDR1 of SEQ ID NO:81, (b) a
HCDR2 of SEQ ID NO:82, (c) a HCDR3 of SEQ ID NO:83; and a light
chain variable region that comprises: (d) a LCDR1 of SEQ ID NO:84,
(e) a LCDR2 of SEQ ID NO:85, and (f) a LCDR3 of SEQ ID NO:86;
[0042] (ix) a heavy chain variable region that comprises (a) a
HCDR1 of SEQ ID NO:113, (b) a HCDR2 of SEQ ID NO:114, (c) a HCDR3
of SEQ ID NO:115; and a light chain variable region that comprises:
(d) a LCDR1 of SEQ ID NO:116, (e) a LCDR2 of SEQ ID NO:117, and (f)
a LCDR3 of SEQ ID NO:118; [0043] (x) a heavy chain variable region
that comprises (a) a HCDR1 of SEQ ID NO:129, (b) a HCDR2 of SEQ ID
NO:130, (c) a HCDR3 of SEQ ID NO:131; and a light chain variable
region that comprises: (d) a LCDR1 of SEQ ID NO:132, (e) a LCDR2 of
SEQ ID NO:133, and (f) a LCDR3 of SEQ ID NO:134; [0044] (xi) a
heavy chain variable region that comprises (a) a HCDR1 of SEQ ID
NO:145, (b) a HCDR2 of SEQ ID NO:146, (c) a HCDR3 of SEQ ID NO:147;
and a light chain variable region that comprises: (d) a LCDR1 of
SEQ ID NO:148, (e) a LCDR2 of SEQ ID NO:149, and (f) a LCDR3 of SEQ
ID NO:150; [0045] (xii) a heavy chain variable region that
comprises (a) a HCDR1 of SEQ ID NO:161, (b) a HCDR2 of SEQ ID
NO:162, (c) a HCDR3 of SEQ ID NO:163; and a light chain variable
region that comprises: (d) a LCDR1 of SEQ ID NO:164, (e) a LCDR2 of
SEQ ID NO:165, and (f) a LCDR3 of SEQ ID NO:166; [0046] (xiii) a
heavy chain variable region that comprises (a) a HCDR1 of SEQ ID
NO:177, (b) a HCDR2 of SEQ ID NO:178, (c) a HCDR3 of SEQ ID NO:179;
and a light chain variable region that comprises: (d) a LCDR1 of
SEQ ID NO:180, (e) a LCDR2 of SEQ ID NO:181, and (f) a LCDR3 of SEQ
ID NO:182; [0047] (xiv) a heavy chain variable region that
comprises (a) a HCDR1 of SEQ ID NO:210, (b) a HCDR2 of SEQ ID
NO:211, (c) a HCDR3 of SEQ ID NO:212; and a light chain variable
region that comprises: (d) a LCDR1 of SEQ ID NO:213, (e) a LCDR2 of
SEQ ID NO:214, and (f) a LCDR3 of SEQ ID NO:215; and [0048] (xv) a
heavy chain variable region that comprises (a) a HCDR1 of SEQ ID
NO:226, (b) a HCDR2 of SEQ ID NO:227, (c) a HCDR3 of SEQ ID NO:228;
and a light chain variable region that comprises: (d) a LCDR1 of
SEQ ID NO:229, (e) a LCDR2 of SEQ ID NO:230, and (f) a LCDR3 of SEQ
ID NO:231.
[0049] An antibody cytokine engrafted protein comprising: [0050]
(i) a heavy chain variable region (VH) that comprises SEQ ID
NO:205, and a light chain variable region (VL) that comprises SEQ
ID NO:206; [0051] (ii) a heavy chain variable region (VH) that
comprises SEQ ID NO: 109, and a light chain variable region (VL)
that comprises SEQ ID NO: 110; [0052] (iii) a heavy chain variable
region (VH) that comprises SEQ ID NO:13, and a light chain variable
region (VL) that comprises SEQ ID NO:14; [0053] (iv) a heavy chain
variable region (VH) that comprises SEQ ID NO:29, and a light chain
variable region (VL) that comprises SEQ ID NO:30; [0054] (v) a
heavy chain variable region (VH) that comprises SEQ ID NO:45, and a
light chain variable region (VL) that comprises SEQ ID NO:46;
[0055] (vi) a heavy chain variable region (VH) that comprises SEQ
ID NO:61, and a light chain variable region (VL) that comprises SEQ
ID NO:62; [0056] (vii) a heavy chain variable region (VH) that
comprises SEQ ID NO:77, and a light chain variable region (VL) that
comprises SEQ ID NO:78; [0057] (viii) a heavy chain variable region
(VH) that comprises SEQ ID NO:93, and a light chain variable region
(VL) that comprises SEQ ID NO:94; [0058] (ix) a heavy chain
variable region (VH) that comprises SEQ ID NO:125, and a light
chain variable region (VL) that comprises SEQ ID NO:126; [0059] (x)
a heavy chain variable region (VH) that comprises SEQ ID NO:141,
and a light chain variable region (VL) that comprises SEQ ID
NO:142; [0060] (xi) a heavy chain variable region (VH) that
comprises SEQ ID NO:157, and a light chain variable region (VL)
that comprises SEQ ID NO:158; [0061] (xii) a heavy chain variable
region (VH) that comprises SEQ ID NO:173, and a light chain
variable region (VL) that comprises SEQ ID NO:174; [0062] (xiii) a
heavy chain variable region (VH) that comprises SEQ ID NO:189, and
a light chain variable region (VL) that comprises SEQ ID NO:190;
[0063] (xiv) a heavy chain variable region (VH) that comprises SEQ
ID NO:222, and a light chain variable region (VL) that comprises
SEQ ID NO:223; and [0064] (xv) a heavy chain variable region (VH)
that comprises SEQ ID NO:238, and a light chain variable region
(VL) that comprises SEQ ID NO:239.
[0065] The antibody cytokine engrafted protein further comprising a
modified Fc region corresponding with reduced effector
function.
[0066] The antibody cytokine engrafted protein wherein the modified
Fc region comprises a mutation selected from one or more of D265A,
P329A, P329G, N297A, L234A, and L235A.
[0067] The antibody cytokine engrafted protein wherein the modified
Fc region is selected from the group consisting of D265A/P329A,
D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0068] An antibody cytokine engrafted protein consisting of a HCDR1
of SEQ ID NO: 193, a HCDR2 of SEQ ID NO:194, a HCDR3 of SEQ ID
NO:195, a LCDR1 of SEQ ID NO:196, a LCDR2 of SEQ ID NO:197, a LCDR3
of SEQ ID NO:198, a modified Fc region containing the mutation
D265A/P329A, and wherein the antibody cytokine engrafted protein
has less activation of T cells or NK cells when compared to
IL10.
[0069] The antibody cytokine engrafted protein wherein the binding
specificity of the CDRs is to a non-human target.
[0070] The antibody cytokine engrafted protein wherein the antibody
cytokine engrafted protein has a longer half-life than rhIL10.
[0071] An isolated nucleic acid comprising: [0072] (i) a heavy
chain variable region encoding polynucleotide sequence of SEQ ID
NO: 246 and a light chain variable region encoding polynucleotide
sequence of SEQ ID NO:247; [0073] (ii) a heavy chain encoding
polynucleotide sequence of SEQ ID NO:248 and a light chain encoding
polynucleotide sequence of SEQ ID NO:249; [0074] (iii) a heavy
chain variable region encoding polynucleotide sequence of SEQ ID
NO: 242 and a light chain variable region encoding polynucleotide
sequence of SEQ ID NO:243; or [0075] (iv) a heavy chain encoding
polynucleotide sequence of SEQ ID NO:244 and a light chain encoding
polynucleotide sequence of SEQ ID NO:245.
[0076] A recombinant host cell suitable for the production of an
antibody cytokine engrafted protein, comprising the nucleic acids
encoding the heavy and light chain polypeptides of the antibody
cytokine engrafted protein, and optionally, secretion signals.
[0077] The host cell wherein the cell is mammalian.
[0078] A pharmaceutical composition comprising the antibody
cytokine engrafted protein and a pharmaceutically acceptable
carrier.
[0079] A method of treatment or prophylaxis of an immune related
disorder in an individual in need thereof, comprising administering
to the individual a therapeutically effective amount of the
antibody cytokine engrafted protein.
[0080] The method wherein the immune related disorder is selected
from the group consisting of: inflammatory bowel disease, Crohn's
disease, ulcerative colitis, rheumatoid arthritis, psoriasis, type
I diabetes, acute pancreatitis, uveitis, Sjogren's disease,
Behcet's disease, sarcoidosis, and graft versus host disease
(GVHD).
[0081] The method wherein the antibody cytokine engrafted protein
is administered in combination with another therapeutic agent.
[0082] The method wherein the therapeutic agent is an anti-TNF
agent selected from the group consisting of: infliximab,
adalimumab, certolizumab, golimumab, natalizumab, and
vedolizumab.
[0083] The method wherein the therapeutic agent is an
aminosalicylate agent selected from the group consisting of:
sulfasalazine, mesalamine, balsalazide, olsalazine and other
derivatives of 5-aminosalicylic acid.
[0084] The method wherein the therapeutic agent is a corticosteroid
selected from the group consisting of: methylprednisolone,
hydrocortisone, prednisone, budenisonide, mesalamine, and
dexamethasone.
[0085] The method wherein the therapeutic agent is an antibacterial
agent.
[0086] A method of activating monocytes in a patient in need
thereof, comprising administering an antibody cytokine engrafted
protein.
[0087] The method wherein monocytes are activated and T cells or NK
cells are not activated.
[0088] The method wherein administration of the antibody cytokine
engrafted protein reduces TNF.alpha. production.
[0089] The use of an antibody cytokine engrafted protein
comprising: [0090] (i) a heavy chain variable region that comprises
(a) a HCDR1 of SEQ ID NO: 193, (b) a HCDR2 of SEQ ID NO:194, (c) a
HCDR3 of SEQ ID NO:195 and a light chain variable region that
comprises: (d) a LCDR1 of SEQ ID NO:196, (e) a LCDR2 of SEQ ID
NO:197, and (f) a LCDR3 of SEQ ID NO:198; and [0091] (ii) a heavy
chain variable region that comprises (a) a HCDR1 of SEQ ID NO:97,
(b) a HCDR2 of SEQ ID NO:98, (c) a HCDR3 of SEQ ID NO:99; and a
light chain variable region that comprises: (d) a LCDR1 of SEQ ID
NO:100, (e) a LCDR2 of SEQ ID NO:101, and (f) a LCDR3 of SEQ ID
NO:102 in the treatment of immune related disorders.
[0092] The antibody cytokine engrafted protein for use as a
therapeutic in treatment of an immune related disorder selected
from the group consisting of: inflammatory bowel disease, Crohn's
disease, ulcerative colitis, rheumatoid arthritis, psoriasis, type
I diabetes, acute pancreatitis, uveitis, Sjogren's disease,
Behcet's disease, sarcoidosis, and graft versus host disease
(GVHD).
[0093] The use wherein the antibody cytokine engrafted protein is
administered in combination with another therapeutic agent.
[0094] The use wherein the therapeutic agent is an anti-TNF agent
selected from the group consisting of: infliximab, adalimumab,
certolizumab, golimumab, natalizumab, and vedolizumab.
[0095] The use wherein the therapeutic agent is an aminosalicylate
agent selected from the group consisting of: sulfasalazine,
mesalamine, balsalazide, olsalazine and other derivatives of
5-aminosalicylic acid.
[0096] The use wherein the therapeutic agent is a corticosteroid
selected from the group consisting of: methylprednisolone,
hydrocortisone, prednisone, budenisonide, mesalamine, and
dexamethasone.
[0097] The use wherein the therapeutic agent is an antibacterial
agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0098] FIG. 1 illustrates the biological effects of IL10, the cell
types it acts on, and whether those biological effects are
anti-inflammatory or pro-inflammatory. Antibody cytokine engrafted
proteins reduce the pro-inflammatory biological effects depicted on
the right side of the diagram.
[0099] FIG. 2 illustrates the structure of an IL10 antibody
cytokine engrafted protein. The insert panel depicts monomeric IL10
inserted into the LCDR1 of an antibody. Dark residues depict
exemplary Fc modifications optionally incorporated in the antibody
cytokine engrafted protein.
[0100] FIG. 3A-3B depicts results of in vitro biological assays of
recombinant human IL10 (rhIL10, gray square) and the IgGIL10M13
antibody cytokine engrafted protein (black triangle). FIG. 3A
illustrates that IgGIL10M13 demonstrated decreased pro-inflammatory
activity as compared to rhIL10 as measured by IFN gamma induction
in CD8 T cell assays. Similar differential activity was found on
human primary NK cells, B cells, and mast cells, as well as using
granzyme-B as a readout measurement. FIG. 3B illustrates that
rhIL10 and IgGIL10M13 demonstrate similar anti-inflammatory
activity as measured by inhibition of TNF.alpha. in whole blood
assays.
[0101] FIG. 4 depicts results of CyTOF analysis of IL10 dependent
pSTAT3 signaling in human whole blood stimulated with equal molar
amounts recombinant human IL10 rhIL10 (left panel) or IgGIL10M13
(right panel). IL10 induces anti-inflammatory activities in
monocytes; and activation of T, B or NK cells induces
pro-inflammatory cytokines. Results of fold change in activity of
cells over unstimulated are depicted by heat map (changes in
shading). Left panel indicates rhIL10 confers stimulation across
all IL10 sensitive cell types (with outline); however, as seen in
the right panel IgGIL10M13 confers less potent stimulation on T, B,
and NK cells, with levels similar or slightly above unstimulated
cells; while a similar potency of stimulation of monocytes
(outlined) and mDC cells was demonstrated with IgG-IL10M and
rhIL10. These relevant cell types (monocytes, mDC) are key cells
for maintenance of gut homeostatis in inflammatory bowel
disease.
[0102] FIG. 5A-5D illustrates improved characteristics of antibody
cytokine engrafted protein IgGIL10M13 in in vivo assays. FIG. 5A-B
depicts results of pharmacokinetic studies of rhIL10 and
IgGIL10M13. Following intravenous administration, IgGIL10M13
demonstrates prolonged pharmacokinetics (half-life) as antibody
cytokine engrafted protein is still detectable after 4.4 days (FIG.
5B), while rhIL10 had a half-life of approximately 1 hour (FIG.
5A). FIG. 5C and 5D depict results of pharmacodynamic assays of in
vivo activity of antibody cytokine engrafted proteins. FIG. 5C
depicts in vivo activity in colon tissue as measured by pSTAT3
signaling seventy-two (72) hours post dosing. FIG. 5D depicts
improved duration of in vivo response of IgGIL10M13 as compared to
rhIL10 as measured by inhibition of TNF.alpha. in response to LPS
challenge following administration of IgGIL10M13.
[0103] FIG. 6 is the results of an LPS challenge model,
demonstrating IgGIL10M13 reduces TNF.alpha. induction 48 hours
after LPS challenge.
[0104] FIG. 7 is a graph representing the improved % CMAX of IL10
antibody cytokine engrafted proteins.
[0105] FIG. 8 depicts CyTOF data of pSTAT3 activity in various
immune cells from healthy subjects and patients when stimulated
with rhIL10 or with IgGIL10M13.
[0106] FIGS. 9A-9C are graphical representations demonstrating
IgGIL10M13 has reduced pro-inflammatory activity in PHA stimulated
human whole blood compared to rhIL10. FIG. 9D shows the graphs of a
titration experiment with rhIL10 and IgGIL10M13.
[0107] FIGS. 10A-10B depict the aggregation properties of IL10 wild
type or monomeric when conjugated via a linker to an Fc, compared
to the aggregation properties of an antibody cytokine engrafted
protein.
[0108] FIG. 11 is ELISA data showing that the IL10 antibody
cytokine engrafted protein still binds to RSV.
[0109] FIG. 12 is a representation of the mechanism of action of an
IL10 antibody cytokine engrafted protein. The left panel shows how
a normal rhIL10 dimer binds IL-10R1, and initiates strong pSTAT3
signaling. The right panel depicts how an IL10 monomer engrafted
into a CDR of an antibody is constrained to have less efficient
binding to IL-10R1 and thus produces a weaker pSTAT3 signal.
[0110] FIG. 13A-13C is the crystal structure resolution of IL10
monomer engrafted into LCDR1 of palivizumab.
DEFINITIONS
[0111] An "antibody" refers to a molecule of the immunoglobulin
family comprising a tetrameric structural unit. Each tetramer is
composed of two identical pairs of polypeptide chains, each pair
having one "light" chain (about 25 kD) and one "heavy" chain (about
50-70 kD), connected through a disulfide bond. Recognized
immunoglobulin genes include the .kappa., .lamda., .alpha.,
.gamma., .delta., .epsilon., and .mu. constant region genes, as
well as the myriad immunoglobulin variable region genes. Light
chains are classified as either .kappa. or .lamda.. Heavy chains
are classified as .gamma., .mu., .alpha., .delta., or .epsilon.,
which in turn define the immunoglobulin classes, IgG, IgM, IgA,
IgD, and IgE, respectively. Antibodies can be of any isotype/class
(e.g., IgG, IgM, IgA, IgD, and IgE), or any subclass (e.g., IgG1,
IgG2, IgG3, IgG4, IgA1, IgA2).
[0112] Both the light and heavy chains are divided into regions of
structural and functional homology. The terms "constant" and
"variable" are used structurally and functionally. The N-terminus
of each chain defines a variable (V) region or domain of about 100
to 110 or more amino acids primarily responsible for antigen
recognition. The terms variable light chain (V.sub.L) and variable
heavy chain (V.sub.H) refer to these regions of light and heavy
chains respectively. The pairing of a V.sub.H and V.sub.L together
forms a single antigen-binding site. In addition to V regions, both
heavy chains and light chains contain a constant (C) region or
domain. A secreted form of a immunoglobulin C region is made up of
three C domains, CH1, CH2, CH3, optionally CH4 (C.mu.), and a hinge
region. A membrane-bound form of an immunoglobulin C region also
has membrane and intracellular domains. Each light chain has a VL
at the N-terminus followed by a constant domain (C) at its other
end. The constant domains of the light chain (CL) and the heavy
chain (CH1, CH2 or CH3) confer important biological properties such
as secretion, transplacental mobility, Fc receptor binding,
complement binding, and the like. By convention, the numbering of
the constant region domains increases as they become more distal
from the antigen binding site or amino-terminus of the antibody.
The N-terminus is a variable region and at the C-terminus is a
constant region; the CH3 and CL domains actually comprise the
carboxy-terminal domains of the heavy and light chain,
respectively. The VL is aligned with the VH and the CL is aligned
with the first constant domain of the heavy chain. As used herein,
an "antibody" encompasses conventional antibody structures and
variations of antibodies. Thus, within the scope of this concept
are full length antibodies, chimeric antibodies, humanized
antibodies, human antibodies, and antibody fragments thereof.
[0113] Antibodies exist as intact immunoglobulin chains or as a
number of well-characterized antibody fragments produced by
digestion with various peptidases. The term "antibody fragment," as
used herein, refers to one or more portions of an antibody that
retain the ability to specifically interact with (e.g., by binding,
steric hindrance, stabilizing/destabilizing, spatial distribution)
an epitope of an antigen. Thus, for example, pepsin digests an
antibody below the disulfide linkages in the hinge region to
produce F(ab)'.sub.2, a dimer of Fab' which itself is a light chain
joined to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2
may be reduced under mild conditions to break the disulfide linkage
in the hinge region, thereby converting the F(ab)'.sub.2 dimer into
an Fab' monomer. The Fab' monomer is essentially Fab with part of
the hinge region. Paul, Fundamental Immunology 3d ed. (1993). While
various antibody fragments are defined in terms of the digestion of
an intact antibody, one of skill will appreciate that such
fragments may be synthesized de novo either chemically or by using
recombinant DNA methodology. As used herein, an "antibody fragment"
refers to one or more portions of an antibody, either produced by
the modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies, that retain binding
specificity and functional activity. Examples of antibody fragments
include Fv fragments, single chain antibodies (ScFv), Fab, Fab', Fd
(Vh and CH1 domains), dAb (Vh and an isolated CDR); and multimeric
versions of these fragments (e.g., F(ab').sub.2,) with the same
binding specificity. Antibody fragments can also be incorporated
into cytokine engrafted proteins to achieve the binding specificity
and activity provided in the present disclosure.
[0114] A "Fab" domain as used herein comprises a heavy chain
variable domain, a constant region CH1 domain, a light chain
variable domain, and a light chain constant region CL domain. The
interaction of the domains is stabilized by a disulfide bond
between the CH1 and CL domains. In some embodiments, the heavy
chain domains of the Fab are in the order, from N-terminus to
C-terminus, VH-CH and the light chain domains of a Fab are in the
order, from N-terminus to C-terminus, VL-CL. In some embodiments,
the heavy chain domains of the Fab are in the order, from
N-terminus to C-terminus, CH-VH and the light chain domains of the
Fab are in the order CL-VL. Although Fabs were historically
identified by papain digestion of an intact immunoglobulin, in the
context of this disclosure, a "Fab" is typically produced
recombinantly by any method. Each Fab fragment is monovalent with
respect to antigen binding, i.e., it has a single antigen-binding
site.
[0115] "Complementarity-determining domains" or
"complementarity-determining regions" ("CDRs") interchangeably
refer to the hypervariable regions of V.sub.L and V.sub.H. CDRs are
the target protein-binding site of antibody chains that harbors
specificity for such target protein. There are three CDRs (CDR1-3,
numbered sequentially from the N-terminus) in each human V.sub.L or
V.sub.H, constituting about 15-20% of the variable domains. CDRs
are structurally complementary to the epitope of the target protein
and are thus directly responsible for the binding specificity. The
remaining stretches of the V.sub.L or V.sub.H, the so-called
framework regions (FR), exhibit less variation in amino acid
sequence (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman &
Co., New York, 2000).
[0116] Positions of CDRs and framework regions can be determined
using various well known definitions in the art, e.g., Kabat,
Chothia, international ImMunoGeneTics database (IMGT) and AbM (see,
e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001);
Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et
al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol.,
227:799-817 (1992); Al-Lazikani et al., J.Mol.Biol., 273:927-748
(1997)).
[0117] Definitions of antigen combining sites are also described in
the following: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000);
and Lefranc, M. P., Nucleic Acids Res., 29:207-209 (2001);
MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et
al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et
al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In
Sternberg M.J.E. (ed.), Protein Structure Prediction, Oxford
University Press, Oxford, 141-172 (1996).
[0118] Under Kabat, CDR amino acid residues in the V.sub.H are
numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the
CDR amino acid residues in the V.sub.L are numbered 24-34 (LCDR1),
50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, CDR amino acids in
the V.sub.H are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102
(HCDR3); and the amino acid residues in V.sub.L are numbered 26-32
(LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR
definitions of both Kabat and Chothia, the CDRs consist of amino
acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in
human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and
89-97 (LCDR3) in human VL.
[0119] An "antibody variable light chain" or an "antibody variable
heavy chain" as used herein refers to a polypeptide comprising the
V.sub.L or V.sub.H, respectively. The endogenous V.sub.L is encoded
by the gene segments V (variable) and J (junctional), and the
endogenous VH by V, D (diversity), and J. Each of V.sub.L or
V.sub.H includes the CDRs as well as the framework regions (FR).
The term "variable region" or "V-region" interchangeably refer to a
heavy or light chain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A
V-region can be naturally occurring, recombinant or synthetic. In
this application, antibody light chains and/or antibody heavy
chains may, from time to time, be collectively referred to as
"antibody chains." As provided and further described herein, an
"antibody variable light chain" or an "antibody variable heavy
chain" and/or a "variable region" and/or an "antibody chain"
optionally comprises a cytokine polypeptide sequence engrafted into
a CDR.
[0120] The C-terminal portion of an immunoglobulin heavy chain as
disclosed herein, comprising, e.g., CH2 and CH3 domains, is the
"Fc" domain. An "Fc region" as used herein refers to the constant
region of an antibody excluding the first constant region (CH1)
immunoglobulin domain. Fc refers to the last two constant region
immunoglobulin domains of IgA, IgD, and IgG, and the last three
constant region immunoglobulin domains of IgE and IgM, and the
flexible hinge N-terminal to these domains. For IgA and IgM Fc may
include the J chain. For IgG, Fc comprises immunoglobulin domains
C.gamma.2 and C.gamma.3 and the hinge between C.gamma.1 and
C.gamma.. It is understood in the art that boundaries of the Fc
region may vary, however, the human IgG heavy chain Fc region is
usually defined to comprise residues C226 or P230 to its
carboxyl-terminus, using the numbering is according to the EU index
as in Kabat et al. (1991, NIH Publication 91-3242, National
Technical Information Service, Springfield, Va.). "Fc region" may
refer to this region in isolation or this region in the context of
an antibody or antibody fragment. "Fc region" includes naturally
occurring allelic variants of the Fc region, e.g., in the CH2 and
CH3 region, including, e.g., modifications that modulate effector
function. Fc regions also include variants that don't result in
alterations to biological function. For example, one or more amino
acids are deleted from the N-terminus or C-terminus of the Fc
region of an immunoglobulin without substantial loss of biological
function. For example, in certain embodiments a C-terminal lysine
is modified replaced or removed. In particular embodiments one or
more C-terminal residues in the Fc region is altered or removed. In
certain embodiments one or more C-terminal residues in the Fc
(e.g., a terminal lysine) is deleted. In certain other embodiments
one or more C-terminal residues in the Fc is substituted with an
alternate amino acid (e.g., a terminal lysine is replaced). Such
variants are selected according to general rules known in the art
so as to have minimal effect on activity (see, e.g., Bowie, et al.,
Science 247:306-1310, 1990). The Fc domain is the portion of the
immunoglobulin (Ig) recognized by cell receptors, such as the FcR,
and to which the complement-activating protein, C1 q, binds. The
lower hinge region, which is encoded in the 5' portion of the CH2
exon, provides flexibility within the antibody for binding to FcR
receptors.
[0121] A "chimeric antibody" is an antibody molecule in which (a)
the constant region, or a portion thereof, is altered, replaced or
exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, and drug; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity.
[0122] A "humanized" antibody is an antibody that retains the
reactivity (e.g., binding specificity, activity) of a non-human
antibody while being less immunogenic in humans. This can be
achieved, for instance, by retaining non-human CDR regions and
replacing remaining parts of an antibody with human counterparts.
See, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA,
81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan,
Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun.,
31(3):169-217 (1994).
[0123] A "human antibody" includes antibodies having variable
regions in which both the framework and CDR regions are derived
from sequences of human origin. Furthermore, if an antibody
contains a constant region, the constant region also is derived
from such human sequences, e.g., human germline sequences, or
mutated versions of human germline sequences or antibody containing
consensus framework sequences derived from human framework
sequences analysis, for example, as described in Knappik et al., J.
Mol. Biol. 296:57-86, 2000). Human antibodies may include amino
acid residues not encoded by human sequences (e.g., mutations
introduced by random or site-specific mutagenesis in vitro or by
somatic mutation in vivo, or a conservative substitution to promote
stability or manufacturing).
[0124] The term "corresponding human germline sequence" refers to a
nucleic acid sequence encoding a human variable region amino acid
sequence or subsequence that shares the highest determined amino
acid sequence identity with a reference variable region amino acid
sequence or subsequence in comparison to all other all other known
variable region amino acid sequences encoded by human germline
immunoglobulin variable region sequences. A corresponding human
germline sequence can also refer to the human variable region amino
acid sequence or subsequence with the highest amino acid sequence
identity with a reference variable region amino acid sequence or
subsequence in comparison to all other evaluated variable region
amino acid sequences. A corresponding human germline sequence can
be framework regions only, complementary determining regions only,
framework and complementary determining regions, a variable segment
(as defined above), or other combinations of sequences or
subsequences that comprise a variable region. Sequence identity can
be determined using the methods described herein, for example,
aligning two sequences using BLAST, ALIGN, or another alignment
algorithm known in the art. The corresponding human germline
nucleic acid or amino acid sequence can have at least about 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity with the reference variable region nucleic acid or amino
acid sequence. Corresponding human germline sequences can be
determined, for example, through the publicly available
international ImMunoGeneTics database (IMGT) and V-base.
[0125] The term "valency" as used herein refers to the number of
potential target binding sites in a polypeptide. Each target
binding site specifically binds one target molecule or a specific
site on a target molecule. When a polypeptide comprises more than
one target binding site, each target binding site may specifically
bind the same or different molecules (e.g., may bind to different
molecules, e.g., different antigens, or different epitopes on the
same molecule). A conventional antibody, for example, has two
binding sites and is bivalent; "trivalent" and "tetravalent" refer
to the presence of three binding sites and four binding sites,
respectively, in an antibody molecule. The antibody cytokine
engrafted protein can be monovalent (i.e., bind one target
molecule), bivalent, or multivalent (i.e., bind more than one
target molecule).
[0126] The phrase "specifically binds" or "binding specificity,"
when used in the context of describing the interaction between the
original antibody target (e.g., an antigen) and an antibody
cytokine engrafted protein before and after the cytokine
engrafting. Under certain designated conditions, an antibody
cytokine engrafted protein with a particular binding specificity
binds to its original target at least two times the background and
does not substantially bind in a significant amount to other
targets present in a sample. In one embodiment, under designated
conditions, an antibody cytokine engrafted protein with a
particular binding specificity binds to its original target at
least ten (10) times the background and does not substantially bind
in a significant amount to other targets present in the sample.
Specific binding of an antibody cytokine engrafted protein under
such conditions can require an antibody cytokine engrafted protein
to have been selected for its specificity to a particular target.
Methods for determining binding specific are, for example,
solid-phase ELISA immunoassays are routinely used to select
antibodies specifically immunoreactive with a protein (see, e.g.,
Harlow & Lane, Using Antibodies, A Laboratory Manual (1998),
for a description of immunoassay formats and conditions that can be
used to determine specific immunoreactivity).
[0127] As used herein, "cytokine-receptor binding specificity" or
"cytokine-receptor specificity" includes antibody cytokine
engrafted proteins that selectively bind to IL10 receptor and does
not include antibody cytokine engrafted proteins that cross-react
with other cytokine receptor superfamily members. In some
embodiments, antibody cytokine engrafted proteins are selected that
selectively bind to human IL10R and cross-react with non-human
primate IL10R (e.g., cynomolgus IL10R). In some embodiments,
antibody engrafted proteins are selected that selectively bind to
human IL1OR1.
[0128] The term "equilibrium dissociation constant (K.sub.D, M)"
refers to the dissociation rate constant (k.sub.d, time.sup.-1)
divided by the association rate constant (k.sub.a, time.sup.-11,
M.sup.-1). Equilibrium dissociation constants can be measured using
any known method in the art. The antibody cytokine engrafted
proteins of the present disclosure generally will have an
equilibrium dissociation constant of less than about 10.sup.-7 or
10.sup.-8M, for example, less than about 10.sup.-9 M or 10.sup.-10
M, in some embodiments, less than about 10.sup.-11 M, 10.sup.-12M
or 10.sup.-13M.
[0129] The term "IL10" or "interleukin 10" or "interleukin-10
(IL-10)", interchangeably, refer to a potent anti-inflammatory
cytokine, capable of down regulating activation of macrophages as
well as reducing antigen presentation and maturation of dendritic
cells. IL10 comprising residues 19-178 of the full length native
human is utilized in construction of the agonist antibody cytokine
engrafted proteins. The human IL10 as disclosed herein has over its
full length at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity with the amino acid sequence
published as GenBank Accession No: NP_000563. The human IL10
nucleic acid encoding for the IL10 protein has over its full length
at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity with the nucleic acid sequence published
under GenBank Accession No: NM_000572.
[0130] "Monomeric IL10" or "IL10M" refers to a molecule has at
least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%,
97%, 98%, 99% or 100% sequence identity to the amino acid sequence
of SEQ ID NO: 209. In some embodiments, the monomeric IL10 molecule
comprises the sequence of SEQ ID NO: 209. In some embodiments, the
monomeric IL10 molecule consists of the sequence of SEQ ID NO:
209.
[0131] The term "agonist" refers to an antibody cytokine engrafted
protein capable of activating a receptor to induce a full or
partial receptor-mediated response. For example, an agonist of
IL10R binds to IL10R and induces IL10R-mediated intracellular
signaling and/or cell activation. The antibody cytokine engrafted
protein stimulates signaling through IL10R similar in some respects
to the native ligand, human IL10. Binding of hIL10 to IL10R induces
NF.kappa.B activation due to degradation of I.kappa.B. In some
embodiments, an antibody cytokine engrafted protein agonist can be
identified by its ability to bind IL10R and induce STAT3
phosphorylation, suppress production of pro-inflammatory cytokines
(e.g. TNF.alpha., IL1, IL6, IL12, IFN.gamma.) and/or
differentiation and proliferation in macrophages; induce T cell
(e.g., CD8.sup.+CTLs or CD4.sup.+ Th cells) proliferation,
survival, cytolytic activity and/or cytokine production (e.g.,
IFN.gamma., IL10, IL-13, TNF.alpha.), or as otherwise described
herein.
[0132] The term "isolated," when applied to a nucleic acid or
protein, denotes that the nucleic acid or protein is essentially
free of other cellular components with which it is associated in
the natural state. It is preferably in a homogeneous state. It can
be in either a dry or aqueous solution. Purity and homogeneity are
typically determined using analytical chemistry techniques such as
polyacrylamide gel electrophoresis or high performance liquid
chromatography. A protein that is the predominant species present
in a preparation is substantially purified. In particular, an
isolated gene is separated from open reading frames that flank the
gene and encode a protein other than the gene of interest. The term
"purified" denotes that a nucleic acid or protein gives rise to
essentially one band in an electrophoretic gel. Particularly, it
means that the nucleic acid or protein is at least 85% pure, more
preferably at least 95% pure, and most preferably at least 99%
pure.
[0133] The term "nucleic acid" or "polynucleotide" refers to
deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and
polymers thereof in either single- or double-stranded form. Unless
specifically limited, the term encompasses nucleic acids containing
known analogues of natural nucleotides that have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions), alleles, orthologs, SNPs, and
complementary sequences as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); and Rossolini et al., Mol. Cell. Probes 8:91-98
(1994)).
[0134] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0135] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refer to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an .alpha.-carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0136] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid that encodes a polypeptide is implicit in each described
sequence.
[0137] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles. The following eight groups each contain amino acids that
are conservative substitutions for one another: 1) Alanine (A),
Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine
(N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
[0138] "Percentage of sequence identity" is determined by comparing
two optimally aligned sequences over a comparison window, wherein
the portion of the polynucleotide sequence in the comparison window
may comprise additions or deletions (i.e., gaps) as compared to the
reference sequence (e.g., a polypeptide), which does not comprise
additions or deletions, for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical nucleic acid base or amino acid residue
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison and multiplying the result by
100 to yield the percentage of sequence identity.
[0139] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same sequences. Two
sequences are "substantially identical" if two sequences have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., at least 85%, 90%, 95%, 96%, 97%, 98% or 99%
sequence identity over a specified region, or, when not specified,
over the entire sequence of a reference sequence), when compared
and aligned for maximum correspondence over a comparison window, or
designated region as measured using one of the following sequence
comparison algorithms or by manual alignment and visual inspection.
The disclosure provides polypeptides or polynucleotides that are
substantially identical to the polypeptides or polynucleotides,
respectively, exemplified herein (e.g., the variable regions
exemplified in any one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:29,
SEQ ID NO:30, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:61, SEQ ID
NO:62, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:93, SEQ ID NO:94, SEQ
ID NO:109, SEQ ID NO:110, SEQ ID NO:125, SEQ ID NO:126, SEQ ID
NO:141, SEQ ID NO:142, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:173,
SEQ ID NO:174, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:205, SEQ ID
NO:206; SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:238 SEQ ID NO:239;
the variable regions exemplified in any one of SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:47, SEQ ID NO:48, SEQ
ID NO:63, SEQ ID NO:64, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:95,
SEQ ID NO:96, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:127, SEQ ID
NO:128, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:159, SEQ ID NO:160,
SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:191, SEQ ID NO:192, SEQ ID
NO:207, SEQ ID NO:208, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:240,
SEQ ID NO:241; the CDRs exemplified in any one of SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:45, SEQ ID NO:46,
SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:77, SEQ ID NO:78, SEQ ID
NO:93, SEQ ID NO:94, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:125,
SEQ ID NO:126, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:157, SEQ ID
NO:158, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:189, SEQ ID NO:190,
SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:222, SEQ ID NO:223, SEQ ID
NO:238 and SEQ ID NO:239. Optionally, the identity exists over a
region that is at least about 15, 25 or 50 nucleotides in length,
or more preferably over a region that is 100 to 500 or 1000 or more
nucleotides in length, or over the full length of the reference
sequence. With respect to amino acid sequences, identity or
substantial identity can exist over a region that is at least 5,
10, 15 or 20 amino acids in length, optionally at least about 25,
30, 35, 40, 50, 75 or 100 amino acids in length, optionally at
least about 150, 200 or 250 amino acids in length, or over the full
length of the reference sequence. With respect to shorter amino
acid sequences, e.g., amino acid sequences of 20 or fewer amino
acids, substantial identity exists when one or two amino acid
residues are conservatively substituted, according to the
conservative substitutions defined herein.
[0140] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0141] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity method of Pearson and Lipman (1988) Proc.
Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel et al., Current Protocols in Molecular Biology
(1995 supplement)).
[0142] Two examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1977)
Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol.
Biol. 215:403-410, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad.
Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0143] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0144] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as described below. Thus, a polypeptide is
typically substantially identical to a second polypeptide, for
example, where the two peptides differ only by conservative
substitutions. Another indication that two nucleic acid sequences
are substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below. Yet another indication that two nucleic acid
sequences are substantially identical is that the same primers can
be used to amplify the sequence.
[0145] The term "antibody cytokine engrafted protein" or "antibody
cytokine graft" or "engrafted" means that at least one cytokine is
incorporated directly within a CDR of the antibody, interrupting
the sequence of the CDR. The cytokine can be incorporated within
HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3. The cytokine can be
incorporated within HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 and
incorporated toward the N-terminal sequence of the CDR or toward
the C-terminal sequence of the CDR. The cytokine incorporated
within a CDR can reduce the specific binding of the antibody
portion to its target protein or the antibody cytokine engrafted
protein can retain its specific binding to its target protein.
[0146] An "immune related disorder" or "immune disease" refers to a
dysfunction of the immune system, in which the body's own immune
system attacks healthy tissue. The dysfunction can be in components
of the immune cells, and includes both overactive and underactive
immune systems.
[0147] The terms "subject," "patient," and "individual"
interchangeably refer to a mammal, for example, a human or a
non-human primate mammal. The mammal can also be a laboratory
mammal, e.g., mouse, rat, rabbit, hamster. In some embodiments, the
mammal can be an agricultural mammal (e.g., equine, ovine, bovine,
porcine, camelid) or domestic mammal (e.g., canine, feline).
[0148] As used herein, the terms "treat," "treating," or
"treatment" of any disease or disorder refer in one embodiment, to
ameliorating the disease or disorder (i.e., slowing or arresting or
reducing the development of the disease or at least one of the
clinical symptoms thereof). In another embodiment, "treat,"
"treating," or "treatment" refers to alleviating or ameliorating at
least one physical parameter including those which may not be
discernible by the patient. In yet another embodiment, "treat,"
"treating," or "treatment" refers to modulating the disease or
disorder, either physically, (e.g., stabilization of a discernible
symptom), physiologically, (e.g., stabilization of a physical
parameter), or both. In yet another embodiment, "treat,"
"treating," or "treatment" or "prophylaxis" refers to preventing or
delaying the onset or development or progression of a disease or
disorder.
[0149] The term "therapeutically acceptable amount" or
"therapeutically effective dose" interchangeably refer to an amount
sufficient to effect the desired result (i.e., a reduction in
inflammation, inhibition of pain, prevention of inflammation,
inhibition or prevention of inflammatory response). In some
embodiments, a therapeutically acceptable amount does not induce or
cause undesirable side effects. A therapeutically acceptable amount
can be determined by first administering a low dose, and then
incrementally increasing that dose until the desired effect is
achieved. A "prophylactically effective dosage," and a
"therapeutically effective dosage," of an IL10 antibody cytokine
engrafted protein can prevent the onset of, or result in a decrease
in severity of, respectively, disease symptoms, including symptoms
associated with immune related disorder.
[0150] The term "co-administer" refers to the simultaneous presence
of two (or more) active agents in an individual. Active agents that
are co-administered can be concurrently or sequentially
delivered.
[0151] As used herein, the phrase "consisting essentially of"
refers to the genera or species of active pharmaceutical agents
included in a method or composition, as well as any inactive
carrier or excipients for the intended purpose of the methods or
compositions. In some embodiments, the phrase "consisting
essentially of" expressly excludes the inclusion of one or more
additional active agents other than an IL10 antibody cytokine
engrafted protein. In some embodiments, the phrase "consisting
essentially of" expressly excludes the inclusion of more additional
active agents other than an IL10 antibody cytokine engrafted
protein and a second co-administered agent.
[0152] The terms "a," "an," and "the" include plural referents,
unless the context clearly indicates otherwise.
DETAILED DESCRIPTION
IL10 Antibody Cytokine Engrafted Proteins
[0153] Interleukin-10 (IL10) is a multifunctional, homodimeric
immunomodulatory cytokine that has both immunostimulatory and
immunosuppressive activities. IL10 inhibits the induction of a
number of proinflammatory cytokines in activated monocytes and
macrophages but is a costimulator for immature and mature
lymphocytes, mast cells and B cells (reviewed in Mosmann, Adv.
Immunol. 1994; 56:1-26; Moore et al., Annu Rev Immunol.
2001;19:683-765). Provided herein are antibody cytokine engrafted
proteins comprising monomeric IL10 engrafted into the
complementarity determining region (CDR) of an antibody. The
antibody cytokine engrafted proteins show suitable properties to be
used in human patients, for example, they retain immunosuppressive
activity similar to that of native or recombinant human IL10,
however, the pro-inflammatory effects are reduced.
[0154] Accordingly, the present disclosure provides antibody
cytokine engrafted proteins that are agonists of the IL10 receptor,
with selective activity profiles. Provided antibody cytokine
engrafted proteins comprise an immunoglobulin heavy chain sequence
and an immunoglobulin light chain sequence. Each immunoglobulin
heavy chain sequence comprises a heavy chain variable region (VH)
and a heavy chain constant region (CH), wherein the heavy chain
constant region consists of CH1, CH2, and CH3 constant regions.
Each immunoglobulin light chain sequence comprises a light chain
variable region (VL) and a light chain constant region (CL). In
each antibody cytokine engrafted protein a monomeric IL10 molecule
is engrafted into a complementarity determining region (CDR) of the
VH or VL region.
[0155] In some embodiments, the antibody cytokine engrafted protein
comprises a monomeric IL10 engrafted into a heavy chain CDR. In
certain embodiments monomeric IL10 is engrafted into heavy chain
complementarity determining region 1 (HCDR1). In certain
embodiments monomeric IL10 is engrafted into heavy chain
complementarity determining region 2 (HCDR2). In certain
embodiments monomeric IL10 is engrafted into heavy chain
complementarity determining region 3 (HCDR3).
[0156] In some embodiments, the antibody cytokine engrafted protein
comprises a monomeric IL10 engrafted into a light chain CDR. In
certain embodiments monomeric IL10 is engrafted into light chain
complementarity determining region 1 (LCDR1). In certain
embodiments monomeric IL10 is engrafted into light chain
complementarity determining region 2 (LCDR2). In certain
embodiments monomeric IL10 is engrafted into light chain
complementarity determining region 3 (LCDR3). In a preferred
embodiment, monomeric IL10 is engrafted into light chain
complementarity determining region 1 (LCDR1).
[0157] In some embodiments, the antibody cytokine engrafted protein
comprises a monomeric IL10 engrafted into a CDR, whereby the IL10
sequence is inserted into the CDR sequence. The engrafted IL10 can
be at or near the N-terminal portion of the CDR, in the center
region of the CDR or at or near the C-terminal portion of the CDR.
In other embodiments, the antibody cytokine engrafted protein
comprises a monomeric IL10 incorporated into a CDR, wherein the
IL10 sequence replaces all or part of a CDR sequence. A replacement
can be at or near the beginning of the CDR, in the middle region of
the CDR or at or near the end of the CDR. A replacement can be as
few as one or two amino acids of a CDR sequence, or as many as an
entire CDR sequence.
[0158] In some embodiments monomeric IL10 is engrafted directly
into a CDR without a peptide linker.
[0159] In some embodiments the antibody cytokine engrafted protein
comprises immunoglobulin heavy chains of an IgG class antibody
heavy chain. In certain embodiments an IgG heavy chain is any one
of an IgG1, an IgG2 or an IgG4 subclass.
[0160] In some embodiments antibody cytokine engrafted proteins
comprise heavy and light chain immunoglobulin sequences selected
from a known, clinically utilized immunoglobulin sequence. In
certain embodiments antibody cytokine engrafted proteins comprise
heavy and light chain immunoglobulin sequences which are humanized
sequences. In other certain embodiments antibody cytokine engrafted
proteins comprise heavy and light chain immunoglobulin sequences
which are human sequences.
[0161] In some embodiments antibody cytokine engrafted proteins
comprise heavy and light chain immunoglobulin sequences selected
from germline immunoglobulin sequences.
[0162] In some embodiments antibody cytokine engrafted proteins
comprise heavy and light chain immunoglobulin sequences having
binding specificity of the immunoglobulin variable domains to a
target distinct from the cytokine receptor binding specificity of
the IL10 monomer. In some embodiments the binding specificity of
the immunoglobulin variable domain is retained in the presence of
the engrafted cytokine. In certain embodiments the antibody binding
specificity is to a non-human antigen. In other embodiments the
binding specificity is to a target having therapeutic utility in
conjunction with IL10 therapy. In certain embodiments modulating
the binding specificity of the immunoglobulin conveys additional
therapeutic benefit to the IL10 component. In certain embodiments
the binding specificity of the immunoglobulin conveys synergistic
activity with IL10 monomer.
[0163] In still other embodiments, the binding specificity of the
immunoglobulin is reduced in the presence of the IL10 engrafted
cytokine.
[0164] In some embodiments, the antibody cytokine engrafted protein
confers anti-inflammatory properties similar to human IL10 or
recombinant human IL10, and the engrafted protein confers reduced
proportional pro-inflammatory activity as compared to human IL10 or
recombinant human IL10. In some embodiments the proportional
pro-inflammatory activity is reduced by at least twenty five
percent (25%) as compared with IL10. In some embodiments the
proportional pro-inflammatory activity is reduced by at least
thirty five percent (35%) as compared with IL10. In some
embodiments the proportional pro-inflammatory activity is reduced
by at least about fifty percent (50%) as compared with IL10. In
some embodiments the proportional pro-inflammatory activity is
reduced by at least about sixty five percent (65%) as compared with
IL10. In some embodiments the proportional pro-inflammatory
activity is reduced by at least about seventy five percent (75%) as
compared with IL10. In some embodiments the proportional
pro-inflammatory activity is reduced by at least about eighty
percent (80%) as compared with IL10. In some embodiments the
proportional pro-inflammatory activity is reduced by more than
eighty percent (80%) as compared with IL10.
[0165] In some embodiments, the antibody cytokine engrafted
proteins comprise a modified immunoglobulin IgG having a modified
Fc conferring modified effector function. In certain embodiments
the modified Fc region comprises a mutation selected from one or
more of D265A, P329A, P329G, N297A, L234A, and L235A. In particular
embodiments the immunoglobulin heavy chain may comprise a mutation
or combination of mutations conferring reduced effector function
selected from any of D265A, P329A, P329G, N297A, D265A/P329A,
D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0166] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain variable region having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a heavy chain variable region of
SEQ ID NO:13 and (ii) a light chain variable region having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a light chain variable region of
SEQ ID NO:14. The immunoglobulin chain is an IgG class selected
from IgG1, IgG2, or IgG4. In certain embodiments the immunoglobulin
heavy chain optionally comprises a mutation or combination of
mutations conferring reduced effector function selected from any of
D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234A/L235A,
P329A/L234A/L235A, and P329G/L234A/L235A.
[0167] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain variable region having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a heavy chain variable region of
SEQ ID NO:29 and (ii) a light chain variable region having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a light chain variable region of
SEQ ID NO:30. The immunoglobulin chain is an IgG class selected
from IgG1, IgG2, or IgG4. In certain embodiments the immunoglobulin
heavy chain optionally comprises a mutation or combination of
mutations conferring reduced effector function selected from any of
D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234A/L235A,
P329A/L234A/L235A, and P329G/L234A/L235A.
[0168] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain variable region having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a heavy chain variable region of
SEQ ID NO:45 and (ii) a light chain variable region having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a light chain variable region of
SEQ ID NO:46. The immunoglobulin chain is an IgG class selected
from IgG1, IgG2, or IgG4. In certain embodiments the immunoglobulin
heavy chain optionally comprises a mutation or combination of
mutations conferring reduced effector function selected from any of
D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234A/L235A,
P329A/L234A/L235A, and P329G/L234A/L235A.
[0169] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain variable region having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a heavy chain variable region of
SEQ ID NO:61 and (ii) a light chain variable region having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a light chain variable region of
SEQ ID NO:62. The immunoglobulin chain is an IgG class selected
from IgG1, IgG2, or IgG4. In certain embodiments the immunoglobulin
heavy chain optionally comprises a mutation or combination of
mutations conferring reduced effector function selected from any of
D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234A/L235A,
P329A/L234A/L235A, and P329G/L234A/L235A.
[0170] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain variable region having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a heavy chain variable region of
SEQ ID NO:77 and (ii) a light chain variable region having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a light chain variable region of
SEQ ID NO:78. The immunoglobulin chain is an IgG class selected
from IgG1, IgG2, or IgG4. In certain embodiments the immunoglobulin
heavy chain optionally comprises a mutation or combination of
mutations conferring reduced effector function selected from any of
D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234A/L235A,
P329A/L234A/L235A, and P329G/L234A/L235A.
[0171] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain variable region having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a heavy chain variable region of
SEQ ID NO:93 and (ii) a light chain variable region having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a light chain variable region of
SEQ ID NO:94. The immunoglobulin chain is an IgG class selected
from IgG1, IgG2, or IgG4. In certain embodiments the immunoglobulin
heavy chain optionally comprises a mutation or combination of
mutations conferring reduced effector function selected from any of
D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234A/L235A,
P329A/L234A/L235A, and P329G/L234A/L235A.
[0172] In some embodiments, the antibody cytokine engrafted protein
comprises each comprising (i) a heavy chain variable region having
at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100% amino acid sequence identity to a heavy chain variable
region of SEQ ID NO:109 and (ii) a light chain variable region
having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% amino acid sequence identity to a light chain
variable region of SEQ ID NO:110. The immunoglobulin chain is an
IgG class selected from IgG1, IgG2, or IgG4. In certain embodiments
the immunoglobulin heavy chain optionally comprises a mutation or
combination of mutations conferring reduced effector function
selected from any of D265A, P329A, P329G, N297A, D265A/P329A,
D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0173] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain variable region having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a heavy chain variable region of
SEQ ID NO:125 and (ii) a light chain variable region having at
least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% amino acid sequence identity to a light chain variable
region of SEQ ID NO:126. The immunoglobulin chain is an IgG class
selected from IgG1, IgG2, or IgG4. In certain embodiments the
immunoglobulin heavy chain optionally comprises a mutation or
combination of mutations conferring reduced effector function
selected from any of D265A, P329A, P329G, N297A, D265A/P329A,
D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0174] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain variable region having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a heavy chain variable region of
SEQ ID NO:141 and (ii) a light chain variable region having at
least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% amino acid sequence identity to a light chain variable
region of SEQ ID NO:142. The immunoglobulin chain is an IgG class
selected from IgG1, IgG2, or IgG4. In certain embodiments the
immunoglobulin heavy chain optionally comprises a mutation or
combination of mutations conferring reduced effector function
selected from any of D265A, P329A, P329G, N297A, D265A/P329A,
D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0175] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain variable region having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a heavy chain variable region of
SEQ ID NO:157 and (ii) a light chain variable region having at
least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% amino acid sequence identity to a light chain variable
region of SEQ ID NO:158. The immunoglobulin chain is an IgG class
selected from IgG1, IgG2, or IgG4. In certain embodiments the
immunoglobulin heavy chain optionally comprises a mutation or
combination of mutations conferring reduced effector function
selected from any of D265A, P329A, P329G, N297A, D265A/P329A,
D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0176] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain variable region having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a heavy chain variable region of
SEQ ID NO:173 and (ii) a light chain variable region having at
least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% amino acid sequence identity to a light chain variable
region of SEQ ID NO:174. The immunoglobulin chain is an IgG class
selected from IgG1, IgG2, or IgG4. In certain embodiments the
immunoglobulin heavy chain optionally comprises a mutation or
combination of mutations conferring reduced effector function
selected from any of D265A, P329A, P329G, N297A, D265A/P329A,
D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0177] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain variable region having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a heavy chain variable region of
SEQ ID NO:189 and (ii) a light chain variable region having at
least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% amino acid sequence identity to a light chain variable
region of SEQ ID NO:190. The immunoglobulin chain is an IgG class
selected from IgG1, IgG2, or IgG4. In certain embodiments the
immunoglobulin heavy chain optionally comprises a mutation or
combination of mutations conferring reduced effector function
selected from any of D265A, P329A, P329G, N297A, D265A/P329A,
D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0178] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain variable region having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a heavy chain variable region of
SEQ ID NO:205 and (ii) a light chain variable region having at
least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% amino acid sequence identity to a light chain variable
region of SEQ ID NO:206. The immunoglobulin chain is an IgG class
selected from IgG1, IgG2, or IgG4. In certain embodiments the
immunoglobulin heavy chain optionally comprises a mutation or
combination of mutations conferring reduced effector function
selected from any of D265A, P329A, P329G, N297A, D265A/P329A,
D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0179] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain variable region having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a heavy chain variable region of
SEQ ID NO:222 and (ii) a light chain variable region having at
least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% amino acid sequence identity to a light chain variable
region of SEQ ID NO:223. The immunoglobulin chain is an IgG class
selected from IgG1, IgG2, or IgG4. In certain embodiments the
immunoglobulin heavy chain optionally comprises a mutation or
combination of mutations conferring reduced effector function
selected from any of D265A, P329A, P329G, N297A, D265A/P329A,
D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0180] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain variable region having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to a heavy chain variable region of
SEQ ID NO:238 and (ii) a light chain variable region having at
least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% amino acid sequence identity to a light chain variable
region of SEQ ID NO:239. The immunoglobulin chain is an IgG class
selected from IgG1, IgG2, or IgG4. In certain embodiments the
immunoglobulin heavy chain optionally comprises a mutation or
combination of mutations conferring reduced effector function
selected from any of D265A, P329A, P329G, N297A, D265A/P329A,
D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0181] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:15 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:16. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0182] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:31 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:32. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0183] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:47 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:48. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0184] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:63 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:64. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0185] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:79 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:80. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0186] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:95 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:96. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0187] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:111 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:112. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0188] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:127 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:128. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0189] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:143 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:144. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0190] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:159 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:160. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0191] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:175 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:176. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0192] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:191 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:192. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0193] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:207 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:208. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0194] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:224 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:225. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
[0195] In some embodiments, the antibody cytokine engrafted protein
comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence
identity to SEQ ID NO:240 and (ii) a light chain having at least
85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
amino acid sequence identity to SEQ ID NO:241. In certain
embodiments the immunoglobulin heavy chain optionally comprises a
mutation or combination of mutations conferring reduced effector
function selected from any of D265A, P329A, P329G, N297A,
D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and
P329G/L234A/L235A.
Engineered and Modified Antibody Cytokine Engrafted Proteins
[0196] Antibody cytokine engrafted proteins are generated by
engrafting a monomeric IL10 sequence into a CDR region of an
immunoglobulin scaffold. Both heavy and light chain immunoglobulin
chains are produced to generate final antibody engrafted proteins.
Antibody cytokine engrafted proteins confer preferred therapeutic
anti-inflammatory properties of IL10; however, antibody cytokine
engrafted proteins have reduced proportional pro-inflammatory
activity as compared with native or recombinant human IL10
(rhIL10).
[0197] To engineer engrafted constructs, monomeric IL10 (IL10M, SEQ
ID NO: 209), comprising residues 19-178 of full length IL10 and a
six amino acid sequence between residues 134 and 135 was engrafted
into a CDR loop of an immunoglobulin scaffold. Engrafted constructs
can be prepared using any of a variety of known immunoglobulin
sequences which have been utilized in clinical settings, known
immunoglobulin sequences which are in current discovery and/or
clinical development, human germline antibody sequences, as well as
sequences of novel antibody immunoglobulin chains. Antibody
cytokine engrafted proteins are produced using standard molecular
biology methodology utilizing recombinant DNA encoding relevant
sequences. Sequences of IL10M in two exemplary scaffolds, referred
to as GFTX1 and GFTX3, and are depicted in TABLE 1. Insertion
points for IL10 monomer were selected to be the mid-point of the
loop based on available structural or homology model data, however,
insertion points may be adjusted toward one or another end of a CDR
loop.
[0198] Thus the present disclosure provides antibody cytokine
engrafted proteins or fragments thereof that cytokine-receptor
specifically bind to an IL10R protein, comprising an IL10M protein
recombinantly inserted into to a heterologous antibody protein or
polypeptide to generate antibody cytokine engrafted proteins. In
particular, the disclosure provides engrafted proteins comprising
an antibody or antigen-binding fragment of an antibody described
herein or any other relevant scaffold antibody polypeptide (e.g., a
full antibody immunoglobulin protein, a Fab fragment, Fd fragment,
Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VL domain, a
VL CDR, etc.) and a heterologous cytokine protein, polypeptide, or
peptide, e.g., IL10M. Methods for conjugating proteins,
polypeptides, or peptides to an antibody or an antibody fragment
are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603,
5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European
Patent Nos. EP 307,434 and EP 367,166; International Publication
Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, Proc.
Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al., 1995, J.
Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad.
Sci. USA 89:11337-11341. Additionally, antibody cytokine engrafted
proteins may be generated through the techniques of gene-shuffling,
motif-shuffling, exon-shuffling, and/or codon-shuffling
(collectively referred to as "DNA shuffling"). DNA shuffling may be
employed to prepare antibody cytokine engrafted proteins and/or to
alter the activities of antibodies or fragments thereof (e.g.,
antibodies or fragments thereof with higher affinities and lower
dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793,
5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al.,
1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends
Biotechnol. 16(2):76-82; Hansson, et al., 1999, J. Mol. Biol.
287:265-76; and Lorenzo and Blasco, 1998, Biotechniques
24(2):308-313 (each of these patents and publications are hereby
incorporated by reference in its entirety). Antibodies or fragments
thereof, or the encoded antibodies or fragments thereof, may be
altered by being subjected to random mutagenesis by error-prone
PCR, random nucleotide insertion or other methods prior to
recombination. A polynucleotide encoding an antibody or fragment
thereof that specifically binds to an antigen protein of interest
may be recombined with one or more components, motifs, sections,
parts, domains, fragments, etc. of one or more heterologous
cytokine molecules, e.g., IL10M, for preparation of antibody
cytokine engrafted proteins as provided herein.
[0199] An antibody Fab contains six CDR loops, 3 in the light chain
(CDRL1, CDRL2, CDRL3) and 3 in the heavy chain (CDRH1, CDRH2,
CDRH3) which can serve as potential insertion sites for a cytokine
protein. Structural and functional considerations are taken into
account in order to determine which CDR loop(s) to insert the
cytokine. As a CDR loop size and conformation vary greatly across
different antibodies, the optimal CDR for insertion may be
determined empirically for each particular antibody/protein
combination. Additionally, since a cytokine protein will be
inserted into a CDR loop, this can put additional constraints on
the structure of the cytokine protein. For example, the amino and
carboxy terminal of the cytokine protein should allow for
possibility to be constrained relatively close in space (.about.25
.ANG.). Additionally, an antibody cytokine engrafted protein should
not rely on oligomerization for biological activity.
[0200] IL10 is a homodimeric cytokine that employs a domain swapped
architecture. In native form, IL10 would not be suitable for
antibody grafting since the domain swapped structure would not be
able to form (i.e. both N and C termini are constrained in the
loop). However, an engineered monomeric IL10 (IL10M), created by
inserting a six residue sequence ((GGGSGG) (SEQ ID NO.251)) between
helices D and E allows helices E and F (which swap into the other
IL10 monomer in the native dimeric structure) to fold back into the
same monomer (Josephson et al J. Biol.Chem. 2000; 275(18) 13552-7)
and create the structure present in the antibody cytokine engrafted
protein as shown in FIG. 2 and FIG. 13. Josephson et al.,
demonstrated that the IL10 monomer with the six amino acid
insertion is more thermostable than the wild-type dimer and is also
able to form a 1:1 interaction with the IL10 receptor chain
(IL10R1). IL10M also has in vitro activity, though it is nine to
eighteen-fold less active than wild-type IL10. Another feature of
IL10M is that the N and C-termini are close to each other in space,
which makes the modified cytokine protein suitable for antibody
grafting.
[0201] An antibody cytokine engrafted protein of the disclosure
further can be prepared using an antibody having one or more of the
CDRs and/or VH and/or VL sequences shown herein (e.g., TABLE 1) as
starting material to engineer a modified antibody cytokine
engrafted protein, which may have altered properties from the
starting antibody. Alternatively, any known antibody sequences may
be utilized as a scaffold to engineer modified antibody cytokine
engrafted proteins. For example, any known, clinically utilized
antibody may be utilized as a starting materials scaffold for
preparation of antibody cytokine engrafted protein. Known
antibodies and corresponding immunoglobulin sequences include,
e.g., palivizumab, alirocumab, mepolizumab, necitumumab, nivolumab,
dinutuximab, secukinumab, evolocumab, blinatumomab, pembrolizumab,
ramucirumab vedolizumab, siltuximab, obinutuzumab, trastuzumab,
raxibacumab, pertuzumab, belimumab, ipilimumab. denosumab,
tocilizumab, ofatumumab, canakinumab, golimumab, ustekinumab,
certolizumab, catumaxomab, eculizumab, ranibizumab, panitumumab,
natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab,
tositumomab, ibritumomab tiuxetan, adalimumab, alemtuzumab,
gemtuzumab, infliximab, basiliximab, daclizumab, rituximab,
abciximab, muromonab, or modifications thereof. Known antibodies
and immunoglobulin sequences also include germline antibody
sequences. Framework sequences can be obtained from public DNA
databases or published references that include germline antibody
gene sequences. For example, germline DNA sequences for human heavy
and light chain variable region genes can be found in the "VBase"
human germline sequence database, as well as in Kabat, E. A., et
al., 1991 Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242; Tomlinson, I. M., et al., 1992 J. fol.
Biol. 227:776-798; and Cox, J. P. L. et al., 1994 Eur. J Immunol.
24:827-836. In still other examples, antibody and corresponding
immunoglobulin sequences from other known entities which may be in
early discovery and/or drug development may be similarly adapted as
starting material to engineer a modified antibody cytokine
engrafted protein .
[0202] A wide variety of antibody/immunoglobulin frameworks or
scaffolds can be employed so long as the resulting polypeptide
includes at least one binding region which accommodates
incorporation of a cytokine (e.g., IL10M). Such frameworks or
scaffolds include the 5 main idiotypes of human immunoglobulins, or
fragments thereof, and include immunoglobulins of other animal
species, preferably having humanized and/or human aspects. Novel
antibodies, frameworks, scaffolds and fragments continue to be
discovered and developed by those skilled in the art.
[0203] Antibodies can be generated using methods that are known in
the art. For preparation of monoclonal antibodies, any technique
known in the art can be used (see, e.g., Kohler & Milstein,
Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72
(1983); Cole et al., Monoclonal Antibodies and Cancer Therapy, pp.
77-96. Alan R. Liss, Inc. 1985). Techniques for the production of
single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to
produce antibodies for use in antibody cytokine engrafted proteins
of this disclosure. Also, transgenic mice, or other organisms such
as other mammals, may be used to express and identify primatized or
humanized or human antibodies. Alternatively, phage display
technology can be used to identify antibodies and heteromeric Fab
fragments that specifically bind to selected antigens for use in
antibody cytokine engrafted proteins. (see, e.g., McCafferty et
al., supra; Marks et al., Biotechnology, 10:779-783, (1992)).
[0204] Methods for primatizing or humanizing non-human antibodies
are well known in the art. Generally, a primatized or humanized
antibody has one or more amino acid residues introduced into it
from a source which is non-primate or non-human. Such non-primate
or non-human amino acid residues are often referred to as import
residues, which are typically taken from an import variable domain.
Humanization can be essentially performed following the method of
Winter and co-workers (see, e.g., Jones et al., Nature 321:522-525
(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et
al., Science 239:1534-1536 (1988) and Presta, Curr. Op. Struct.
Biol. 2:593-596 (1992)), by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
primatized or humanized antibodies are typically primate or human
antibodies in which some complementary determining region ("CDR")
residues and possibly some framework ("FR") residues are
substituted by residues from analogous sites in an originating
species (e.g., rodent antibodies) to confer binding
specificity.
[0205] Alternatively or additionally, an in vivo method for
replacing a nonhuman antibody variable region with a human variable
region in an antibody while maintaining the same or providing
better binding characteristics relative to that of the nonhuman
antibody can be utilized to convert non-human antibodies into
engineered human antibodies. See, e.g., U.S. Patent Publication No.
20050008625, U.S. Patent Publication No. 2005/0255552.
Alternatively, human V segment libraries can be generated by
sequential cassette replacement in which only part of the reference
antibody V segment is initially replaced by a library of human
sequences; and identified human "cassettes" supporting binding in
the context of residual reference antibody amino acid sequences are
then recombined in a second library screen to generate completely
human V segments (see, U.S. Patent Publication No.
2006/0134098).
[0206] Various antibodies or antigen-binding fragments for use in
preparation of antibody cytokine engrafted proteins can be produced
by enzymatic or chemical modification of the intact antibodies, or
synthesized de novo using recombinant DNA methodologies (e.g.,
single chain Fv), or identified using phage display libraries (see,
e.g., McCafferty et al., Nature 348:552-554, 1990). For example,
minibodies can be generated using methods described in the art,
e.g., Vaughan and Sollazzo, Comb Chem High Throughput Screen.
4:417-30 2001. Bispecific antibodies can be produced by a variety
of methods including engrafted of hybridomas or linking of Fab'
fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp.
Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148,
1547-1553 (1992). Single chain antibodies can be identified using
phage display libraries or ribosome display libraries, gene
shuffled libraries. Such libraries can be constructed from
synthetic, semi-synthetic or native and immunocompetent sources.
Selected immunoglobulin sequences may thus be utilized in
preparation of antibody cytokine engrafted proteins as provided
herein.
[0207] Antibodies or antigen-binding molecules of use in the
present disclosure further include bispecific antibodies. A
bispecific or bifunctional antibody is an artificial hybrid
antibody having two different heavy/light chain pairs and two
different binding sites. Other antigen-binding fragments or
antibody portions include bivalent scFv (diabody), bispecific scFv
antibodies where the antibody molecule recognizes two different
epitopes, single binding domains (dAbs), and minibodies. Selected
immunoglobulin sequences can be utilized in preparation of antibody
cytokine engrafted proteins as provided herein.
[0208] Antigen-binding fragments of antibodies e.g., a Fab
fragment, scFv, can be used as building blocks to construct
antibody cytokine engrafted proteins, and optionally include
multivalent formats. In some embodiments, such multivalent
molecules comprise a constant region of an antibody (e.g., Fc).
[0209] Antibody cytokine engrafted proteins can be engineered by
modifying one or more residues within one or both variable regions
(i.e., VH and/or VL) of an antibody, for example within one or more
CDR regions, and such adapted VH and/or VL region sequences
utilized for incorporation of a cytokine for preparation of
antibody cytokine engrafted protein. One type of variable region
engineering that can be performed is CDR grafting. Antibodies
interact with target antigens predominantly through amino acid
residues that are located in the six heavy and light chain
complementarity determining regions (CDRs). For this reason, the
amino acid sequences within CDRs are more diverse between
individual antibodies than sequences outside of CDRs. CDR sequences
are responsible for most antibody-antigen interactions, it is
possible to express recombinant antibodies that mimic the
properties of a specific antibody by constructing expression
vectors that include CDR sequences from a specific antibody grafted
onto framework sequences from a different antibody with different
properties (see, e.g., Riechmann, L. et al., 1998 Nature
332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C.
et al., 1989 Proc. Natl. Acad., U.S.A. 86:10029-10033; U.S. Pat.
No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089;
5,693,762 and 6,180,370 to Queen et al.). In certain instances it
is beneficial to mutate residues within the framework regions to
maintain or enhance the antigen binding ability of the antibody
(see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and
6,180,370 to Queen et al).
[0210] In some aspects mutation of amino acid residues within the
VH and/or VL CDR1, CDR2, and/or CDR3 regions to thereby improve one
or more binding properties (e.g., affinity) of the antibody of
interest, known as "affinity maturation," can be beneficial, e.g.,
to optimize antigen binding of an antibody in conjunction with the
context of the cytokine engrafted protein. Site-directed
mutagenesis or PCR-mediated mutagenesis can be performed to
introduce the mutation(s) and the effect on antibody binding, or
other functional property of interest, can be evaluated in in vitro
or in vivo assays as described herein and provided in the Examples
and/or alternative or additional assays known in the art.
Conservative modifications can be introduced. The mutations may be
amino acid substitutions, additions or deletions. Moreover,
typically no more than one, two, three, four or five residues
within a CDR region are altered.
[0211] Engineered antibodies or antibody fragments include those in
which modifications have been made to framework residues within VH
and/or VL, e.g. to improve the properties of the antibody. In some
embodiments such framework modifications are made to decrease
immunogenicity of the antibody. For example, one approach is to
"backmutate" one or more framework residues to the corresponding
germline sequence. More specifically, an antibody that has
undergone somatic mutation contains framework residues that differ
from germline sequence from which the antibody is derived. Such
residues can be identified by comparing the antibody framework
sequences to the germline sequences from which the antibody is
derived. To return the framework region sequences to their germline
configuration, the somatic mutations can be "backmutated" to the
germline sequence by, for example, site-directed mutagenesis. Such
"backmutated" antibodies are also intended to be encompassed by the
disclosure. Additional framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T cell epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in U.S. Patent Publication No. 2003/0153043 by Carr et al.
[0212] Constant regions of the antibodies or antibody fragments
utilized for preparation of the antibody cytokine engrafted
proteins can be any type or subtype, as appropriate, and can be
selected to be from the species of the subject to be treated by the
present methods (e.g., human, non-human primate or other mammal,
for example, agricultural mammal (e.g., equine, ovine, bovine,
porcine, camelid), domestic mammal (e.g., canine, feline) or rodent
(e.g., rat, mouse, hamster, rabbit). In some embodiments antibodies
utilized in antibody cytokine engrafted proteins are engineered to
generate humanized or Humaneered.RTM. antibodies. In some
embodiments antibodies utilized in antibody cytokine engrafted
proteins are human antibodies. In some embodiments, antibody
constant region isotype is IgG, for example, IgG1, IgG2, IgG3,
IgG4. In certain embodiments the constant region isotype is IgGi.
In some embodiments, antibody cytokine engrafted proteins comprise
an IgG. In some embodiments, antibody cytokine engrafted proteins
comprise an IgG1 Fc. In some embodiments, antibody cytokine
engrafted proteins comprise an IgG2 Fc.
[0213] In addition or alternative to modifications made within
framework or CDR regions, antibodies or antibody fragments utilized
in preparation of antibody cytokine engrafted proteins can be
engineered to include modifications within an Fc region, typically
to alter one or more functional properties of the antibody, such
as, e.g., serum half-life, complement fixation, Fc receptor
binding, and/or antigen-dependent cellular cytotoxicity.
Furthermore, an antibody or antibody fragment can be chemically
modified (e.g., one or more chemical moieties can be attached to
the antibody) or be modified to alter its glycosylation, again to
alter one or more functional properties of the antibody cytokine
engrafted protein.
[0214] In one embodiment, a hinge region of CH1 is modified such
that the number of cysteine residues in the hinge region is
altered, e.g., increased or decreased. For example, by the approach
is described further in U.S. Pat. No. 5,677,425 by Bodmer et al.
wherein the number of cysteine residues in the hinge region of CH1
is altered to, for example, facilitate assembly of the light and
heavy chains or to increase or decrease the stability of the
antibody cytokine engrafted protein. In another embodiment, an Fc
hinge region of an antibody is mutated to alter the biological
half-life of the antibody cytokine engrafted protein. More
specifically, one or more amino acid mutations are introduced into
the CH2-CH3 domain interface region of the Fc-hinge fragment such
that the antibody cytokine engrafted protein has impaired
Staphylococcyl protein A (SpA) binding relative to native Fc-hinge
domain SpA binding. This approach is described in further detail in
U.S. Pat. No. 6,165,745 by Ward et al.
[0215] The present disclosure provides for antibody cytokine
engrafted proteins that specifically bind to IL10R protein which
have an extended half-life in vivo. In another embodiment, an
antibody cytokine engrafted protein is modified to increase its
biological half-life. Various approaches are possible. Antibody
cytokine engrafted proteins having an increased half-life in vivo
can also be generated introducing one or more amino acid
modifications (i.e., substitutions, insertions or deletions) into
an IgG constant domain, or FcRn binding fragment thereof
(preferably a Fc or hinge Fc domain fragment). For example, one or
more of the following mutations can be introduced: T252L, T254S,
T256F, as described in U.S. Pat. No. 6,277,375 to Ward. See, e.g.,
International Publication No. WO 98/23289; International
Publication No. WO 97/34631; and U.S. Pat. No. 6,277,375.
Alternatively, to increase the biological half-life, the antibody
cytokine engrafted protein is altered within the CH1 or CL region
to contain a salvage receptor binding epitope taken from two loops
of a CH2 domain of an Fc region of an IgG, as described in U.S.
Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. In yet other
embodiments, the Fc region is altered by replacing at least one
amino acid residue with a different amino acid residue to alter the
effector functions of the antibody cytokine engrafted protein. For
example, one or more amino acids can be replaced with a different
amino acid residue such that the antibody cytokine engrafted
protein has an altered affinity for an effector ligand but retains
antigen-binding ability of the parent antibody. The effector ligand
to which affinity is altered can be, for example, an Fc receptor
(FcR) or the C1 component of complement. This approach is described
in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both
by Winter et al.
[0216] In another embodiment, one or more amino acids selected from
amino acid residues can be replaced with a different amino acid
residue such that the antibody cytokine engrafted protein has
altered C1q binding and/or reduced or abolished complement
dependent cytotoxicity (CDC). This approach is described in further
detail in U.S. Pat. No. 6,194,551 by Idusogie et al.
[0217] Antibody cytokine engrafted proteins containing such
mutations mediate reduced or no antibody-dependent cellular
cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In
some embodiments, amino acid residues L234 and L235 of the IgG1
constant region are substituted to Ala234 and Ala235. In some
embodiments, amino acid residue N267 of the IgG1 constant region is
substituted to Ala267.
[0218] In another embodiment, one or more amino acid residues are
altered to thereby alter the ability of the antibody cytokine
engrafted protein to fix complement. This approach is described
further in PCT Publication WO 94/29351 by Bodmer et al.
[0219] In yet another embodiment, an Fc region is modified to
increase the ability of the antibody to mediate antibody dependent
cellular cytotoxicity (ADCC) and/or to increase the affinity of the
antibody cytokine engrafted protein for an Fey receptor by
modifying one or more amino acids. This approach is described
further in PCT Publication W000/42072 by Presta.
[0220] Moreover, binding sites on human IgG1 for Fc.gamma.R1,
Fc.gamma.RII, Fc.gamma.RIII and FcRn have been mapped and variants
with improved binding have been described (see Shields, R. L. et
al., 2001 J. Biol. Chem. 276:6591-6604).
[0221] In still another embodiment, glycosylation of an antibody
cytokine engrafted protein is modified. For example, an
aglycoslated antibody cytokine engrafted protein can be made (i.e.,
the antibody cytokine engrafted protein lacks glycosylation).
Glycosylation can be altered to, for example, increase the affinity
of the antibody for "antigen." Such carbohydrate modifications can
be accomplished by, for example, altering one or more sites of
glycosylation within the antibody sequence. For example, one or
more amino acid substitutions can be made that result in
elimination of one or more variable region framework glycosylation
sites to thereby eliminate glycosylation at that site. Such
aglycosylation may increase the affinity of the antibody for
antigen. Such an approach is described in further detail in U.S.
Pat. Nos. 5,714,350 and 6,350,861 by Co et al.
[0222] Additionally or alternatively, an antibody cytokine
engrafted protein can be made that has an altered type of
glycosylation, such as a hypofucosylated antibody cytokine
engrafted protein having reduced amounts of fucosyl residues or an
antibody having increased bisecting GlcNac structures. Such altered
glycosylation patterns have been demonstrated to increase the ADCC
ability of antibodies. Such carbohydrate modifications can be
accomplished by, for example, expressing the antibody cytokine
engrafted protein in a host cell with altered glycosylation
machinery. Cells with altered glycosylation machinery have been
described in the art and can be used as host cells in which to
express recombinant antibody cytokine engrafted proteins to thereby
produce an antibody cytokine engrafted protein with altered
glycosylation. For example, EP 1,176,195 by Hang et al. describes a
cell line with a functionally disrupted FUT8 gene, which encodes a
fucosyl transferase, such that antibody cytokine engrafted proteins
expressed in such a cell line exhibit hypofucosylation. PCT
Publication WO 03/035835 by Presta describes a variant CHO cell
line, Lec13 cells, with reduced ability to attach fucose to
Asn(297)-linked carbohydrates, also resulting in hypofucosylation
of antibody cytokine engrafted proteins expressed in that host cell
(see also Shields, R. L. et al., 2002 J. Biol. Chem.
277:26733-26740). PCT Publication WO 99/54342 by Umana et al.
describes cell lines engineered to express glycoprotein-modifying
glycosyl transferases (e.g., beta(1,4)-N
acetylglucosaminyltransferase III (GnTIII)) such that antibody
cytokine engrafted proteins expressed in the engineered cell lines
exhibit increased bisecting GlcNac structures which results in
increased ADCC activity of the antibodies (see also Umana et al.,
1999 Nat. Biotech. 17:176-180).
[0223] In some embodiments, one or more domains, or regions, of an
antibody cytokine engrafted protein are connected via a linker, for
example, a peptide linker, such as those that are well known in the
art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci.
USA 90:6444-6448; Poljak, R J., et al. (1994) Structure
2:1121-1123). A peptide linker may vary in length, e.g., a linker
can be 1-100 amino acids in length, typically a linker is from five
to 50 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, or 50 amino acids in length.
[0224] In some embodiments, monomeric IL10 is incorporated into the
CDR sequence optionally with one or more peptide linker sequences.
In certain embodiments one or more peptide linkers is independently
selected from a (Gly.sub.n-Ser).sub.m sequence, a
(Gly.sub.n-Ala).sub.m sequence, or any combination of a
(Gly.sub.n-Ser).sub.m/(Gly.sub.n-Ala).sub.m sequence, wherein each
n is independently an integer from 1 to 5 and each m is
independently an integer from 0 to 10. In some embodiments a
peptide linker is (Gly.sub.4-Ser).sub.m wherein m is an integer
from 0 to 10. In some embodiments a peptide linker is
(Gly.sub.4-Ala).sub.m wherein m is an integer from 0 to 10.
Examples of linkers include, but are not limited to, certain
embodiments one or more linkers include G.sub.4S repeats, e.g., the
Gly-Ser linker GGGGS (SEQ ID NO:252), or (GGGGS)m wherein m is a
positive integer equal to or greater than 1. For example, m=1, m=2,
m=3. m=4, m-=5 and m=6, m=7, m=8, m=9 and m=10. In some
embodiments, the linker includes multiple repeats of GGGGS (SEQ ID
NO:252), including, but is not limited to (GGGGS).sub.3 or
(GGGGS).sub.4. In some embodiments, Ser can be replaced with Ala
e.g., linkers G/A such as (GGGGA) (SEQ ID NO: 253), or
(GGGGA).sub.m wherein m is a positive integer equal to or greater
than 1. In some embodiments, the linker includes multiple repeats
of GGGGA (SEQ ID NO:253). In other embodiments, a linker includes
combinations and multiples of GGGGS (SEQ ID NO:252) and GGGGA (SEQ
ID NO:253).
[0225] Moreover, the antibody cytokine engrafted proteins can be
linked to marker sequences, such as a peptide to facilitate
purification of antibody cytokine engrafted proteins. In preferred
embodiments, a marker amino acid sequence is a hexa-histidine
peptide, such as the tag provided in a pQE vector (QIAGEN, Inc.,
9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of
which are commercially available. As described in Gentz et al.,
1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance,
hexa-histidine provides for convenient purification of the
engrafted protein. Other peptide tags useful for purification
include, but are not limited to, the hemagglutinin ("HA") tag,
which corresponds to an epitope derived from the influenza
hemagglutinin protein (Wilson et al., 1984, Cell 37:767), and the
"flag" tag.
[0226] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
Assays for Antibody Cytokine Engrafted Protein Activity
[0227] Assays for identifying antibody cytokine engrafted proteins
are known in the art and are described herein. The antibody
cytokine engrafted proteins bind to IL10 receptor (IL10R), and
promote, induce and stimulate intracellular signaling resulting in
anti-inflammatory effects as well as immunostimulatory effects.
[0228] Binding of the antibody cytokine engrafted proteins to
IL-10R can be determined using any method known in the art. For
example, binding to IL-10R can be determined using known
techniques, including without limitation ELISA, Western blots,
surface plasmon resonance (e.g., BIAcore), and flow cytometry.
[0229] Intracellular signaling through IL-10R can be measured using
any method known in the art. For example, activation through IL-10R
promotes STAT3 phosphorylation and signaling. Methods for measuring
STAT3 activation are standard in the art (e.g., phosphorylation
status of STAT3 protein, reporter gene assays, downstream signaling
assays, etc.). Activation through IL-10R has anti-inflammatory
effects, including suppression of pro-inflammatory cytokines as
well as differentiation and proliferation of macrophages.
Additionally, activation through IL-10R has immunostimulatory
effects including stimulation of B cell proliferation, mast cell
proliferation (e.g., MC/9), and natural killer (NK) cell
proliferation and proliferation of activated CD8.sup.+ T cells, as
well as induction of certain pro-inflammatory cytokines. Methods
for measuring proliferation of cells are standard in the art (e.g.,
.sup.3H-thymidine incorporation assays, CFSE labeling). Methods for
measuring cytokine production are well known in the art (e.g.,
ELISA assays, ELISpot assays). In performing in vitro assays, test
cells or culture supernatant from test cells contacted with an
agonist antibody cytokine engrafted proteins can be compared to
control cells or culture supernatants from control cells that have
not been contacted with an agonist antibody cytokine engrafted
proteins and/or those that have been contacted with recombinant
human IL10 (rhIL10).
[0230] IL10 receptor agonist activity of the antibody cytokine
engrafted proteins can also be measured ex vivo and/or in vivo. In
some aspects, methods for measuring STAT3 activation across various
cell types ex vivo from animals treated with antibody cytokine
engrafted proteins as compared to untreated control animals and/or
animals similarly treated with rhIL10 may be used to show
differential activity of the antibody engrafted proteins across
cell types. Preferred antibody cytokine engrafted proteins have the
ability to activate and expand monocytes. For example, in vivo
activation and expansion of monocytes can be measured using any
method known in the art, e.g., by flow cytometry. Antibody cytokine
engrafted proteins can be therapeutically useful in suppressing
levels of pro-inflammatory response following stimulation. Levels
of pro-inflammatory cytokines can be measured using any method
known in the art in samples isolated from animals treated with an
antibody cytokine engrafted protein before, during and/or after
treatment with a stimulatory agent (eg., LPS), and results may be
compared to non-treated control animals and/or animals similarly
treated with recombinant human IL10(rhIL10) therapy. Preferred
agonist antibody cytokine engrafted proteins can be therapeutically
useful in preventing, reducing, inhibiting or eliminating
inflammatory bowel disease, Crohn's disease, ulcerative colitis,
rheumatoid arthritis, psoriasis, type I diabetes, acute
pancreatitis, uveitis, Sjogren's disease, Behcet's disease,
sarcoidosis, and graft versus host disease (GVHD). The efficacy of
the agonist antibody cytokine engrafted proteins can be determined
by administering a therapeutically effective amount of the antibody
cytokine engrafted protein to a subject and comparing the subject
before and after administration of the antibody cytokine engrafted
protein. Efficacy of the agonist antibody cytokine engrafted
protein in therapy for inflammatory bowel disease, Crohn's disease,
ulcerative colitis, rheumatoid arthritis, psoriasis, type I
diabetes, acute pancreatitis, uveitis, Sjogren's disease, Behcet's
disease, sarcoidosis, and graft versus host disease (GVHD) also can
be determined by administering a therapeutically effective amount
of an antibody cytokine engrafted protein to a test subject and
comparing the test subject to a control subject who has not been
administered the antibody cytokine engrafted protein and/or
comparison to a subject similarly treated with rhIL10.
Polynucleotides Encoding Antibody Cytokine Engrafted Proteins
[0231] In another aspect, isolated nucleic acids encoding heavy and
light chain proteins of the antibody cytokine engrafted proteins
are provided. Antibody cytokine engrafted proteins can be produced
by any means known in the art, including but not limited to,
recombinant expression, chemical synthesis, and enzymatic digestion
of antibody tetramers. Recombinant expression can be from any
appropriate host cells known in the art, for example, mammalian
host cells, bacterial host cells, yeast host cells, insect host
cells, etc.
[0232] Provided herein are polynucleotides that encode the variable
regions exemplified in any one of SEQ ID NO:109, SEQ ID NO:110, SEQ
ID NO:205, and SEQ ID NO:206; the variable regions exemplified in
any one of SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:207, SEQ ID
NO:208, the CDRs exemplified in any one of SEQ ID NO:109, SEQ ID
NO:110, SEQ ID NO:205, and SEQ ID NO:206; the heavy and light
chains exemplified in any one of SEQ ID NO:111, SEQ ID NO:112, SEQ
ID NO:207 and SEQ ID NO:208.
[0233] The disclosure thus provides polynucleotides encoding the
heavy and/or light chain polypeptides of the antibody cytokine
engrafted proteins described herein, e.g., polynucleotides encoding
heavy or light chain variable regions or segments comprising the
complementary determining regions as described herein. In some
embodiments, the polynucleotide encoding the heavy chain variable
regions comprises a sequence having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid
sequence identity with a polynucleotide of SEQ ID NO:242. In some
embodiments, the polynucleotide encoding the light chain variable
regions comprises a sequence having at least 85%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid
sequence identity with a polynucleotide of SEQ ID NO:243.
[0234] In some embodiments, the polynucleotide encoding the heavy
chain variable regions comprises a sequence having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
nucleic acid sequence identity with a polynucleotide of SEQ ID
NO:246. In some embodiments, the polynucleotide encoding the light
chain variable regions comprises a sequence having at least 85%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
nucleic acid sequence identity with a polynucleotide of SEQ ID
NO:247.
[0235] In some embodiments, the polynucleotide encoding the heavy
chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% nucleic acid sequence identity with a
polynucleotide of SEQ ID NO:244. In some embodiments, the
polynucleotide encoding the light chain has at least 85%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid
sequence identity with a polynucleotide of SEQ ID NO:245.
[0236] In some embodiments, the polynucleotide encoding the heavy
chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% nucleic acid sequence identity with a
polynucleotide of SEQ ID NO:248. In some embodiments, the
polynucleotide encoding the light chain has at least 85%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid
sequence identity with a polynucleotide selected of SEQ ID
NO:249.
[0237] Polynucleotides can encode the variable region sequence of
an antibody cytokine engrafted protein. They can also encode both a
variable region and a constant region of the antibody cytokine
engrafted protein. Some of the polynucleotide sequences encode a
polypeptide that comprises variable regions of both the heavy chain
and the light chain of one of the antibody cytokine engrafted
proteins. Some other polynucleotides encode two polypeptide
segments that respectively are substantially identical to the
variable regions of the heavy chain and the light chain of one of
the antibody protein engrafted proteins.
[0238] In certain embodiments polynucleotides or nucleic acids
comprise DNA. In other embodiments polynucleotides or nucleic acids
comprise RNA, which may be single stranded or double stranded.
[0239] In some embodiments a recombinant host cell comprising the
nucleic acids encoding one or more cytokine engrafted protein or
fragment thereof, and optionally, secretion signals are provided.
In certain embodiments a recombinant host cell comprises a vector
encoding an antibody cytokine engrafted protein or fragment thereof
and secretion signals. In other certain embodiments a recombinant
host cell comprises one or more vectors encoding two immunoglobulin
protein chains of the antibody cytokine engrafted protein and
secretion signals. In some embodiments a recombinant host cell
comprises a single vector encoding two immunoglobulin protein
chains of the antibody cytokine engrafted protein and secretion
signals. In some embodiments a recombinant host cell comprises two
vectors, one encoding a heavy chain immunoglobulin protein chain,
and another encoding a light chain immunoglobulin protein chain of
the heterodimer of the antibody cytokine engrafted protein, with
each including secretion signals. A recombinant host cell may be a
prokaryotic or eukaryotic cell. In some embodiments a host cell is
a eukaryotic cell line. In some embodiments a host cell is a
mammalian cell line.
[0240] Additionally provided are methods for producing the antibody
cytokine engrafted proteins. In some embodiments the method
comprises the steps of (i) culturing a host cell comprising one or
more vectors encoding immunoglobulin protein chains of an antibody
cytokine engrafted protein under conditions suitable for
expression, formation, and secretion of the antibody cytokine
engrafted protein and (ii) recovering the antibody cytokine
engrafted protein.
[0241] The polynucleotide sequences can be produced by de novo
solid-phase DNA synthesis or by PCR mutagenesis of an existing
sequence (e.g., sequences as described herein) encoding a
polypeptide chain of an antibody cytokine engrafted protein. Direct
chemical synthesis of nucleic acids can be accomplished by methods
known in the art, such as the phosphotriester method of Narang et
al., Meth. Enzymol. 68:90, 1979; the phosphodiester method of Brown
et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite
method of Beaucage et al., Tetra. Lett., 22:1859, 1981; and the
solid support method of U.S. Pat. No. 4,458,066. Introducing
mutations to a polynucleotide sequence by PCR can be performed as
described in, e.g., PCR Technology: Principles and Applications for
DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992;
PCR Protocols: A Guide to Methods and Applications, Innis et al.
(Ed.), Academic Press, San Diego, Calif., 1990; Mattila et al.,
Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and
Applications 1:17, 1991.
[0242] Also provided in the disclosure are expression vectors and
host cells for producing the antibody cytokine engrafted proteins
described above. Various expression vectors can be employed to
express polynucleotides encoding the immunoglobulin polypeptide
chains, or fragments, of the antibody cytokine engrafted proteins.
Both viral-based and nonviral expression vectors can be used to
produce the immunoglobulin proteins in a mammalian host cell.
Nonviral vectors and systems include plasmids, episomal vectors,
typically with an expression cassette for expressing a protein or
RNA, and human artificial chromosomes (see, e.g., Harrington et
al., Nat Genet 15:345, 1997). For example, nonviral vectors useful
for expression of the antibody cytokine engrafted protein
polynucleotides and polypeptides in mammalian (e.g., human) cells
include pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C
(Invitrogen, San Diego, Calif.), MPSV vectors, and numerous other
vectors known in the art for expressing other proteins. Useful
viral vectors include vectors based on retroviruses, adenoviruses,
adeno-associated viruses, herpes viruses, vectors based on SV40,
papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and
Semliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu.
Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143,
1992.
[0243] The choice of expression vector depends on the intended host
cells in which the vector is to be expressed. Typically, the
expression vectors contain a promoter and other regulatory
sequences (e.g., enhancers) that are operably linked to the
polynucleotides encoding the antibody cytokine engrafted protein.
In some embodiments, an inducible promoter is employed to prevent
expression of inserted sequences except under inducing conditions.
Inducible promoters include, e.g., arabinose, lacZ, metallothionein
promoter or a heat shock promoter. Cultures of transformed
organisms can be expanded under noninducing conditions without
biasing the population for coding sequences whose expression
products are better tolerated by the host cells. In addition to
promoters, other regulatory elements may also be required or
desired for efficient expression of an immunoglobulin chain or
fragment of the antibody cytokine engrafted proteins. These
elements typically include an ATG initiation codon and adjacent
ribosome binding site or other sequences. In addition, the
efficiency of expression may be enhanced by the inclusion of
enhancers appropriate to the cell system in use (see, e.g., Scharf
et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et
al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer
or CMV enhancer may be used to increase expression in mammalian
host cells.
[0244] Expression vectors can also provide a secretion signal
sequence to form a heterologous protein in addition to sequences
encoded by the antibody cytokine engrafted protein sequences. More
often, the inserted immunoglobulin sequences of the antibody
cytokine engrafted proteins are operably linked to a signal
sequence before inclusion in the vector. Vectors to be used to
receive sequences encoding immunoglobulin light and heavy chain
variable domains sometimes also encode constant regions or parts
thereof. Such vectors allow expression of the variable regions as
antibody cytokine engrafted proteins with the constant regions,
thereby leading to production of intact antibody cytokine engrafted
proteins or fragments thereof. Typically, such constant regions are
human.
[0245] Host cells for harboring and expressing the antibody
cytokine engrafted protein chains can be either prokaryotic or
eukaryotic. E. coli is one prokaryotic host useful for cloning and
expressing the polynucleotides of the present disclosure. Other
microbial hosts suitable for use include bacilli, such as Bacillus
subtilis, and other enterobacteriaceae, such as Salmonella,
Serratia, and various Pseudomonas species. In these prokaryotic
hosts, one can also make expression vectors, which typically
contain expression control sequences compatible with the host cell
(e.g., an origin of replication). In addition, any number of a
variety of well-known promoters will be present, such as the
lactose promoter system, a tryptophan (trp) promoter system, a
beta-lactamase promoter system, or a promoter system from phage
lambda. The promoters typically control expression, optionally with
an operator sequence, and have ribosome binding site sequences and
the like, for initiating and completing transcription and
translation. Other microbes, such as yeast, can also be employed to
express antibody cytokine engrafted proteins. Insect cells in
combination with baculovirus vectors can also be used.
[0246] In some preferred embodiments, mammalian host cells are used
to express and produce the antibody cytokine engrafted proteins of
the present disclosure. For example, they can be a mammalian cell
line harboring an exogenous expression vector. These include any
normal mortal or normal or abnormal immortal animal or human cell.
For example, a number of suitable host cell lines capable of
secreting intact immunoglobulins have been developed, including the
CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell
lines, transformed B-cells and hybridomas. The use of mammalian
tissue cell culture to express polypeptides is discussed generally
in, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y.,
N.Y., 1987. Expression vectors for mammalian host cells can include
expression control sequences, such as an origin of replication, a
promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev.
89:49-68, 1986), and necessary processing information sites, such
as ribosome binding sites, RNA splice sites, polyadenylation sites,
and transcriptional terminator sequences. These expression vectors
usually contain promoters derived from mammalian genes or from
mammalian viruses. Suitable promoters may be constitutive, cell
type-specific, stage-specific, and/or modulatable or regulatable.
Useful promoters include, but are not limited to, the
metallothionein promoter, the constitutive adenovirus major late
promoter, the dexamethasone-inducible MMTV promoter, the SV40
promoter, the MRP polIII promoter, the constitutive MPSV promoter,
the tetracycline-inducible CMV promoter (such as the human
immediate-early CMV promoter), the constitutive CMV promoter, and
promoter-enhancer combinations known in the art.
[0247] Methods for introducing expression vectors containing the
polynucleotide sequences of interest vary depending on the type of
cellular host. For example, calcium chloride transfection is
commonly utilized for prokaryotic cells, whereas calcium phosphate
treatment or electroporation may be used for other cellular hosts
(see generally Sambrook et al., supra). Other methods include,
e.g., electroporation, calcium phosphate treatment,
liposome-mediated transformation, injection and microinjection,
ballistic methods, virosomes, immunoliposomes, polycation:nucleic
acid conjugates, naked DNA, artificial virions, engrafted to the
herpes virus structural protein VP22 (Elliot and O'Hare, Cell
88:223, 1997), agent-enhanced uptake of DNA, and ex vivo
transduction. For long-term, high-yield production of recombinant
proteins, stable expression will often be desired. For example,
cell lines which stably express antibody cytokine engrafted protein
immunoglobulin chains can be prepared using expression vectors
which contain viral origins of replication or endogenous expression
elements and a selectable marker gene. Following introduction of
the vector, cells may be allowed to grow for 1-2 days in an
enriched media before they are switched to selective media. The
purpose of the selectable marker is to confer resistance to
selection, and its presence allows growth of cells which
successfully express the introduced sequences in selective media.
Resistant, stably transfected cells can be proliferated using
tissue culture techniques appropriate to the cell type.
Compositions Comprising Antibody Cytokine Engrafted Proteins
[0248] Provided are pharmaceutical compositions comprising an
antibody cytokine engrafted protein formulated together with a
pharmaceutically acceptable carrier. Optionally, pharmaceutical
compositions additionally contain other therapeutic agents that are
suitable for treating or preventing a given disorder.
Pharmaceutically acceptable carriers enhance or stabilize the
composition, or facilitate preparation of the composition.
Pharmaceutically acceptable carriers include solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like that are physiologically
compatible.
[0249] A pharmaceutical composition of the present disclosure can
be administered by a variety of methods known in the art. Route
and/or mode of administration vary depending upon the desired
results. It is preferred that administration be by parenteral
administration (e.g., selected from any of intravenous,
intramuscular, intraperitoneal, intrathecal, intraarterial, or
subcutaneous), or administered proximal to the site of the target.
A pharmaceutically acceptable carrier is suitable for
administration by any one or more of intravenous, intramuscular,
intraperitoneal, intrathecal, intraarterial, subcutaneous,
intranasal, inhalational, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of
administration, active compound, e.g., antibody cytokine engrafted
protein, may be coated in a material to protect the compound from
the action of acids and other natural conditions that may
inactivate the compound. In some embodiments the pharmaceutical
composition is formulated for intravenous administration. In some
embodiments the pharmaceutical composition is formulation for
subcutaneous administration.
[0250] An antibody cytokine engrafted protein, alone or in
combination with other suitable components, can be made into
aerosol formulations (i.e., they can be "nebulized") to be
administered via inhalation. Aerosol formulations can be placed
into pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like.
[0251] In some embodiments, a pharmaceutical composition is sterile
and fluid. Proper fluidity can be maintained, for example, by use
of coating such as lecithin, by maintenance of required particle
size in the case of dispersion and by use of surfactants. In many
cases, it is preferable to include isotonic agents, for example,
sugars, polyalcohols such as mannitol or sorbitol, and sodium
chloride in the composition. Long-term absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate or gelatin. In certain embodiments compositions can be
prepared for storage in a lyophilized form using appropriate
excipients (e.g., sucrose).
[0252] Pharmaceutical compositions can be prepared in accordance
with methods well known and routinely practiced in the art.
Pharmaceutically acceptable carriers are determined in part by the
particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions of the present disclosure. Applicable methods for
formulating an antibody cytokine engrafted protein and determining
appropriate dosing and scheduling can be found, for example, in
Remington: The Science and Practice of Pharmacy, 21.sup.st Ed.,
University of the Sciences in Philadelphia, Eds., Lippincott
Williams & Wilkins (2005); and in Martindale: The Complete Drug
Reference, Sweetman, 2005, London: Pharmaceutical Press., and in
Martindale, Martindale: The Extra Pharmacopoeia, 31st Edition.,
1996, Amer Pharmaceutical Assn, and Sustained and Controlled
Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker,
Inc., New York, 1978, each of which are hereby incorporated herein
by reference. Pharmaceutical compositions are preferably
manufactured under GMP conditions. Typically, a therapeutically
effective dose or efficacious dose of an antibody cytokine
engrafted protein is employed in the pharmaceutical compositions.
An antibody cytokine engrafted protein is formulated into
pharmaceutically acceptable dosage form by conventional methods
known to those of skill in the art. Dosage regimens are adjusted to
provide the desired response (e.g., a therapeutic response). In
determining a therapeutically or prophylactically effective dose, a
low dose can be administered and then incrementally increased until
a desired response is achieved with minimal or no undesired side
effects. For example, a single bolus may be administered, several
divided doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subjects to be treated; each unit contains a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier.
[0253] Actual dosage levels of active ingredients in the
pharmaceutical compositions of the present disclosure can be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level
depends upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present disclosure
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors.
Co-Formulation with Second Agent
[0254] In some embodiments, the pharmacological compositions
comprise a mixture of an antibody cytokine engrafted protein and
one or more additional pharmacological agent(s). Exemplary second
agents for inclusion in mixtures with the present antibody cytokine
engrafted protein include without limitation anti-inflammatory
agents, immunomodulatory agents, aminosalicylates, and antibiotics.
Appropriate selection may depend on preferred formulation, dosage
and/or delivery method.
[0255] In some embodiments an antibody cytokine engrafted protein
is co-formulated (i.e., provided as a mixture or prepared in a
mixture) with an anti-inflammatory agent. In particular
embodiments, corticosteroid anti-inflammatory agents can be used in
conjunction with the antibody cytokine engrafted protein.
Corticosteroids for use can be selected from any of
methylprednisolone, hydrocortisone, prednisone, budenisonide,
mesalamine, and dexamethasone. Appropriate selection will depend on
formulation and delivery preferences.
[0256] In some embodiments, an antibody cytokine engrafted protein
is co-formulated with an immunomodulatory agent. In particular
embodiments, the immunomodulatory agent is selected from any of
6-mercaptopurine, azathioprine, cyclosporine A, tacrolimus, and
methotrexate. In a particular embodiment, the immunomodulatory
agent is selected from an anti-TNF agent (e.g., infliximab,
adalimumab, certolizumab, golimumab), natalizumab, and
vedolizumab.
[0257] In some embodiments an antibody cytokine engrafted protein
is co-formulated with an aminosalicylate agent. In particular
embodiments, an aminosalicylate is selected from sulfasalazine,
mesalamine, balsalazide, olsalazine or other derivatives of
5-aminosalicylic acid.
[0258] In some embodiments an antibody cytokine engrafted protein
is co-formulated with an antibacterial agent. Exemplary
antibacterial agents include without limitation sulfonamides (e.g.,
sulfanilamide, sulfadiazine, sulfamethoxazole, sulfisoxazole,
sulfacetamide), trimethoprim, quinolones (e.g., nalidixic acid,
cinoxacin, norfloxacin, ciprofloxacin, ofloxacin, sparfloxacin,
fleroxacin, perloxacin, levofloxacin, garenoxacin and
gemifloxacin), methenamine, nitrofurantoin, penicillins (e.g.,
penicillin G, penicillin V, methicilin oxacillin, cloxacillin,
dicloxacillin, nafcilin, ampicillin, amoxicillin, carbenicillin,
ticarcillin, mezlocillin, and piperacillin), cephalosporins (e.g.,
cefazolin, cephalexin, cefadroxil, cefoxitin, cefaclor, cefprozil,
cefuroxime, cefuroxime acetil, loracarbef, cefotetan, ceforanide,
cefotaxime, cefpodoxime proxetil, cefibuten, cefdinir, cefditoren
pivorxil, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, and
cefepine), carbapenems (e.g., imipenem, aztreonam), and
aminoglycosides (e.g., neomycin, kanamycin, streptomycin,
gentamicin, toramycin, netilmicin, and amikacin).
Articles of Manufacture/Kits
[0259] In some aspects, an antibody cytokine engrafted protein is
provided in an article of manufacture (i.e., a kit). The antibody
cytokine engrafted protein is generally in a vial or a container.
Thus, an article of manufacture comprises a container and a label
or package insert, on or associated with the container. Suitable
containers include, for example, a bottle, vial, syringe, solution
bag, etc. As appropriate, the antibody cytokine engrafted protein
can be in liquid or dried (e.g., lyophilized) form. The container
holds a composition which, by itself or combined with another
composition, is effective for preparing a composition for treating,
preventing and/or ameliorating an immune related disorder. The
label or package insert indicates the composition is used for
treating, preventing and/or ameliorating an immune related
disorder. Articles of manufacture (kits) comprising an antibody
cytokine engrafted protein, as described herein, optionally contain
one or more additional agent. In some embodiments, an article of
manufacture (kit) contains antibody cytokine engrafted protein and
a pharmaceutically acceptable diluent. In some embodiments an
antibody cytokine engrafted protein is provided in an article of
manufacture (kit) with one or more additional active agent in the
same formulation (e.g., as mixtures). In some embodiments an
antibody cytokine engrafted protein is provided in an article of
manufacture (kit) with a second or third agent in separate
formulations (e.g., in separate containers). In certain embodiments
an article of manufacture (kit) contains aliquots of the antibody
cytokine engrafted protein, wherein the aliquot provides for one or
more doses. In some embodiments aliquots for multiple
administrations are provided, wherein doses are uniform or varied.
In particular embodiments varied dosing regimens are escalating or
decreasing, as appropriate. In some embodiments dosages of an
antibody cytokine engrafted protein and a second agent are
independently uniform or independently varying. In certain
embodiments, an article of manufacture (kit) comprises an
additional agent selected from any of an anti-inflammatory agent,
immunomodulatory agent, aminosalicylate, and antibiotic. Selection
of one or more additional agent will depend on the dosage,
delivery, and disease condition to be treated.
Methods of Treatment of and use of Antibody Cytokine Engrafted
Proteins in Immune Related Disorders
Conditions Subject to Treatment or Prevention
[0260] Antibody cytokine engrafted proteins find use in the
treatment, amelioration or prophylaxis of immune related disorders.
In one aspect, the disclosure provides methods of treatment of
immune related disorders in an individual in need thereof,
comprising administering to the individual a therapeutically
effective amount of an antibody cytokine engrafted protein as
described herein. The disclosure also provides in one aspect, an
antibody cytokine engrafted protein for use as a therapeutic agent.
In some embodiment an antibody cytokine engrafted protein is
provided for use as a therapeutic agent in the treatment or
prophylaxis of an immune related disorder in an individual. In some
embodiments use of an antibody cytokine engrafted protein is
provided for manufacture of a medicament for treatment of an immune
related disorder in an individual in need thereof. In a further
aspect, the disclosure provides a composition comprising such an
antibody cytokine engrafted protein for use in treating or
ameliorating an immune related disorder in an individual in need
thereof.
[0261] For therapeutic purposes, an individual can have an immune
related disorder. For preventative or prophylactic purposes, an
individual may be in remission from an active state of immune
related disorder or may anticipate future onset. In some
embodiments, the patient has an immune related disorder, is
suspected of having an immune related disorder, or is in remission
from an immune related disorder. Immune related disorders subject
to treatment with the antibody cytokine engrafted protein usually
derive benefit from activation of IL10 receptor signaling, as
described herein. Immune related disorders subject to treatment
include without limitation: inflammatory bowel disease, Crohn's
disease, ulcerative colitis, rheumatoid arthritis, psoriasis, type
I diabetes, acute pancreatitis, uveitis, Sjogren's disease,
Behcet's disease, sarcoidosis, and graft versus host disease
(GVHD).
Administration of Antibody Cytokine Engrafted Proteins
[0262] A physician or veterinarian can start doses of an antibody
cytokine engrafted protein employed in a pharmaceutical composition
at levels lower than that required to achieve the desired
therapeutic effect and gradually increase the dosage until the
desired effect is achieved. In general, effective doses of the
compositions of the present disclosure vary depending upon many
different factors, including the specific disease or condition to
be treated, means of administration, target site, physiological
state of the patient, whether a patient is human or an animal,
other medications administered, and whether treatment is
prophylactic or therapeutic. Treatment dosages typically require
titration to optimize safety and efficacy. For administration with
an antibody cytokine engrafted protein, dosage ranges from about
0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host
body weight. For example dosages can be 1 mg/kg body weight or 10
mg/kg body weight or within the range of 1-10 mg/kg. Dosing can be
daily, weekly, bi-weekly, monthly, or more or less often, as needed
or desired. An exemplary treatment regime entails administration
once weekly, once per every two weeks or once a month or once every
3 to 6 months.
[0263] The antibody cytokine engrafted protein can be administered
in single or divided doses. An antibody cytokine engrafted protein
is usually administered on multiple occasions. Intervals between
single dosages can be weekly, bi-weekly, monthly or yearly, as
needed or desired. Intervals can also be irregular as indicated by
measuring blood levels of antibody cytokine engrafted protein in
the patient. In some methods, dosage is adjusted to achieve a
plasma antibody cytokine engrafted protein concentration of 1-1000
.mu.g/ml and in some methods 25-300 .mu.g/ml. Alternatively,
antibody cytokine engrafted protein can be administered as a
sustained release formulation, in which case less frequent
administration is required. Dosage and frequency vary depending on
the half-life of the antibody cytokine engrafted protein in the
patient. In general, antibody cytokine engrafted proteins show
longer half-life than that of native cytokines (e.g. IL10). Dosage
and frequency of administration can vary depending on whether
treatment is prophylactic or therapeutic. In general for
prophylactic applications, a relatively low dosage is administered
at relatively infrequent intervals over a long period of time. Some
patients continue to receive treatment for the duration of their
lives. In general for therapeutic applications, a relatively high
dosage in relatively short intervals is sometimes required until
progression of the disease is reduced or terminated, and preferably
until the patient shows partial or complete amelioration of
symptoms of disease. Thereafter, a patient can be administered a
prophylactic regime.
Co-Administration with a Second Agent
[0264] In some embodiments, an antibody cytokine engrafted protein
is co-administered with one or more additional pharmacological
agent(s). In some embodiments, an antibody cytokine engrafted
protein and an additional one or more agent(s) are administered as
a mixture. In some embodiments, an antibody cytokine engrafted
protein and an additional one or more agent(s) are administered as
separate formulations. In certain embodiments where separate
formulations are utilized, administration is concurrent. In certain
embodiments where separate formulations are utilized,
administration is sequential. In certain embodiments where separate
formulations are utilized, administration is via the same route. In
certain embodiments where separate formulations are utilized,
administration is via different routes. Exemplary additional agents
for co-administration with an antibody cytokine engrafted protein
include without limitation anti-inflammatory agents,
immunomodulatory agents, aminosalicylates, and antibiotics.
Appropriate selection may depend on preferred formulation, dosage
and/or delivery method. The antibody cytokine engrafted proteins
also find use in combination therapies with additional established
procedures for treating immune related disorder conditions, e.g.,
surgery.
[0265] In some embodiments, an antibody cytokine engrafted protein
is co-administered with an anti-inflammatory agent. In particular
embodiments corticosteroid anti-inflammatory agents may be used in
conjunction with the antibody cytokine engrafted protein.
Corticosteroids for use may be selected from any of
methylprednisolone, hydrocortisone, prednisone, budenisonide,
mesalamine, and dexamethasone. Appropriate selection will depend on
formulation and delivery preferences.
[0266] In some embodiments, an antibody cytokine engrafted protein
is co-administered with an immunomodulatory agent. In certain
embodiments, the immunomodulatory agent is selected from any of
6-mercaptopurine, azathioprine, cyclosporine A, tacrolimus, and
methotrexate. In particular embodiments, an immunomodulatory agent
is selected from an anti-TNF agent (e.g., infliximab, adalimumab,
certolizumab, golimumab), natalizumab, and vedolizumab.
[0267] In some embodiments an antibody cytokine engrafted protein
is co-administered with an aminosalicylate agent. In particular
embodiments the aminosalicylate agent is selected from
sulfasalazine, mesalamine, balsalazide, olsalazine or other
derivatives of 5-aminosalicylic acid.
[0268] In some embodiments an antibody cytokine engrafted protein
is co-administered with an antibacterial agent. Exemplary
antibacterial agents include without limitation sulfonamides (e.g.,
sulfanilamide, sulfadiazine, sulfamethoxazole, sulfisoxazole,
sulfacetamide), trimethoprim, quinolones (e.g., nalidixic acid,
cinoxacin, norfloxacin, ciprofloxacin, ofloxacin, sparfloxacin,
fleroxacin, perloxacin, levofloxacin, garenoxacin and
gemifloxacin), methenamine, nitrofurantoin, penicillins (e.g.,
penicillin G, penicillin V, methicilin oxacillin, cloxacillin,
dicloxacillin, nafcilin, ampicillin, amoxicillin, carbenicillin,
ticarcillin, mezlocillin, and piperacillin), cephalosporins (e.g.,
cefazolin, cephalexin, cefadroxil, cefoxitin, cefaclor, cefprozil,
cefuroxime, cefuroxime acetil, loracarbef, cefotetan, ceforanide,
cefotaxime, cefpodoxime proxetil, cefibuten, cefdinir, cefditoren
pivorxil, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, and
cefepine), carbapenems (e.g., imipenem, aztreonam), and
aminoglycosides (e.g., neomycin, kanamycin, streptomycin,
gentamicin, toramycin, netilmicin, and amikacin).
EXAMPLES
Example 1
Creation of Immunoglobulin-IL10 Engrafted Constructs
[0269] Engrafted constructs IgGIL10M1, IgGIL10M2, IgGIL10M3, IgGIL
10M4, IgGIL10M5, IgGIL10M6, IgGIL10M7, IgGIL10M8, IgGIL10M9,
IgGIL10M10, IgGIL10M11, IgGIL10M12, IgGIL10M13, IgGIL10M14, and
IgGIL10M15 were generated by engineering a monomeric IL10 sequence
into CDR regions of various immunoglobulin scaffolds, then both
heavy and light chain immunoglobulin chains were produced to
generate final protein constructs. IgGIL10M engrafted constructs
confer preferred therapeutic anti-inflammatory properties of IL10;
however, IgGIL10M engrafted constructs have reduced proportional
pro-inflammatory activity as compared with rhIL10.
[0270] To create antibody cytokine engrafted proteins, monomeric
IL10 (IL10M), comprising residues 19-178 of full length IL10 with a
six amino acid linker between residues 134 and 135 (SEQ ID NO: 209)
was inserted into various CDR loops of immunoglobulin chain
scaffold. Engrafted constructs were prepared using a variety of
known immunoglobulin sequences which have been utilized in clinical
settings as well as germline antibody sequences. Sequences of IL10M
in two exemplary scaffolds, referred to as GFTX1 and GFTX3, are
depicted in TABLE 1. Insertion points were selected to be the
mid-point of the CDR loop based on available structural or homology
model data. Antibody cytokine engrafted proteins were produced
using standard molecular biology methodology utilizing recombinant
DNA encoding the relevant sequences.
[0271] For example, a variable region of each antibody containing
IL10M inserted into one of the six CDRs was synthesized. DNA
encoding variable region was amplified via PCR and the resulting
fragment was sub-cloned into vector containing either the light
chain constant region or the heavy chain constant and Fc regions.
In this manner IL10M antibody cytokine engrafted proteins were made
corresponding to insertion of IL10M into each of the 6 CDRs (L1,
L2, L3, H1, H2, H3). Resulting constructs are shown in TABLE 1.
Transfections of the appropriate combination of heavy and light
chain vectors results in the expression of a recombinant antibody
with two grafted IL10M molecules (one IL10 monomer in each Fab
arm). See FIG. 2.
[0272] The selection of which CDR is chosen for cytokine
engraftment is chosen on the parameters of: the required biology,
the biophysical properties and a favorable development profile. At
this time, modeling software is only partially useful in predicting
which CDR and which location within the CDR will provide the
desired parameters, so therefore all six possible antibody cytokine
grafts are made and then evaluated in biological assays. If the
required biological activity was achieved, then the biophysical
properties such as structural resolution of the antibody cytokine
engrafted molecule was resolved.
[0273] By virtue of the grafting of IL10 into a CDR, the antibody
portion of the antibody cytokine engrafted protein presents the
IL10 monomer with a unique structure which influences the binding
to the IL10 receptor as discussed below. There are no off-target
effects due to the antibody portion. In addition, the Fc portion of
the antibody cytokine engrafted protein has been modified to be
fully silent regarding ADCC (Antibody Dependent Cell-mediated
Cytotoxicity) and CDC (Complement-Dependent Cytotoxicity).
[0274] In summary, the insertion point in each CDR was chosen on a
structural basis, with the hypothesis that grafting into the CDR
would provide some level of steric hindrance to individual subunits
of the IL10 receptor. The final selection of which CDR graft is
best for a particular cytokine is based on desired biology and
biophysical properties. The nature of the cytokine receptor, the
cytokine/receptor interactions and the mechanism of signaling also
played a role and this was resolved by comparing each individual
antibody cytokine engrafted molecule for their respective
properties. For example, engrafting of IL10 into the light chain
CDR1 (CDRL1) produced the desired biological activity of activating
monocytes but not other cells such as NK cells. This was seen in
the exemplary antibody cytokine engrafted proteins IgGIL 10M7 and
IgGIL 10M13.
TABLE-US-00001 TABLE 1 SEQ ID NO: Description Comments 1 CDRH1 of
GFTFSSY GFTX1 IgGIL10M1 (Chothia) 2 CDRH2 of SGSGGS GFTX1 IgGIL10M1
(Chothia) 3 CDRH3 of TRTKRF GFTX1 IgGIL10M1 (Chothia) 4 CDRL1 of
SQSVSPGQGTQSENSCTHFPGNLPNML GFTX1 IgGIL10M1
RDLRDAFSRVKTFFQMKDQLDNLLLK Monomeric (Chothia)
ESLLEDFKGYLGCQALSEMIQFYLEEV IL10 grafted
MPQAENQDPDIKAHVNSLGENLKTLRL into RLRRCHRFLPCENGGGSGGKSKAVEQ CDRL1.
VKNAFNKLQEKGIYKAMSEFDIFINYIE AYMTMKIRNSSSY IL10 is bolded,
underlined 5 CDRL2 of GAS GFTX1 IgGIL10M1 (Chothia) 6 CDRL3 of
YGSSPL GFTX1 IgGIL10M1 (Chothia) 7 CDRH1 of SYAMS GFTX1 IgGIL10M1
(Kabat) 8 CDRH2 of AISGSGGSTYYGDSVKG GFTX1 IgGIL10M1 (Kabat) 9
CDRH3 of TRTKRF GFTX1 IgGIL10M1 (Kabat) 10 CDRL1 of
RASQSVSPGQGTQSENSCTHFPGNLPN GFTX1 IgGIL10M1 (Kabat)
MLRDLRDAFSRVKTFFQMKDQLDNLL Monomeric LKESLLEDFKGYLGCQALSEMIQFYLE
IL10 grafted EVMPQAENQDPDIKAHVNSLGENLKTL into
RLRLRRCHRFLPCEGGGSGGNKSKAV CDRL1. EQVKNAFNKLQEKGIYKAMSEFDIFIN IL10
is YIEAYMTMKIRNSSSYLA bolded, underlined 11 CDRL2 of GASSRAT GFTX1
IgGIL10M1 (Kabat) 12 CDRL3 of QQYGSSPLT GFTX1 IgGIL10M1 (Kabat) 13
VH of IgGIL10M1 EVQLLESGGGLVQPGGSLRLSCAASGFTF GFTX1
SSYAMSWVRQAPGKGLEWVSAISGSGGS TYYGDSVKGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCARTRTKRFWGQGTLVT VSS 14 VL of IgGIL10M1
EIVLTQSPGTLSLSPGERATLSCRASQSVS GFTX1 PGQGTQSENSCTHFPGNLPNMLRDLRD
Monomeric IL10 AFSRVKTFFQMKDQLDNLLLKESLLED grafted into
FKGYLGCQALSEMIQFYLEEVMPQAEN CDRL1. QDPDIKAHVNSLGENLKTLRLRLRRCH
RFLPCENGGGSGGKSKAVEQVKNAFN KLQEKGIYKAMSEFDIFINYIE
AYMTMKIRNSSSYLAWYQQKPGQAPRL LIYGASSRATGIPDRFSGSGSGTDFTLTISR
LEPEDFAVYYCQQYGSSPLTFGGGTKVEI K 15 Heavy chain of
EVQLLESGGGLVQPGGSLRLSCAASGFTF GFTX1 IgGIL10M1
SSYAMSWVRQAPGKGLEWVSAISGSGGS TYYGDSVKGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCARTRTKRFWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK 16 Light chain of EIVLTQSPGTLSLSPGERATLSCRASQSVS
GFTX1 IgGIL10M1 PGQGTQSENSCTHFPGNLPNMLRDLRD Monomeric
AFSRVKTFFQMKDQLDNLLLKESLLED IL10 grafted
FKGYLGCQALSEMIQFYLEEVMPQAEN into QDPDIKAHVNSLGENLKTLRLRLRRCH CDRL1.
RFLPCENGGGSGGKSKAVEQVKNAFN KLQEKGIYKAMSEFDIFINYIEAYMTM
KIRNSSSYLAWYQQKPGQAPRLLIYGASS RATGIPDRFSGSGSGTDFTLTISRLEPEDFA
VYYCQQYGSSPLTFGGGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPRE
AKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQ
GLSSPVTKSFNRGEC 17 CDRH1 of GFTFSSY GFTX1 IgGIL10M2 (Chothia) 18
CDRH2 of SGSGGS GFTX1 IgGIL10M2 (Chothia) 19 CDRH3 of TRTKRF GFTX1
IgGIL10M2 (Chothia) 20 CDRL1 of SQSVSSSY GFTX1 IgGIL10M2 (Chothia)
21 CDRL2 of GASSSPGQGTQSENSCTHFPGNLPNML GFTX1 IgGIL10M2
RDLRDAFSRVKTFFQMKDQLDNLLLK Monomeric (Chothia)
ESLLEDFKGYLGCQALSEMIQFYLEEV IL10 grafted
MPQAENQDPDIKAHVNSLGENLKTLRL into RLRRCHRFLPCENGGGSGGKSKAVEQ CDRL2.
VKNAFNKLQEKGIYKAMSEFDIFINYIE IL10 is AYMTMKIRN bolded, underlined
22 CDRL3 of YGSSPL GFTX1 IgGIL10M2 (Chothia) 23 CDRH1 of SYAMS
GFTX1 IgGIL10M2 (Kabat) 24 CDRH2 of AISGSGGSTYYGDSVKG GFTX1
IgGIL10M2 (Kabat) 25 CDRH3 of TRTKRF GFTX1 IgGIL10M2 (Kabat) 26
CDRL1 of RASQSVSSSYLA GFTX1 IgGIL10M2 (Kabat) 27 CDRL2 of
GASSSPGQGTQSENSCTHFPGNLPNML GFTX1 IgGIL10M2 (Kabat)
RDLRDAFSRVKTFFQMKDQLDNLLLK Monomeric ESLLEDFKGYLGCQALSEMIQFYLEEV
IL10 grafted MPQAENQDPDIKAHVNSLGENLKTLRL into
RLRRCHRFLPCENGGGSGGKSKAVEQ CDRL2. VKNAFNKLQEKGIYKAMSEFDIFINYIE IL10
is AYMTMKIRNRAT bolded, underlined 28 CDRL3 of QQYGSSPLT GFTX1
IgGIL10M2 (Kabat) 29 VH of IgGIL10M2 EVQLLESGGGLVQPGGSLRLSCAASGFTF
GFTX1 SSYAMSWVRQAPGKGLEWVSAISGSGGS TYYGDSVKGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCARTRTKRFWGQGTLVT VSS 30 VL of IgGIL10M2
EIVLTQSPGTLSLSPGERATLSCRASQSVS GFTX1 SSYLAWYQQKPGQAPRLLIYGASSSPGQ
Monomeric GTQSENSCTHFPGNLPNMLRDLRDAFS IL10 grafted
RVKTFFQMKDQLDNLLLKESLLEDFK into GYLGCQALSEMIQFYLEEVMPQAENQ CDRL2.
DPDIKAHVNSLGENLKTLRLRLRRCHR FLPCENGGGSGGKSKAVEQVKNAFNK
LQEKGIYKAMSEFDIFINYIEAYMTMKI RNRATGIPDRFSGSGSGTDFTLTISRLEPE
DFAVYYCQQYGSSPLTFGGGTKVEIK 31 Heavy chain of
EVQLLESGGGLVQPGGSLRLSCAASGFTF GFTX1 IgGIL10M2
SSYAMSWVRQAPGKGLEWVSAISGSGGS TYYGDSVKGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCARTRTKRFWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK 32 Light chain of EIVLTQSPGTLSLSPGERATLSCRASQSVS
GFTX1 IgGIL10M2 SSYLAWYQQKPGQAPRLLIYGASSSPGQ Monomeric
GTQSENSCTHFPGNLPNMLRDLRDAFS IL10 grafted RVKTFFQMKDQLDNLLLKESLLEDFK
into GYLGCQALSEMIQFYLEEVMPQAENQ CDRL2. DPDIKAHVNSLGENLKTLRLRLRRCHR
IL10 is FLPCENGGGSGGKSKAVEQVKNAFNK bolded,
LQEKGIYKAMSEFDIFINYIEAYMTMKI underlined
RNRATGIPDRFSGSGSGTDFTLTISRLEPE DFAVYYCQQYGSSPLTFGGGTKVEIKRTV
AAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC 33 CDRH1 of GFTFSSY
GFTX1 IgGIL10M3 (Chothia) 34 CDRH2 of SGSGGS GFTX1 IgGIL10M3
(Chothia) 35 CDRH3 of TRTKRF GFTX1 IgGIL10M3 (Chothia) 36 CDRL1 of
SQSVSSSY GFTX1 IgGIL10M3 (Chothia) 37 CDRL2 of GAS GFTX1 IgGIL10M3
(Chothia) 38 CDRL3 of YGSSPGQGTQSENSCTHFPGNLPNMLR GFTX1 IgGIL10M3
DLRDAFSRVKTFFQMKDQLDNLLLKES Monomeric (Chothia)
LLEDFKGYLGCQALSEMIQFYLEEVMP IL10 grafted
QAENQDPDIKAHVNSLGENLKTLRLRL into RRCHRFLPCENGGGSGGKSKAVEQVK CDRL3.
NAFNKLQEKGIYKAMSEFDIFINYIEAY IL10 is MTMKIRNSPL bolded, underlined
39 CDRH1 of SYAMS GFTX1 IgGIL10M3 (Kabat) 40 CDRH2 of
AISGSGGSTYYGDSVKG GFTX1 IgGIL10M3 (Kabat) 41 CDRH3 of TRTKRF GFTX1
IgGIL10M3 (Kabat) 42 CDRL1 of RASQSVSSSYLA GFTX1 IgGIL10M3
(Kabat)
43 CDRL2 of GASSRAT GFTX1 IgGIL10M3 (Kabat) 44 CDRL3 of
QQYGSSPGQGTQSENSCTHFPGNLPNM GFTX1 IgGIL10M3 (Kabat)
LRDLRDAFSRVKTFFQMKDQLDNLLL Monomeric KESLLEDFKGYLGCQALSEMIQFYLEE
IL10 grafted VMPQAENQDPDIKAHVNSLGENLKTLR into
LRLRRCHRFLPCENGGGSGGKSKAVE CDRL3. QVKNAFNKLQEKGIYKAMSEFDIFINYI IL10
is EAYMTMKIRNSPLT bolded, underlined 45 VH of IgGIL10M3
EVQLLESGGGLVQPGGSLRLSCAASGFTF GFTX1 SSYAMSWVRQAPGKGLEWVSAISGSGGS
TYYGDSVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCARTRTKRFWGQGTLVT VSS 46 VL
of IgGIL10M3 EIVLTQSPGTLSLSPGERATLSCRASQSVS GFTX1
SSYLAWYQQKPGQAPRLLIYGASSRATGI Monomeric
PDRFSGSGSGTDFTLTISRLEPEDFAVYYC IL10 grafted
QQYGSSPGQGTQSENSCTHFPGNLPNM into LRDLRDAFSRVKTFFQMKDQLDNLLL CDRL3.
KESLLEDFKGYLGCQALSEMIQFYLEE IL10 is VMPQAENQDPDIKAHVNSLGENLKTLR
bolded, LRLRRCHRFLPCENGGGSGGKSKAVE underlined
QVKNAFNKLQEKGIYKAMSEFDIFINYI EAYMTMKIRNSPLTFGGGTKVEIK 47 Heavy
chain of EVQLLESGGGLVQPGGSLRLSCAASGFTF GFTX1 IgGIL10M3
SSYAMSWVRQAPGKGLEWVSAISGSGGS TYYGDSVKGRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCARTRTKRFWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK 48 Light chain of EIVLTQSPGTLSLSPGERATLSCRASQSVS
GFTX1 IgGIL10M3 SSYLAWYQQKPGQAPRLLIYGASSRATGI Monomeric
PDRFSGSGSGTDFTLTISRLEPEDFAVYYC IL10 grafted
QQYGSSPGQGTQSENSCTHFPGNLPNM into LRDLRDAFSRVKTFFQMKDQLDNLLL CDRL3.
KESLLEDFKGYLGCQALSEMIQFYLEE VMPQAENQDPDIKAHVNSLGENLKTLR
LRLRRCHRFLPCENGGGSGGKSKAVE QVKNAFNKLQEKGIYKAMSEFDIFINYI
EAYMTMKIRNSPLTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYP
REAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVT
HQGLSSPVTKSFNRGEC 49 CDRH1 of GFTFSPGQGTQSENSCTHFPGNLPNML GFTX1
IgGIL10M4 RDLRDAFSRVKTFFQMKDQLDNLLLK Monomeric (Chothia)
ESLLEDFKGYLGCQALSEMIQFYLEEV IL10 grafted
MPQAENQDPDIKAHVNSLGENLKTLRL into RLRRCHRFLPCENGGGSGGKSKAVEQ CDRH1.
VKNAFNKLQEKGIYKAMSEFDIFINYIE IL10 is AYMTMKIRNSSY bolded,
underlined 50 CDRH2 of SGSGGS GFTX1 IgGIL10M4 (Chothia) 51 CDRH3 of
TRTKRF GFTX1 IgGIL10M4 (Chothia) 52 CDRL1 of SQSVSSSY GFTX1
IgGIL10M4 (Chothia) 53 CDRL2 of GAS GFTX1 IgGIL10M4 (Chothia) 54
CDRL3 of YGSSPL GFTX1 IgGIL10M4 (Chothia) 55 CDRH1 of
SPGQGTQSENSCTHFPGNLPNMLRDLR GFTX1 IgGIL10M4 (Kabat)
DAFSRVKTFFQMKDQLDNLLLKESLLE Monomeric DFKGYLGCQALSEMIQFYLEEVMPQAE
IL10 grafted NQDPDIKAHVNSLGENLKTLRLRLRRC into
HRFLPCENGGGSGGKSKAVEQVKNAF CDRH1. NKLQEKGIYKAMSEFDIFINYIEAYMT IL10
is MKIRNSSYAMS bolded, underlined 56 CDRH2 of AISGSGGSTYYGDSVKG
GFTX1 IgGIL10M4 (Kabat) 57 CDRH3 of TRTKRF GFTX1 IgGIL10M4 (Kabat)
58 CDRL1 of RASQSVSSSYLA GFTX1 IgGIL10M4 (Kabat) 59 CDRL2 of
GASSRAT GFTX1 IgGIL10M4 (Kabat) 60 CDRL3 of QQYGSSPLT GFTX1
IgGIL10M4 (Kabat) 61 VH of IgGIL10M4 EVQLLESGGGLVQPGGSLRLSCAASGFTF
GFTX1 SPGQGTQSENSCTHFPGNLPNMLRDLR Monomeric
DAFSRVKTFFQMKDQLDNLLLKESLLE IL10 grafted
DFKGYLGCQALSEMIQFYLEEVMPQAE into CDRH1 NQDPDIKAHVNSLGENLKTLRLRLRRC
HRFLPCENGGGSGGKSKAVEQVKNAF NKLQEKGIYKAMSEFDIFINYIEAYMT
MKIRNSSYAMSWVRQAPGKGLEWVSAI SGSGGSTYYGDSVKGRFTISRDNSKNTLY
LQMNSLRAEDTAVYYCARTRTKRFWGQ GTLVTVSS 62 VL of IgGIL10M4
EIVLTQSPGTLSLSPGERATLSCRASQSVS GFTX1 SSYLAWYQQKPGQAPRLLIYGASSRATGI
PDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPLTFGGGTKVEIK 63 Heavy chain
of EVQLLESGGGLVQPGGSLRLSCAASGFTF GFTX1 IgGIL10M4
SPGQGTQSENSCTHFPGNLPNMLRDLR Monomeric DAFSRVKTFFQMKDQLDNLLLKESLLE
IL10 grafted DFKGYLGCQALSEMIQFYLEEVMPQAE into CDRH1
NQDPDIKAHVNSLGENLKTLRLRLRRC HRFLPCENGGGSGGKSKAVEQVKNAF
NKLQEKGIYKAMSEFDIFINYIEAYMT MKIRNSSYAMSWVRQAPGKGLEWVSAI
SGSGGSTYYGDSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCARTRTKRFWGQ
GTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 64 Light chain
of EIVLTQSPGTLSLSPGERATLSCRASQSVS GFTX1 IgGIL10M4
SSYLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAVYYC
QQYGSSPLTFGGGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC 65 CDRH1 of GFTFSSY GFTX1 IgGIL10M5 (Chothia) 66 CDRH2
of SGSGSPGQGTQSENSCTHFPGNLPNML GFTX1 IgGIL10M5
RDLRDAFSRVKTFFQMKDQLDNLLLK Monomeric (Chothia)
ESLLEDFKGYLGCQALSEMIQFYLEEV IL10 grafted
MPQAENQDPDIKAHVNSLGENLKTLRL into RLRRCHRFLPCENGGGSGGKSKAVEQ CDRH2.
VKNAFNKLQEKGIYKAMSEFDIFINYIE IL10 is AYMTMKIRNGS bolded, underlined
67 CDRH3 of TRTKRF GFTX1 IgGIL10M5 (Chothia) 68 CDRL1 of SQSVSSSY
GFTX1 IgGIL10M5 (Chothia) 69 CDRL2 of GAS GFTX1 IgGIL10M5 (Chothia)
70 CDRL3 of YGSSPL GFTX1 IgGIL10M5 (Chothia) 71 CDRH1 of SYAMS
GFTX1 IgGIL10M5 (Kabat) 72 CDRH2 of AISGSGSPGQGTQSENSCTHFPGNLPNM
GFTX1 IgGIL10M5 (Kabat) LRDLRDAFSRVKTFFQMKDQLDNLLL Monomeric
KESLLEDFKGYLGCQALSEMIQFYLEE IL10 grafted
VMPQAENQDPDIKAHVNSLGENLKTLR into LRLRRCHRFLPCENGGGSGGKSKAVE CDRH2.
QVKNAFNKLQEKGIYKAMSEFDIFINYI IL10 is EAYMTMKIRNGSTYYGDSVKG bolded,
underlined 73 CDRH3 of TRTKRF GFTX1 IgGIL10M5 (Kabat) 74 CDRL1 of
RASQSVSSSYLA GFTX1 IgGIL10M5 (Kabat) 75 CDRL2 of GASSRAT GFTX1
IgGIL10M5 (Kabat) 76 CDRL3 of QQYGSSPLT GFTX1 IgGIL10M5 (Kabat) 77
VH of IgGIL10M5 EVQLLESGGGLVQPGGSLRLSCAASGFTF GFTX1
SSYAMSWVRQAPGKGLEWVSAISGSGSP Monomeric GQGTQSENSCTHFPGNLPNMLRDLRDA
FSRVKTFFQMKDQLDNLLLKESLLEDF KGYLGCQALSEMIQFYLEEVMPQAEN
QDPDIKAHVNSLGENLKTLRLRLRRCH IL10 grafted RFLPCENGGGSGGKSKAVEQVKNAFN
into CDRH2 KLQEKGIYKAMSEFDIFINYIEAYMTM
KIRNGSTYYGDSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCARTRTKRFWGQ GTLVTVSS
78 VL of IgGIL10M5 EIVLTQSPGTLSLSPGERATLSCRASQSVS GFTX1
SSYLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAVYYC
QQYGSSPLTFGGGTKVEIK 79 Heavy chain of EVQLLESGGGLVQPGGSLRLSCAASGFTF
GFTX1 IgGIL10M5 SSYAMSWVRQAPGKGLEWVSAISGSGSP Monomeric
GQGTQSENSCTHFPGNLPNMLRDLRDA IL10 grafted
FSRVKTFFQMKDQLDNLLLKESLLEDF into CDRH2 KGYLGCQALSEMIQFYLEEVMPQAEN
QDPDIKAHVNSLGENLKTLRLRLRRCH RFLPCENGGGSGGKSKAVEQVKNAFN
KLQEKGIYKAMSEFDIFINYIEAYMTM KIRNGSTYYGDSVKGRFTISRDNSKNTLY
LQMNSLRAEDTAVYYCARTRTKRFWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 80 Light chain
of EIVLTQSPGTLSLSPGERATLSCRASQSVS GFTX1 IgGIL10M5
SSYLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAVYYC
QQYGSSPLTFGGGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC 81 CDRH1 of GFTFSSY GFTX1 IgGIL10M6 (Chothia) 82 CDRH2
of SGSGGS GFTX1 IgGIL10M6 (Chothia) 83 CDRH3 of
TRTSPGQGTQSENSCTHFPGNLPNMLR GFTX1 IgGIL10M6
DLRDAFSRVKTFFQMKDQLDNLLLKES Monomeric (Chothia)
LLEDFKGYLGCQALSEMIQFYLEEVMP IL10 grafted
QAENQDPDIKAHVNSLGENLKTLRLRL into RRCHRFLPCENGGGSGGKSKAVEQVK CDRH3.
NAFNKLQEKGIYKAMSEFDIFINYIEAY IL10 is MTMKIRNKRF bolded, underlined
84 CDRL1 of SQSVSSSY GFTX1 IgGIL10M6 (Chothia) 85 CDRL2 of GAS
GFTX1 IgGIL10M6 (Chothia) 86 CDRL3 of YGSSPL GFTX1 IgGIL10M6
(Chothia) 87 CDRH1 of SYAMS GFTX1 IgGIL10M6 (Kabat) 88 CDRH2 of
AISGSGGSTYYGDSVKG GFTX1 IgGIL10M6 (Kabat) 89 CDRH3 of
TRTSPGQGTQSENSCTHFPGNLPNMLR GFTX1 IgGIL10M6 (Kabat)
DLRDAFSRVKTFFQMKDQLDNLLLKES Monomeric LLEDFKGYLGCQALSEMIQFYLEEVMP
IL10 grafted QAENQDPDIKAHVNSLGENLKTLRLRL into
RRCHRFLPCENGGGSGGKSKAVEQVK CDRH3. NAFNKLQEKGIYKAMSEFDIFINYIEAY IL10
is MTMKIRNKRF bolded, underlined 90 CDRL1 of RASQSVSSSYLA GFTX1
IgGIL10M6 (Kabat) 91 CDRL2 of GASSRAT GFTX1 IgGIL10M6 (Kabat) 92
CDRL3 of QQYGSSPLT GFTX1 IgGIL10M6 (Kabat) 93 VH of IgGIL10M6
EVQLLESGGGLVQPGGSLRLSCAASGFTF GFTX1 SSYAMSWVRQAPGKGLEWVSAISGSGGS
Monomeric TYYGDSVKGRFTISRDNSKNTLYLQMNS IL10 grafted
LRAEDTAVYYCARTRTSPGQGTQSENSC into CDRH3 THFPGNLPNMLRDLRDAFSRVKTFFQM
KDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVNS
LGENLKTLRLRLRRCHRFLPCENGGGS GGKSKAVEQVKNAFNKLQEKGIYKAM
SEFDIFINYIEAYMTMKIRNKRFWGQGT LVTVSS 94 VL of IgGIL10M6
EIVLTQSPGTLSLSPGERATLSCRASQSVS GFTX1 SSYLAWYQQKPGQAPRLLIYGASSRATGI
PDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPLTFGGGTKVEIK 95 Heavy chain
of EVQLLESGGGLVQPGGSLRLSCAASGFTF GFTX1 IgGIL10M6
SSYAMSWVRQAPGKGLEWVSAISGSGGS Monomeric TYYGDSVKGRFTISRDNSKNTLYLQMNS
IL10 grafted LRAEDTAVYYCARTRTSPGQGTQSENSC into CDRH3
THFPGNLPNMLRDLRDAFSRVKTFFQM KDQLDNLLLKESLLEDFKGYLGCQALS
EMIQFYLEEVMPQAENQDPDIKAHVNS LGENLKTLRLRLRRCHRFLPCENGGGS
GGKSKAVEQVKNAFNKLQEKGIYKAM SEFDIFINYIEAYMTMKIRNKRFWGQGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 96 Light chain
of EIVLTQSPGTLSLSPGERATLSCRASQSVS GFTX1 IgGIL10M6
SSYLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAVYYC
QQYGSSPLTFGGGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC 97 CDRH1 of GFSLSTSGM GFTX3 IgGIL10M7 (Chothia) 98 CDRH2
of WWDDK GFTX3 IgGIL10M7 (Chothia) 99 CDRH3 of SMITNWYFDV GFTX3
IgGIL10M7 (Chothia) 100 CDRL1 of QLSSPGQGTQSENSCTHFPGNLPNMLR
Monomeric IgGIL10M7 DLRDAFSRVKTFFQMKDQLDNLLLKES IL10 grafted
(Chothia) LLEDFKGYLGCQALSEMIQFYLEEVMP into
QAENQDPDIKAHVNSLGENLKTLRLRL CDRL1. RRCHRFLPCENGGGSGGKSKAVEQVK IL10
is NAFNKLQEKGIYKAMSEFDIFINYIEAY bolded, MTMKIRNVGY underlined 101
CDRL2 of DTS GFTX3 IgGIL10M7 (Chothia) 102 CDRL3 of GSGYPF GFTX3
IgGIL10M7 (Chothia) 103 CDRH1 of TSGMSVG GFTX3 IgGIL10M7 (Kabat)
104 CDRH2 of DIWWDDKKDYNPSLKS GFTX3 IgGIL10M7 (Kabat) 105 CDRH3 of
SMITNWYFDV GFTX3 IgGIL10M7 (Kabat) 106 CDRL1 of
KAQLSSPGQGTQSENSCTHFPGNLPNM Monomeric IgGIL10M7 (Kabat)
LRDLRDAFSRVKTFFQMKDQLDNLLL IL10 grafted KESLLEDFKGYLGCQALSEMIQFYLEE
into VMPQAENQDPDIKAHVNSLGENLKTLR CDRL1. LRLRRCHRFLPCENGGGSGGKSKAVE
IL10 is QVKNAFNKLQEKGIYKAMSEFDIFINYI bolded, EAYMTMKIRNVGYMH
underlined 107 CDRL2 of DTSKLAS GFTX3 IgGIL10M7 (Kabat) 108 CDRL3
of FQGSGYPFT GFTX3 IgGIL10M7 (Kabat) 109 VH of IgGIL10M7
QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 TSGMSVGWIRQPPGKALEWLADIWWDD
KKDYNPSLKSRLTISKDTSANQVVLKVTN MDPADTATYYCARSMITNWYFDVWGAG TTVTVSS
110 VL of IgGIL10M7 DIQMTQSPSTLSASVGDRVTITCKAQLSSP Monomeric
GQGTQSENSCTHFPGNLPNMLRDLRDA IL10 grafted
FSRVKTFFQMKDQLDNLLLKESLLEDF into KGYLGCQALSEMIQFYLEEVMPQAEN CDRL1.
QDPDIKAHVNSLGENLKTLRLRLRRCH RFLPCENGGGSGGKSKAVEQVKNAFN
KLQEKGIYKAMSEFDIFINYIEAYMTM KIRNVGYMHWYQQKPGKAPKLLIYDTS
KLASGVPSRFSGSGSGTAFTLTISSLQPDD FATYYCFQGSGYPFTFGGGTKLEIK 111 Heavy
chain of QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 IgGIL10M7
TSGMSVGWIRQPPGKALEWLADIWWDD KKDYNPSLKSRLTISKDTSANQVVLKVTN
MDPADTATYYCARSMITNWYFDVWGAG TTVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK 112 Light chain of
DIQMTQSPSTLSASVGDRVTITCKAQLSSP Monomeric IgGIL10M7
GQGTQSENSCTHFPGNLPNMLRDLRDA IL10 grafted
FSRVKTFFQMKDQLDNLLLKESLLEDF into KGYLGCQALSEMIQFYLEEVMPQAEN CDRL1.
QDPDIKAHVNSLGENLKTLRLRLRRCH RFLPCENGGGSGGKSKAVECIVKNAFN
KLQEKGIYKAMSEFDIFINYIEAYMTM KIRNVGYMHWYQQKPGKAPKLLIYDTS
KLASGVPSRFSGSGSGTAFTLTISSLQPDD FATYYCFQGSGYPFTFGGGTKLEIKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC 113 CDRH1 of
GFSLSTSGM GFTX3 IgGIL10M8 (Chothia) 114 CDRH2 of WWDDK GFTX3
IgGIL10M8 (Chothia) 115 CDRH3 of SMITNWYFDV GFTX3 IgGIL10M8
(Chothia) 116 CDRL1 of QLSVGY GFTX3 IgGIL10M8 (Chothia) 117 CDRL2
of DTSPGQGTQSENSCTHFPGNLPNMLRD GFTX3 IgGIL10M8
LRDAFSRVKTFFQMKDQLDNLLLKESL Monomeric (Chothia)
LEDFKGYLGCQALSEMIQFYLEEVMPQ IL10 grafted
AENQDPDIKAHVNSLGENLKTLRLRLR into RCHRFLPCENGGGSGGKSKAVEQVKN CDRL2.
AFNKLQEKGIYKAMSEFDIFINYIEAYM IL10 is TMKIRNS bolded, underlined 118
CDRL3 of GSGYPF GFTX3 IgGIL10M8 (Chothia) 119 CDRH1 of TSGMSVG
GFTX3 IgGIL10M8 (Kabat) 120 CDRH2 of DIWWDDKKDYNPSLKS GFTX3
IgGIL10M8 (Kabat)
121 CDRH3 of SMITNWYFDV GFTX3 IgGIL10M8 (Kabat) 122 CDRL1 of
KAQLSVGYMH GFTX3 IgGIL10M8 (Kabat) 123 CDRL2 of
DTSPGQGTQSENSCTHFPGNLPNMLRD GFTX3 IgGIL10M8 (Kabat)
LRDAFSRVKTFFQMKDQLDNLLLKESL Monomeric LEDFKGYLGCQALSEMIQFYLEEVMPQ
IL10 grafted AENQDPDIKAHVNSLGENLKTLRLRLR into
RCHRFLPCENGGGSGGKSKAVEQVKN CDRL2. AFNKLQEKGIYKAMSEFDIFINYIEAYM IL10
is TMKIRNSKLAS bolded, underlined 124 CDRL3 of FQGSGYPFT GFTX3
IgGIL10M8 (Kabat) 125 VH of IgGIL10M8
QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 TSGMSVGWIRQPPGKALEWLADIWWDD
KKDYNPSLKSRLTISKDTSANQVVLKVTN MDPADTATYYCARSMITNWYFDVWGAG TTVTVSS
126 VL of IgGIL10M8 DIQMTQSPSTLSASVGDRVTITCKAQLSV GFTX3
GYMHWYQQKPGKAPKLLIYDTSPGQGT Monomeric QSENSCTHFPGNLPNMLRDLRDAFSRV
IL10 grafted KTFFQMKDQLDNLLLKESLLEDFKGYL into
GCQALSEMIQFYLEEVMPQAENQDPDI CDRL2. KAHVNSLGENLKTLRLRLRRCHRFLPC
ENGGGSGGKSKAVEQVKNAFNKLQEK GIYKAMSEFDIFINYIEAYMTMKIRNSK
LASGVPSRFSGSGSGTAFTLTISSLQPDDF ATYYCFQGSGYPFTFGGGTKLEIK 127 Heavy
chain of QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 IgGIL10M8
TSGMSVGWIRQPPGKALEWLADIWWDD KKDYNPSLKSRLTISKDTSANQVVLKVTN
MDPADTATYYCARSMITNWYFDVWGAG TTVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK 128 Light chain of
DIQMTQSPSTLSASVGDRVTITCKAQLSV GFTX3 IgGIL10M8
GYMHWYQQKPGKAPKLLIYDTSPGQGT Monomeric QSENSCTHFPGNLPNMLRDLRDAFSRV
IL10 grafted KTFFQMKDQLDNLLLKESLLEDFKGYL into
GCQALSEMIQFYLEEVMPQAENQDPDI CDRL2. KAHVNSLGENLKTLRLRLRRCHRFLPC
ENGGGSGGKSKAVEQVKNAFNKLQEK GIYKAMSEFDIFINYIEAYMTMKIRNSK
LASGVPSRFSGSGSGTAFTLTISSLQPDDF ATYYCFQGSGYPFTFGGGTKLEIKRTVAA
PSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC 129 CDRH1 of
GFSLSTSGM GFTX3 IgGIL10M9 (Chothia) 130 CDRH2 of WWDDK GFTX3
IgGIL10M9 (Chothia) 131 CDRH3 of SMITNWYFDV GFTX3 IgGIL10M9
(Chothia) 132 CDRL1 of QLSVGY GFTX3 IgGIL10M9 (Chothia) 133 CDRL2
of DTS GFTX3 IgGIL10M9 (Chothia) 134 CDRL3 of
GSGSPGQGTQSENSCTHFPGNLPNMLR GFTX3 IgGIL10M9
DLRDAFSRVKTFFQMKDQLDNLLLKES Monomeric (Chothia)
LLEDFKGYLGCQALSEMIQFYLEEVMP IL10 grafted
QAENQDPDIKAHVNSLGENLKTLRLRL into RRCHRFLPCENGGGSGGKSKAVEQVK CDRL3.
NAFNKLQEKGIYKAMSEFDIFINYIEAY IL10 is MTMKIRNYPF bolded, underlined
135 CDRH1 of TSGMSVG GFTX3 IgGIL10M9 (Kabat) 136 CDRH2 of
DIWWDDKKDYNPSLKS GFTX3 IgGIL10M9 (Kabat) 137 CDRH3 of SMITNWYFDV
GFTX3 IgGIL10M9 (Kabat) 138 CDRL1 of KAQLSVGYMH GFTX3 IgGIL10M9
(Kabat) 139 CDRL2 of DTSKLAS GFTX3 IgGIL10M9 (Kabat) 140 CDRL3 of
FQGSGSPGQGTQSENSCTHFPGNLPNM GFTX3 IgGIL10M9 (Kabat)
LRDLRDAFSRVKTFFQMKDQLDNLLL Monomeric KESLLEDFKGYLGCQALSEMIQFYLEE
IL10 grafted VMPQAENQDPDIKAHVNSLGENLKTLR into
LRLRRCHRFLPCENGGGSGGKSKAVE CDRL3. QVKNAFNKLQEKGIYKAMSEFDIFINYI IL10
is EAYMTMKIRNYPFT bolded, underlined 141 VH of IgGIL10M9
QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 TSGMSVGWIRQPPGKALEWLADIWWDD
KKDYNPSLKSRLTISKDTSANQVVLKVTN MDPADTATYYCARSMITNWYFDVWGAG TTVTVSS
142 VL of IgGIL10M9 DIQMTQSPSTLSASVGDRVTITCKAQLSV GFTX3
GYMHWYQQKPGKAPKLLIYDTSKLASG Monomeric
VPSRFSGSGSGTAFTLTISSLQPDDFATYY IL10 grafted
CFQGSGSPGQGTQSENSCTHFPGNLPN into MLRDLRDAFSRVKTFFQMKDQLDNLL CDRL3.
LKESLLEDFKGYLGCQALSEMIQFYLE EVMPQAENQDPDIKAHVNSLGENLKTL
RLRLRRCHRFLPCENGGGSGGKSKAV EQVKNAFNKLQEKGIYKAMSEFDIFIN
YIEAYMTMKIRNYPFTFGGGTKLEIK 143 Heavy chain of
QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 IgGIL10M9
TSGMSVGWIRQPPGKALEWLADIWWDD KKDYNPSLKSRLTISKDTSANQVVLKVTN
MDPADTATYYCARSMITNWYFDVWGAG TTVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK 144 Light chain of
DIQMTQSPSTLSASVGDRVTITCKAQLSV GFTX3 IgGIL10M9
GYMHWYQQKPGKAPKLLIYDTSKLASG Monomeric
VPSRFSGSGSGTAFTLTISSLQPDDFATYY IL10 grafted
CFQGSGSPGQGTQSENSCTHFPGNLPN into MLRDLRDAFSRVKTFFQMKDQLDNLL CDRL3.
LKESLLEDFKGYLGCQALSEMIQFYLE EVMPQAENQDPDIKAHVNSLGENLKTL
RLRLRRCHRFLPCENGGGSGGKSKAV EQVKNAFNKLQEKGIYKAMSEFDIFIN
YIEAYMTMKIRNYPFTFGGGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNF
YPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC 145 CDRH1 of GFSLSPGQGTQSENSCTHFPGNLPNML GFTX3
IgGIL10M10 RDLRDAFSRVKTFFQMKDQLDNLLLK Monomeric (Chothia)
ESLLEDFKGYLGCQALSEMIQFYLEEV IL10 grafted
MPQAENQDPDIKAHVNSLGENLKTLRL into RLRRCHRFLPCENGGGSGGKSKAVEQ CDRH1.
VKNAFNKLQEKGIYKAMSEFDIFINYIE IL10 is AYMTMKIRNSTSGM bolded,
underlined 146 CDRH2 of WWDDK GFTX3 IgGIL10M10 (Chothia) 147 CDRH3
of SMITNWYFDV GFTX3 IgGIL10M10 (Chothia) 148 CDRL1 of QLSVGY GFTX3
IgGIL10M10 (Chothia) 149 CDRL2 of DTS GFTX3 IgGIL10M10 (Chothia)
150 CDRL3 of GSGYPF GFTX3 IgGIL10M10 (Chothia) 151 CDRH1 of
SPGQGTQSENSCTHFPGNLPNMLRDLR GFTX3 IgGIL10M10 (Kabat)
DAFSRVKTFFQMKDQLDNLLLKESLLE Monomeric DFKGYLGCQALSEMIQFYLEEVMPQAE
IL10 grafted NQDPDIKAHVNSLGENLKTLRLRLRRC into
HRFLPCENGGGSGGKSKAVEQVKNAF CDRH1. NKLQEKGIYKAMSEFDIFINYIEAYMT IL10
is MKIRNSTSGMSVG bolded, underlined 152 CDRH2 of DIWWDDKKDYNPSLKS
GFTX3 IgGIL10M10 (Kabat) 153 CDRH3 of SMITNWYFDV GFTX3 IgGIL10M10
(Kabat) 154 CDRL1 of KAQLSVGYMH GFTX3 IgGIL10M10 (Kabat) 155 CDRL2
of DTSKLAS GFTX3 IgGIL10M10 (Kabat) 156 CDRL3 of FQGSGYPFT GFTX3
IgGIL10M10 (Kabat) 157 VH of IgGIL10M10
QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 PGQGTQSENSCTHFPGNLPNMLRDLRD
Monomeric AFSRVKTFFQMKDQLDNLLLKESLLED IL10 grafted
FKGYLGCQALSEMIQFYLEEVMPQAEN into CDRH1 QDPDIKAHVNSLGENLKTLRLRLRRCH
RFLPCENGGGSGGKSKAVEQVKNAFN KLQEKGIYKAMSEFDIFINYIEAYMTM
KIRNSTSGMSVGWIRQPPGKALEWLADI WWDDKKDYNPSLKSRLTISKDTSANQVV
LKVTNMDPADTATYYCARSMITNWYFD VWGAGTTVTVSS 158 VL of IgGIL10M10
DIQMTQSPSTLSASVGDRVTITCKAQLSV GFTX3 GYMHWYQQKPGKAPKLLIYDTSKLASG
VPSRFSGSGSGTAFTLTISSLQPDDFATYY CFQGSGYPFTFGGGTKLEIK 159 Heavy chain
of QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 IgGIL10M10
PGQGTQSENSCTHFPGNLPNMLRDLRD Monomeric AFSRVKTFFQMKDQLDNLLLKESLLED
IL10 grafted FKGYLGCQALSEMIQFYLEEVMPQAEN into CDRH1
QDPDIKAHVNSLGENLKTLRLRLRRCH RFLPCENGGGSGGKSKAVEQVKNAFN
KLQEKGIYKAMSEFDIFINYIEAYMTM KIRNSTSGMSVGWIRQPPGKALEWLADI
WWDDKKDYNPSLKSRLTISKDTSANQVV LKVTNMDPADTATYYCARSMITNWYFD
VWGAGTTVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEK
TISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK 160 Light
chain of DIQMTQSPSTLSASVGDRVTITCKAQLSV GFTX3 IgGIL10M10
GYMHWYQQKPGKAPKLLIYDTSKLASG VPSRFSGSGSGTAFTLTISSLQPDDFATYY
CFQGSGYPFTFGGGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC 161 CDRH1 of GFSLSTSGM GFTX3 IgGIL10M11 (Chothia) 162
CDRH2 of WWDSPGQGTQSENSCTHFPGNLPNML GFTX3 IgGIL10M11
RDLRDAFSRVKTFFQMKDQLDNLLLK Monomeric (Chothia)
ESLLEDFKGYLGCQALSEMIQFYLEEV IL10 grafted
MPQAENQDPDIKAHVNSLGENLKTLRL into RLRRCHRFLPCENGGGSGGKSKAVEQ CDRH2.
VKNAFNKLQEKGIYKAMSEFDIFINYIE IL10 is AYMTMKIRNDK bolded, underlined
163 CDRH3 of SMITNWYFDV GFTX3 IgGIL10M11 (Chothia) 164 CDRL1 of
QLSVGY GFTX3 IgGIL10M11 (Chothia) 165 CDRL2 of DTS GFTX3 IgGIL10M11
(Chothia) 166 CDRL3 of GSGYPF GFTX3 IgGIL10M11 (Chothia) 167 CDRH1
of TSGMSVG GFTX3 IgGIL10M11 (Kabat) 168 CDRH2 of
DIWWDSPGQGTQSENSCTHFPGNLPNM GFTX3 IgGIL10M11 (Kabat)
LRDLRDAFSRVKTFFQMKDQLDNLLL Monomeric KESLLEDFKGYLGCQALSEMIQFYLEE
IL10 grafted VMPQAENQDPDIKAHVNSLGENLKTLR into
LRLRRCHRFLPCENGGGSGGKSKAVE CDRH2. QVKNAFNKLQEKGIYKAMSEFDIFINYI IL10
is EAYMTMKIRNDKKDYNPSLKS bolded, underlined 169 CDRH3 of SMITNWYFDV
GFTX3 IgGIL10M11 (Kabat) 170 CDRL1 of KAQLSVGYMH GFTX3 IgGIL10M11
(Kabat) 171 CDRL2 of DTSKLAS GFTX3 IgGIL10M11 (Kabat) 172 CDRL3 of
FQGSGYPFT GFTX3 IgGIL10M11 (Kabat) 173 VH of IgGIL10M11
QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 TSGMSVGWIRQPPGKALEWLADIWWDSP
Monomeric GQGTQSENSCTHFPGNLPNMLRDLRDA IL10 grafted
FSRVKTFFQMKDQLDNLLLKESLLEDF into CDRH2 KGYLGCQALSEMIQFYLEEVMPQAEN
QDPDIKAHVNSLGENLKTLRLRLRRCH RFLPCENGGGSGGKSKAVEQVKNAFN
KLQEKGIYKAMSEFDIFINYIEAYMTM KIRNDKKDYNPSLKSRLTISKDTSANQVV
LKVTNMDPADTATYYCARSMITNWYFD VWGAGTTVTVSS 174 VL of IgGIL10M11
DIQMTQSPSTLSASVGDRVTITCKAQLSV GFTX3 GYMHWYQQKPGKAPKLLIYDTSKLASG
VPSRFSGSGSGTAFTLTISSLQPDDFATYY CFQGSGYPFTFGGGTKLEIK 175 Heavy chain
of QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 IgGIL10M11
TSGMSVGWIRQPPGKALEWLADIWWDSP Monomeric GQGTQSENSCTHFPGNLPNMLRDLRDA
IL10 grafted FSRVKTFFQMKDQLDNLLLKESLLEDF into CDRH2
KGYLGCQALSEMIQFYLEEVMPQAEN QDPDIKAHVNSLGENLKTLRLRLRRCH
RFLPCENGGGSGGKSKAVEQVKNAFN KLQEKGIYKAMSEFDIFINYIEAYMTM
KIRNDKKDYNPSLKSRLTISKDTSANQVV LKVTNMDPADTATYYCARSMITNWYFD
VWGAGTTVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEK
TISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK 176 Light
chain of DIQMTQSPSTLSASVGDRVTITCKAQLSV GFTX3 IgGIL10M11
GYMHWYQQKPGKAPKLLIYDTSKLASG VPSRFSGSGSGTAFTLTISSLQPDDFATYY
CFQGSGYPFTFGGGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC 177 CDRH1 of GFSLSTSGM GFTX3 IgGIL10M12 (Chothia) 178
CDRH2 of WWDDK GFTX3 IgGIL10M12 (Chothia) 179 CDRH3 of
SMITSPGQGTQSENSCTHFPGNLPNMLR GFTX3 IgGIL10M12
DLRDAFSRVKTFFQMKDQLDNLLLKES Monomeric (Chothia)
LLEDFKGYLGCQALSEMIQFYLEEVMP IL10 grafted
QAENQDPDIKAHVNSLGENLKTLRLRL into RRCHRFLPCENGGGSGGKSKAVEQVK CDRH3.
NAFNKLQEKGIYKAMSEFDIFINYIEAY IL10 is MTMKIRNNWYFDV bolded,
underlined 180 CDRL1 of QLSVGY GFTX3 IgGIL10M12 (Chothia) 181 CDRL2
of DTS GFTX3 IgGIL10M12 (Chothia) 182 CDRL3 of GSGYPF GFTX3
IgGIL10M12 (Chothia) 183 CDRH1 of TSGMSVG GFTX3 IgGIL10M12 (Kabat)
184 CDRH2 of DIWWDDKKDYNPSLKS GFTX3 IgGIL10M12 (Kabat) 185 CDRH3 of
into SMITSPGQGTQSENSCTHFPGNLPNMLR GFTX3 IgGIL10M12 (Kabat)
DLRDAFSRVKTFFQMKDQLDNLLLKES Monomeric LLEDFKGYLGCQALSEMIQFYLEEVMP
IL10 grafted QAENQDPDIKAHVNSLGENLKTLRLRL CDRH3.
RRCHRFLPCENGGGSGGKSKAVEQVK IL10 is NAFNKLQEKGIYKAMSEFDIFINYIEAY
bolded, MTMKIRNNWYFDV underlined 186 CDRL1 of KAQLSVGYMH GFTX3
IgGIL10M12 (Kabat) 187 CDRL2 of DTSKLAS GFTX3 IgGIL10M12 (Kabat)
188 CDRL3 of FQGSGYPFT GFTX3 IgGIL10M12 (Kabat) 189 VH of
IgGIL10M12 QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3
TSGMSVGWIRQPPGKALEWLADIWWDD Monomeric KKDYNPSLKSRLTISKDTSANQVVLKVTN
IL10 grafted MDPADTATYYCARSMITSPGQGTQSENS
CTHFPGNLPNMLRDLRDAFSRVKTFFQ MKDQLDNLLLKESLLEDFKGYLGCQA
LSEMIQFYLEEVMPQAENQDPDIKAHV NSLGENLKTLRLRLRRCHRFLPCENGG into CDRH3
GSGGKSKAVEQVKNAFNKLQEKGIYK AMSEFDIFINYIEAYMTMKIRNNWYFD VWGAGTTVTVSS
190 VL of IgGIL10M12 DIQMTQSPSTLSASVGDRVTITCKAQLSV GFTX3
GYMHWYQQKPGKAPKLLIYDTSKLASG VPSRFSGSGSGTAFTLTISSLQPDDFATYY
CFQGSGYPFTFGGGTKLEIK 191 Heavy chain of
QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 IgGIL10M12
TSGMSVGWIRQPPGKALEWLADIWWDD Monomeric KKDYNPSLKSRLTISKDTSANQVVLKVTN
IL10 grafted MDPADTATYYCARSMITSPGQGTQSENS into CDRH3
CTHFPGNLPNMLRDLRDAFSRVKTFFQ MKDQLDNLLLKESLLEDFKGYLGCQA
LSEMIQFYLEEVMPQAENQDPDIKAHV NSLGENLKTLRLRLRRCHRFLPCENGG
GSGGKSKAVEQVKNAFNKLQEKGIYK AMSEFDIFINYIEAYMTMKIRNNWYFD
VWGAGTTVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEK
TISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK 192 Light
chain of DIQMTQSPSTLSASVGDRVTITCKAQLSV GFTX3 IgGIL10M12
GYMHWYQQKPGKAPKLLIYDTSKLASG VPSRFSGSGSGTAFTLTISSLQPDDFATYY
CFQGSGYPFTFGGGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC 193 CDRH1 of GFSLSTSGM GFTX3 IgGIL10M13 (Chothia) 194
CDRH2 of WWDDK GFTX3 IgGIL10M13 (Chothia) 195 CDRH3 of SMITNWYFDV
GFTX3 IgGIL10M13 (Chothia) 196 CDRL1 of QLSSPGQGTQSENSCTHFPGNLPNMLR
Monomeric IgGIL10M13 DLRDAFSRVKTFFQMKDQLDNLLLKES IL10 grafted
(Chothia) LLEDFKGYLGCQALSEMIQFYLEEVMP into
QAENQDPDIKAHVNSLGENLKTLRLRL CDRL1. RRCHRFLPCENGGGSGGKSKAVEQVK IL10
is NAFNKLQEKGIYKAMSEFDIFINYIEAY bolded, MTMKIRNVGY underlined 197
CDRL2 of DTS GFTX3 IgGIL10M13 (Chothia) 198 CDRL3 of GSGYPF GFTX3
IgGIL10M13 (Chothia)
199 CDRH1 of TSGMSVG GFTX3 IgGIL10M13 (Kabat) 200 CDRH2 of
DIWWDDKKDYNPSLKS GFTX3 IgGIL10M13 (Kabat) 201 CDRH3 of SMITNWYFDV
GFTX3 IgGIL10M13 (Kabat) 202 CDRL1 of KAQLSSPGQGTQSENSCTHFPGNLPNM
Monomeric IgGIL10M13 (Kabat) LRDLRDAFSRVKTFFQMKDQLDNLLL IL10
grafted KESLLEDFKGYLGCQALSEMIQFYLEE into CDRL1
VMPQAENQDPDIKAHVNSLGENLKTLR IL10 is LRLRRCHRFLPCENGGGSGGKSKAVE
bolded, QVKNAFNKLQEKGIYKAMSEFDIFINYI underlined EAYMTMKIRNVGYMH
GFTX3 203 CDRL2 of DTSKLAS GFTX3 IgGIL10M13 (Kabat) 204 CDRL3 of
FQGSGYPFT GFTX3 IgGIL10M13 (Kabat) 205 VH of IgGIL10M13
QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 TSGMSVGWIRQPPGKALEWLADIWWDD
KKDYNPSLKSRLTISKDTSANQVVLKVTN MDPADTATYYCARSMITNWYFDVWGAG TTVTVSS
206 VL of IgGIL10M13 DIQMTQSPSTLSASVGDRVTITCKAQLSSP Monomeric
GQGTQSENSCTHFPGNLPNMLRDLRDA IL10 grafted
FSRVKTFFQMKDQLDNLLLKESLLEDF into CDRL1 KGYLGCQALSEMIQFYLEEVMPQAEN
GFTX3 QDPDIKAHVNSLGENLKTLRLRLRRCH RFLPCENGGGSGGKSKAVECIVKNAFN
KLQEKGIYKAMSEFDIFINYIEAYMTM KIRNVGYMHWYQQKPGKAPKLLIYDTS
KLASGVPSRFSGSGSGTAFTLTISSLQPDD FATYYCFQGSGYPFTFGGGTKLEIK 207 Heavy
chain of QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 IgGIL10M13
TSGMSVGWIRQPPGKALEWLADIWWDD KKDYNPSLKSRLTISKDTSANQVVLKVTN
MDPADTATYYCARSMITNWYFDVWGAG TTVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVVVAVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALAAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK 208 Light chain of
DIQMTQSPSTLSASVGDRVTITCKAQLSSP Monomeric IgGIL10M13
GQGTQSENSCTHFPGNLPNMLRDLRDA IL10 grafted
FSRVKTFFQMKDQLDNLLLKESLLEDF into CDRL1 KGYLGCQALSEMIQFYLEEVMPQAEN
GFTX3 QDPDIKAHVNSLGENLKTLRLRLRRCH RFLPCENGGGSGGKSKAVEQVKNAFN
KLQEKGIYKAMSEFDIFINYIEAYMTM KIRNVGYMHWYQQKPGKAPKLLIYDTS
KLASGVPSRFSGSGSGTAFTLTISSLQPDD FATYYCFQGSGYPFTFGGGTKLEIKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC 209 Monomeric IL10
SPGQGTQSENSCTHFPGNLPNMLRDLRD Mature form (IL10M)
AFSRVKTFFQMKDQLDNLLLKESLLEDFK of IL10, GYLGCQALSEMIQFYLEEVMPQAENQDP
with an DIKAHVNSLGENLKTLRLRLRRCHRFLPC internal
ENGGGSGGKSKAVEQVKNAFNKLQEKGI G3SG2 YKAMSEFDIFINYIEAYMTMKIRN spacer
210 CDRH1 of GFSLSTSGM GFTX3 IgGIL10M14 (Chothia) 211 CDRH2 of
WWDDK GFTX3 IgGIL10M14 (Chothia) 212 CDRH3 of SMITNWYFDV GFTX3
IgGIL10M14 (Chothia) 213 CDRL1 of QLSSPGQGTQSENSCTHFPGNLPNMLR
Monomeric IgGIL10M14 DLRDAFSRVKTFFQMKDQLDNLLLKES IL10 grafted
(Chothia) LLEDFKGYLGCQALSEMIQFYLEEVMP into
QAENQDPDIKAHVNSLGENLKTLRLRL CDRL1. RRCHRFLPCENGGGSGGKSKAVEQVK IL10
is NAFNKLQEKGIYKAMSEFDIFINYIEAY bolded, MTMKIRNVGY underlined GFTX3
214 CDRL2 of DTS GFTX3 IgGIL10M14 (Chothia) 215 CDRL3 of GSGYPF
GFTX3 IgGIL10M14 (Chothia) 216 CDRH1 of TSGMSVG GFTX3 IgGIL10M14
(Kabat) 217 CDRH2 of DIWWDDKKDYNPSLKS GFTX3 IgGIL10M14 (Kabat) 218
CDRH3 of SMITNWYFDV GFTX3 IgGIL10M14 (Kabat) 219 CDRL1 of
KAQLSSPGQGTQSENSCTHFPGNLPNM Monomeric IgGIL10M14 (Kabat)
LRDLRDAFSRVKTFFQMKDQLDNLLL IL10 grafted KESLLEDFKGYLGCQALSEMIQFYLEE
into VMPQAENQDPDIKAHVNSLGENLKTLR CDRL1. LRLRRCHRFLPCENGGGSGGKSKAVE
IL10 is QVKNAFNKLQEKGIYKAMSEFDIFINYI bolded, EAYMTMKIRNVGYMH
underlined GFTX3 220 CDRL2 of DTSKLAS GFTX3 IgGIL10M14 (Kabat) 221
CDRL3 of FQGSGYPFT GFTX3 IgGIL10M14 (Kabat) 222 VH of IgGIL10M14
QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 TSGMSVGWIRQPPGKALEWLADIWWDD
KKDYNPSLKSRLTISKDTSANQVVLKVTN MDPADTATYYCARSMITNWYFDVWGAG TTVTVSS
223 VL of IgGIL10M14 DIQMTQSPSTLSASVGDRVTITCKAQLSSP Monomeric
GQGTQSENSCTHFPGNLPNMLRDLRDA IL10 grafted
FSRVKTFFQMKDQLDNLLLKESLLEDF into CDRL1 KGYLGCQALSEMIQFYLEEVMPQAEN
GFTX3 QDPDIKAHVNSLGENLKTLRLRLRRCH RFLPCENGGGSGGKSKAVEQVKNAFN
KLQEKGIYKAMSEFDIFINYIEAYMTM KIRNVGYMHWYQQKPGKAPKLLIYDTS
KLASGVPSRFSGSGSGTAFTLTISSLQPDD FATYYCFQGSGYPFTFGGGTKLEIK 224 Heavy
chain of QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 IgGIL10M14
TSGMSVGWIRQPPGKALEWLADIWWDD LALA KKDYNPSLKSRLTISKDTSANQVVLKVTN
MDPADTATYYCARSMITNWYFDVWGAG TTVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK 225 Light chain of
DIQMTQSPSTLSASVGDRVTITCKAQLSSP GFTX3 IgGIL10M14
GQGTQSENSCTHFPGNLPNMLRDLRDA LALA FSRVKTFFQMKDQLDNLLLKESLLEDF
Monomeric KGYLGCQALSEMIQFYLEEVMPQAEN IL10 grafted
QDPDIKAHVNSLGENLKTLRLRLRRCH into RFLPCENGGGSGGKSKAVEQVKNAFN CDRL1.
KLQEKGIYKAMSEFDIFINYIEAYMTM KIRNVGYMHWYQQKPGKAPKLLIYDTS
KLASGVPSRFSGSGSGTAFTLTISSLQPDD FATYYCFQGSGYPFTFGGGTKLEIKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC 226 CDRH1 of
GFSLSTSGM GFTX3 IgGIL10M15 (Chothia) 227 CDRH2 of WWDDK GFTX3
IgGIL10M15 (Chothia) 228 CDRH3 of SMITNWYFDV GFTX3 IgGIL10M15
(Chothia) 229 CDRL1 of QLSSPGQGTQSENSCTHFPGNLPNMLR Monomeric
IgGIL10M15 DLRDAFSRVKTFFQMKDQLDNLLLKES IL10 grafted (Chothia)
LLEDFKGYLGCQALSEMIQFYLEEVMP into QAENQDPDIKAHVNSLGENLKTLRLRL CDRL1.
RRCHRFLPCENGGGSGGKSKAVEQVK IL10 is NAFNKLQEKGIYKAMSEFDIFINYIEAY
bolded, MTMKIRNVGY underlined 230 CDRL2 of DTS GFTX3 IgGIL10M15
(Chothia) 231 CDRL3 of GSGYPF GFTX3 IgGIL10M15 (Chothia) 232 CDRH1
of TSGMSVG GFTX3 IgGIL10M15 (Kabat) 233 CDRH2 of DIWWDDKKDYNPSLKS
GFTX3 IgGIL10M15 (Kabat) 234 CDRH3 of SMITNWYFDV GFTX3 IgGIL10M15
(Kabat) 235 CDRL1 of KAQLSSPGQGTQSENSCTHFPGNLPNM Monomeric
IgGIL10M15 (Kabat) LRDLRDAFSRVKTFFQMKDQLDNLLL IL10 grafted
KESLLEDFKGYLGCQALSEMIQFYLEE into CDRL1 VMPQAENQDPDIKAHVNSLGENLKTLR
IL10 is LRLRRCHRFLPCENGGGSGGKSKAVE bolded,
QVKNAFNKLQEKGIYKAMSEFDIFINYI underlined EAYMTMKIRNVGYMH GFTX3 236
CDRL2 of DTSKLAS GFTX3 IgGIL10M15 (Kabat) 237 CDRL3 of FQGSGYPFT
GFTX3 IgGIL10M15 (Kabat) 238 VH of IgGIL10M15
QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 TSGMSVGWIRQPPGKALEWLADIWWDD
KKDYNPSLKSRLTISKDTSANQVVLKVTN MDPADTATYYCARSMITNWYFDVWGAG TTVTVSS
239 VL of IgGIL10M15 DIQMTQSPSTLSASVGDRVTITCKAQLSSP Monomeric
GQGTQSENSCTHFPGNLPNMLRDLRDA IL10 grafted
FSRVKTFFQMKDQLDNLLLKESLLEDF into CDRL1 KGYLGCQALSEMIQFYLEEVMPQAEN
IL10 is QDPDIKAHVNSLGENLKTLRLRLRRCH bolded,
RFLPCENGGGSGGKSKAVEQVKNAFN underlined KLQEKGIYKAMSEFDIFINYIEAYMTM
GFTX3 KIRNVGYMHWYQQKPGKAPKLLIYDTS KLASGVPSRFSGSGSGTAFTLTISSLQPDD
FATYYCFQGSGYPFTFGGGTKLEIK 240 Heavy chain of
QVTLRESGPALVKPTQTLTLTCTFSGFSLS GFTX3 IgGIL10M15
TSGMSVGWIRQPPGKALEWLADIWWDD NEM
KKDYNPSLKSRLTISKDTSANQVVLKVTN MDPADTATYYCARSMITNWYFDVWGAG
TTVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYASTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWVSNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCS VIHEALHNHYTQKSLSLSPGK 241 Light chain
of DIQMTQSPSTLSASVGDRVTITCKAQLSSP GFTX3 IgGIL10M15
GQGTQSENSCTHFPGNLPNMLRDLRDA NEM FSRVKTFFQMKDQLDNLLLKESLLEDF
Monomeric KGYLGCQALSEMIQFYLEEVMPQAEN IL10 grafted
QDPDIKAHVNSLGENLKTLRLRLRRCH into RFLPCENGGGSGGKSKAVEQVKNAFN CDRL1.
KLQEKGIYKAMSEFDIFINYIEAYMTM IL10 is KIRNVGYMHWYQQKPGKAPKLLIYDTS
bolded, KLASGVPSRFSGSGSGTAFTLTISSLQPDD underlined
FATYYCFQGSGYPFTFGGGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYP
REAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVT
HQGLSSPVTKSFNRGEC 242 VH of IgGIL10M7 CAGGTCACACTGAGAGAGTCAGGCCCT
GCCCTGGTCAAGCCTACTCAGACCCTGA CCCTGACCTGCACCTTTAGCGGCTTTAG
CCTGAGCACTAGCGGAATGAGCGTGGG CTGGATTAGACAGCCCCCTGGTAAAGC
CCTGGAGTGGCTGGCCGATATTTGGTGG GACGATAAGAAGGACTATAACCCTAGC
CTGAAGTCTAGGCTGACTATCTCTAAGG ACACTAGCGCTAATCAGGTGGTGCTGA
AAGTGACTAATATGGACCCCGCCGACA CCGCTACCTACTACTGCGCTAGATCTAT
GATCACTAACTGGTACTTCGACGTGTGG GGCGCTGGCACTACCGTGACCGTGTCTA GC 243 VL
of IgGIL10M7 GATATTCAGATGACTCAGTCACCTAGCA
CCCTGAGCGCTAGTGTGGGCGATAGAG TGACTATCACCTGTAAAGCTCAGCTGTC
TAGCCCAGGTCAGGGAACTCAGTCAGA GAATAGCTGCACTCACTTCCCCGGTAAC
CTGCCTAATATGCTGAGAGATCTGAGG GACGCCTTCTCTAGGGTCAAGACCTTCT
TTCAGATGAAGGATCAGCTGGATAACC TGCTGCTGAAAGAGTCACTGCTGGAGG
ACTTTAAGGGCTACCTGGGCTGTCAGGC CCTGAGCGAGATGATTCAGTTCTACCTG
GAAGAAGTGATGCCCCAGGCCGAGAAT CAGGACCCCGATATTAAGGCTCACGTG
AACTCACTGGGCGAGAACCTGAAAACC CTGAGACTGAGGCTGAGGCGGTGTCAC
CGGTTTCTGCCCTGCGAGAACGGCGGA GGTAGCGGCGGTAAATCTAAGGCCGTG
GAACAGGTCAAAAACGCCTTTAACAAG CTGCAGGAAAAGGGAATCTATAAGGCT
ATGAGCGAGTTCGACATCTTTATTAACT ATATCGAGGCCTATATGACTATGAAGA
TTAGGAACGTGGGCTATATGCACTGGT ATCAGCAGAAGCCCGGTAAAGCCCCTA
AGCTGCTGATCTACGACACCTCTAAGCT GGCTAGTGGCGTGCCCTCTAGGTTTAGC
GGTAGCGGTAGTGGCACCGCCTTCACC CTGACTATCTCTAGCCTGCAGCCCGACG
ACTTCGCTACCTACTACTGTTTTCAGGG TAGCGGCTACCCCTTCACCTTCGGCGGA
GGCACTAAGCTGGAGATTAAG 244 Heavy Chain of
CAGGTCACACTGAGAGAGTCAGGCCCT IgGIL10M7 GCCCTGGTCAAGCCTACTCAGACCCTGA
CCCTGACCTGCACCTTTAGCGGCTTTAG CCTGAGCACTAGCGGAATGAGCGTGGG
CTGGATTAGACAGCCCCCTGGTAAAGC CCTGGAGTGGCTGGCCGATATTTGGTGG
GACGATAAGAAGGACTATAACCCTAGC CTGAAGTCTAGGCTGACTATCTCTAAGG
ACACTAGCGCTAATCAGGTGGTGCTGA AAGTGACTAATATGGACCCCGCCGACA
CCGCTACCTACTACTGCGCTAGATCTAT GATCACTAACTGGTACTTCGACGTGTGG
GGCGCTGGCACTACCGTGACCGTGTCTA GCGCTAGCACTAAGGGCCCAAGTGTGT
TTCCCCTGGCCCCCAGCAGCAAGTCTAC TTCCGGCGGAACTGCTGCCCTGGGTTGC
CTGGTGAAGGACTACTTCCCCGAGCCC GTGACAGTGTCCTGGAACTCTGGGGCTC
TGACTTCCGGCGTGCACACCTTCCCCGC CGTGCTGCAGAGCAGCGGCCTGTACAG
CCTGAGCAGCGTGGTGACAGTGCCCTC CAGCTCTCTGGGAACCCAGACCTATATC
TGCAACGTGAACCACAAGCCCAGCAAC ACCAAGGTGGACAAGAGAGTGGAGCCC
AAGAGCTGCGACAAGACCCACACCTGC CCCCCCTGCCCAGCTCCAGAACTGCTGG
GAGGGCCTTCCGTGTTCCTGTTCCCCCC CAAGCCCAAGGACACCCTGATGATCAG
CAGGACCCCCGAGGTGACCTGCGTGGT GGTGGACGTGTCCCACGAGGACCCAGA
GGTGAAGTTCAACTGGTACGTGGACGG CGTGGAGGTGCACAACGCCAAGACCAA
GCCCAGAGAGGAGCAGTACAACAGCAC CTACAGGGTGGTGTCCGTGCTGACCGTG
CTGCACCAGGACTGGCTGAACGGCAAA GAATACAAGTGCAAAGTCTCCAACAAG
GCCCTGCCAGCCCCAATCGAAAAGACA ATCAGCAAGGCCAAGGGCCAGCCACGG
GAGCCCCAGGTGTACACCCTGCCCCCC AGCCGGGAGGAGATGACCAAGAACCAG
GTGTCCCTGACCTGTCTGGTGAAGGGCT TCTACCCCAGCGATATCGCCGTGGAGTG
GGAGAGCAACGGCCAGCCCGAGAACAA CTACAAGACCACCCCCCCAGTGCTGGA
CAGCGACGGCAGCTTCTTCCTGTACAGC AAGCTGACCGTGGACAAGTCCAGGTGG
CAGCAGGGCAACGTGTTCAGCTGCAGC GTGATGCACGAGGCCCTGCACAACCAC
TACACCCAGAAGTCCCTGAGCCTGAGC CCCGGCAAG 245 Light Chain of
GATATTCAGATGACTCAGTCACCTAGCA IgGIL10M7 CCCTGAGCGCTAGTGTGGGCGATAGAG
TGACTATCACCTGTAAAGCTCAGCTGTC TAGCCCAGGTCAGGGAACTCAGTCAGA
GAATAGCTGCACTCACTTCCCCGGTAAC CTGCCTAATATGCTGAGAGATCTGAGG
GACGCCTTCTCTAGGGTCAAGACCTTCT TTCAGATGAAGGATCAGCTGGATAACC
TGCTGCTGAAAGAGTCACTGCTGGAGG ACTTTAAGGGCTACCTGGGCTGTCAGGC
CCTGAGCGAGATGATTCAGTTCTACCTG GAAGAAGTGATGCCCCAGGCCGAGAAT
CAGGACCCCGATATTAAGGCTCACGTG AACTCACTGGGCGAGAACCTGAAAACC
CTGAGACTGAGGCTGAGGCGGTGTCAC CGGTTTCTGCCCTGCGAGAACGGCGGA
GGTAGCGGCGGTAAATCTAAGGCCGTG GAACAGGTCAAAAACGCCTTTAACAAG
CTGCAGGAAAAGGGAATCTATAAGGCT ATGAGCGAGTTCGACATCTTTATTAACT
ATATCGAGGCCTATATGACTATGAAGA TTAGGAACGTGGGCTATATGCACTGGT
ATCAGCAGAAGCCCGGTAAAGCCCCTA AGCTGCTGATCTACGACACCTCTAAGCT
GGCTAGTGGCGTGCCCTCTAGGTTTAGC GGTAGCGGTAGTGGCACCGCCTTCACC
CTGACTATCTCTAGCCTGCAGCCCGACG ACTTCGCTACCTACTACTGTTTTCAGGG
TAGCGGCTACCCCTTCACCTTCGGCGGA GGCACTAAGCTGGAGATTAAGCGTACG
GTGGCCGCTCCCAGCGTGTTCATCTTCC CCCCCAGCGACGAGCAGCTGAAGAGCG
GCACCGCCAGCGTGGTGTGCCTGCTGA ACAACTTCTACCCCCGGGAGGCCAAGG
TGCAGTGGAAGGTGGACAACGCCCTGC AGAGCGGCAACAGCCAGGAGAGCGTCA
CCGAGCAGGACAGCAAGGACTCCACCT ACAGCCTGAGCAGCACCCTGACCCTGA
GCAAGGCCGACTACGAGAAGCATAAGG TGTACGCCTGCGAGGTGACCCACCAGG
GCCTGTCCAGCCCCGTGACCAAGAGCTT CAACAGGGGCGAGTGC 246 VH of IgGIL10M13
CAGGTCACACTGAGAGAGTCAGGCCCT GCCCTGGTCAAGCCTACTCAGACCCTGA
CCCTGACCTGCACCTTTAGCGGCTTTAG CCTGAGCACTAGCGGAATGAGCGTGGG
CTGGATTAGACAGCCCCCTGGTAAAGC CCTGGAGTGGCTGGCCGATATTTGGTGG
GACGATAAGAAGGACTATAACCCTAGC CTGAAGTCTAGGCTGACTATCTCTAAGG
ACACTAGCGCTAATCAGGTGGTGCTGA AAGTGACTAATATGGACCCCGCCGACA
CCGCTACCTACTACTGCGCTAGATCTAT GATCACTAACTGGTACTTCGACGTGTGG
GGCGCTGGCACTACCGTGACCGTGTCTA GC 247 VL of IgGIL10M13
GATATTCAGATGACTCAGTCACCTAGCA CCCTGAGCGCTAGTGTGGGCGATAGAG
TGACTATCACCTGTAAAGCTCAGCTGTC TAGCCCAGGTCAGGGAACTCAGTCAGA
GAATAGCTGCACTCACTTCCCCGGTAAC CTGCCTAATATGCTGAGAGATCTGAGG
GACGCCTTCTCTAGGGTCAAGACCTTCT TTCAGATGAAGGATCAGCTGGATAACC
TGCTGCTGAAAGAGTCACTGCTGGAGG ACTTTAAGGGCTACCTGGGCTGTCAGGC
CCTGAGCGAGATGATTCAGTTCTACCTG GAAGAAGTGATGCCCCAGGCCGAGAAT
CAGGACCCCGATATTAAGGCTCACGTG AACTCACTGGGCGAGAACCTGAAAACC
CTGAGACTGAGGCTGAGGCGGTGTCAC CGGTTTCTGCCCTGCGAGAACGGCGGA
GGTAGCGGCGGTAAATCTAAGGCCGTG GAACAGGTCAAAAACGCCTTTAACAAG
CTGCAGGAAAAGGGAATCTATAAGGCT ATGAGCGAGTTCGACATCTTTATTAACT
ATATCGAGGCCTATATGACTATGAAGA TTAGGAACGTGGGCTATATGCACTGGT
ATCAGCAGAAGCCCGGTAAAGCCCCTA AGCTGCTGATCTACGACACCTCTAAGCT
GGCTAGTGGCGTGCCCTCTAGGTTTAGC GGTAGCGGTAGTGGCACCGCCTTCACC
CTGACTATCTCTAGCCTGCAGCCCGACG ACTTCGCTACCTACTACTGTTTTCAGGG
TAGCGGCTACCCCTTCACCTTCGGCGGA GGCACTAAGCTGGAGATTAAG 248 Heavy Chain
of CAGGTCACACTGAGAGAGTCAGGCCCT IgGIL10M13
GCCCTGGTCAAGCCTACTCAGACCCTGA CCCTGACCTGCACCTTTAGCGGCTTTAG
CCTGAGCACTAGCGGAATGAGCGTGGG CTGGATTAGACAGCCCCCTGGTAAAGC
CCTGGAGTGGCTGGCCGATATTTGGTGG GACGATAAGAAGGACTATAACCCTAGC
CTGAAGTCTAGGCTGACTATCTCTAAGG ACACTAGCGCTAATCAGGTGGTGCTGA
AAGTGACTAATATGGACCCCGCCGACA CCGCTACCTACTACTGCGCTAGATCTAT
GATCACTAACTGGTACTTCGACGTGTGG GGCGCTGGCACTACCGTGACCGTGTCTA
GCGCTAGCACTAAGGGCCCCTCCGTGTT CCCTCTGGCCCCTTCCAGCAAGTCTACC
TCCGGCGGCACAGCTGCTCTGGGCTGCC TGGTCAAGGACTACTTCCCTGAGCCTGT
GACAGTGTCCTGGAACTCTGGCGCCCTG ACCTCTGGCGTGCACACCTTCCCTGCCG
TGCTGCAGTCCTCCGGCCTGTACTCCCT GTCCTCCGTGGTCACAGTGCCTTCAAGC
AGCCTGGGCACCCAGACCTATATCTGC AACGTGAACCACAAGCCTTCCAACACC
AAGGTGGACAAGCGGGTGGAGCCTAAG TCCTGCGACAAGACCCACACCTGTCCTC
CCTGCCCTGCTCCTGAACTGCTGGGCGG CCCTTCTGTGTTCCTGTTCCCTCCAAAG
CCCAAGGACACCCTGATGATCTCCCGG ACCCCTGAAGTGACCTGCGTGGTGGTG
GCCGTGTCCCACGAGGATCCTGAAGTG AAGTTCAATTGGTACGTGGACGGCGTG
GAGGTGCACAACGCCAAGACCAAGCCT CGGGAGGAACAGTACAACTCCACCTAC
CGGGTGGTGTCCGTGCTGACCGTGCTGC ACCAGGACTGGCTGAACGGCAAAGAGT
ACAAGTGCAAAGTCTCCAACAAGGCCC TGGCCGCCCCTATCGAAAAGACAATCT
CCAAGGCCAAGGGCCAGCCTAGGGAAC CCCAGGTGTACACCCTGCCACCCAGCC
GGGAGGAAATGACCAAGAACCAGGTGT CCCTGACCTGTCTGGTCAAGGGCTTCTA
CCCTTCCGATATCGCCGTGGAGTGGGA GTCTAACGGCCAGCCTGAGAACAACTA
CAAGACCACCCCTCCTGTGCTGGACTCC GACGGCTCCTTCTTCCTGTACTCCAAAC
TGACCGTGGACAAGTCCCGGTGGCAGC AGGGCAACGTGTTCTCCTGCTCCGTGAT
GCACGAGGCCCTGCACAACCACTACAC CCAGAAGTCCCTGTCCCTGTCTCCCGGC AAG 249
Light Chain of GATATTCAGATGACTCAGTCACCTAGCA IgGIL10M13
CCCTGAGCGCTAGTGTGGGCGATAGAG TGACTATCACCTGTAAAGCTCAGCTGTC
TAGCCCAGGTCAGGGAACTCAGTCAGA GAATAGCTGCACTCACTTCCCCGGTAAC
CTGCCTAATATGCTGAGAGATCTGAGG GACGCCTTCTCTAGGGTCAAGACCTTCT
TTCAGATGAAGGATCAGCTGGATAACC TGCTGCTGAAAGAGTCACTGCTGGAGG
ACTTTAAGGGCTACCTGGGCTGTCAGGC CCTGAGCGAGATGATTCAGTTCTACCTG
GAAGAAGTGATGCCCCAGGCCGAGAAT CAGGACCCCGATATTAAGGCTCACGTG
AACTCACTGGGCGAGAACCTGAAAACC CTGAGACTGAGGCTGAGGCGGTGTCAC
CGGTTTCTGCCCTGCGAGAACGGCGGA GGTAGCGGCGGTAAATCTAAGGCCGTG
GAACAGGTCAAAAACGCCTTTAACAAG CTGCAGGAAAAGGGAATCTATAAGGCT
ATGAGCGAGTTCGACATCTTTATTAACT ATATCGAGGCCTATATGACTATGAAGA
TTAGGAACGTGGGCTATATGCACTGGT ATCAGCAGAAGCCCGGTAAAGCCCCTA
AGCTGCTGATCTACGACACCTCTAAGCT GGCTAGTGGCGTGCCCTCTAGGTTTAGC
GGTAGCGGTAGTGGCACCGCCTTCACC CTGACTATCTCTAGCCTGCAGCCCGACG
ACTTCGCTACCTACTACTGTTTTCAGGG TAGCGGCTACCCCTTCACCTTCGGCGGA
GGCACTAAGCTGGAGATTAAGCGTACG GTGGCCGCTCCCAGCGTGTTCATCTTCC
CCCCCAGCGACGAGCAGCTGAAGAGCG GCACCGCCAGCGTGGTGTGCCTGCTGA
ACAACTTCTACCCCCGGGAGGCCAAGG TGCAGTGGAAGGTGGACAACGCCCTGC
AGAGCGGCAACAGCCAGGAGAGCGTCA CCGAGCAGGACAGCAAGGACTCCACCT
ACAGCCTGAGCAGCACCCTGACCCTGA GCAAGGCCGACTACGAGAAGCATAAGG
TGTACGCCTGCGAGGTGACCCACCAGG GCCTGTCCAGCCCCGTGACCAAGAGCTT
CAACAGGGGCGAGTGC 250 Monomeric IL10 AGTCCCGGTCAGGGAACTCAGTCAGAG
AATAGCTGCACTCACTTCCCCGGTAACC TGCCTAATATGCTGAGAGATCTGAGGG
ACGCCTTCTCTAGGGTCAAGACCTTCTT TCAGATGAAGGATCAGCTGGATAACCT
GCTGCTGAAAGAGTCACTGCTGGAGGA CTTTAAGGGCTACCTGGGCTGTCAGGCC
CTGAGCGAGATGATTCAGTTCTACCTGG AAGAAGTGATGCCCCAGGCCGAGAATC
AGGACCCCGATATTAAGGCTCACGTCA ACTCACTGGGCGAGAACCTGAAAACCC
TGAGACTGAGGCTGAGGCGGTGTCACC GGTTTCTGCCCTGCGAGAACGGCGGAG
GTAGCGGCGGTAAATCTAAGGCCGTGG AACAGGTCAAAAACGCCTTTAACAAGC
TGCAGGAAAAGGGAATCTATAAGGCTA TGAGCGAGTTCGACATCTTTATTAACTA
TATCGAGGCCTATATGACTATGAAGATT AGGAAC 251 linker GGGSGG 252 linker
GGGGS 253 linker GGGGA
Example 2
Antibody Cytokine Engrafted Proteins have Anti-Inflammatory
Activity
[0275] Using an assay developed in support of rhIL10's
pro-inflammatory activity in the clinic (Lauw et al., J Immunol.
2000; 165(5):2783-9), the pro-inflammatory activity of IgGIL10M13
in human whole blood was assessed. In order to assess
pro-inflammatory activity, antibody cytokine engrafted proteins
were profiled for their ability to induce interferon gamma
(IFN.gamma.) or granzyme B in activated primary human CD8 T cells.
It was found that antibody cytokine engrafted proteins such as
IgGIL10M13 demonstrated significantly less pro-inflammatory
activity than recombinant human IL10 (rhIL10) as measured by
IFN.gamma. production. This data is shown in FIG. 3A. Similar
results were found in assays measuring granzyme B (data not shown),
as well as with other exemplary antibody cytokine engrafted
proteins (IgGIL10M7). The significantly decreased pro-inflammatory
activity demonstrated by IgGIL10M13 as compared to rhIL10 indicates
it would be superior to rhIL10 for treating immune related
disorders, as IgGIL10M13 could be administered over a broader dose
range.
[0276] To examine anti-inflammatory activity, antibody cytokine
engrafted proteins and rhIL10 were tested for their ability to
inhibit LPS-induced TNF.alpha. in human whole blood. This data is
shown in FIG. 3B, wherein increasing concentrations of either
rhIL10 or IgGIL10M13 reduced TNF.alpha. production. Note that the
rhIL10 and IgGIL10M13 curves are similar, indicating that both
molecules had potent anti-inflammatory activity.
[0277] In summary, these results show that antibody cytokine
engrafted proteins have the desired properties of having
anti-inflammatory properties similar to IL10, but without the dose
limiting, and unwanted pro-inflammatory properties.
Example 3
IL10 Dependent Signaling
[0278] In vitro signaling studies in human PBMCs and whole blood
indicate that antibody cytokine engrafted proteins such as
IgGIL10M13 had a more specific signaling profile when compared to
rhIL10. Using CyTOF, a FACS based method that utilizes mass
spectrometry, antibody cytokine engrafted protein signaling in
multiple different cell populations in whole blood was assessed by
pSTAT3 detection (FIG. 4). Antibody cytokine engrafted proteins
such IgGIL10M13 induced a pSTAT3 signal only on monocytes,
macrophages and plasmacytoid dendritic cells above .mu.M
concentrations (up to 1.8 .mu.M). All of these cell types are known
to have increased expression of IL10 receptor. rhIL10 induced a
pSTAT3 signal on monocytes, but also on additional cell types such
as T cells, B cells, and NK cells. This was seen even at low nM
concentrations of rhIL10. In whole blood treated with rhIL10 at a
concentration of 100nM, the strongest pSTAT3 signal was seen on
monocytes and myeloid dendritic cells with additional moderate
activation of T, NK, B cells, and Granulocytes. The functional
consequences of pSTAT3 signaling leads to increased production of
IFN.gamma. and Granzyme B from CD8 T cells and NK cells. There is
also proliferation of B cells in response to rhIL10 signaling. This
pro-inflammatory activity of rhIL10 in human whole blood is
observed at exposures less than 5-fold above the anti-inflammatory
IC90. The more selective cellular profile of antibody cytokine
engrafted proteins such as IgGIL10M13 resulted in reduced
pro-inflammatory activity leading to better anti-inflammatory
efficacy.
Example 4
Antibody Cytokine Engrafted Protein Signaling in Various
Species
[0279] rhIL10 potently inhibits LPS-induced pro-inflammatory
cytokine production in human monocytes, PBMCs, and whole blood. The
antibody cytokine engrafted protein IgGIL10M13 exhibits pM potency
on target cells, although 10-fold less potent than rhIL10. Table 2
is a potency comparison for IL10 or IgGIL10M13 activity in human
whole blood as well as whole blood of selected toxicity
species.
[0280] Potency calculations are based on ex vivo whole blood assays
from either mouse, cynomolgus monkey or human. For each species
tested, IgGIL10M13 or rhIL10 were titrated and assessed for ability
to inhibit LPS-induced TNF.alpha. production. IC50s were calculated
as the level of molecule that gave rise to 50% inhibition of total
TNF.alpha. signal. IC90s and IC30s were calculated taking into
account Hill slope value for each assay with the following
equation: logEC50=logECF-(1/HillSlope)*lob(F/100-F)), where ECF is
the concentration that gives a response of F percent of total
TNF.alpha. signal.
TABLE-US-00002 TABLE 2 IgGIL10M13 (CV %) IL10 (CV %) Mouse IC30 2.2
pM (pooled blood) 0.57 pM (pooled blood) IC50 12 pM 1.7 pM IC90 108
pM 15 pM Cyno IC30 4.13 pM (48% n = 3) 0.44 pM (28% n = 3) IC50
6.67 pM (53%) 0.65 pM (31%) IC90 24 pM (73%) 1.9 pM (43%) Human
IC30 10.8 pM (76% n = 48) 1.3 pM (96% n = 48) IC50 25.2 pM (76%)
2.8 pM (98%) IC90 262 pM (94%) 22.8 pM (79%)
Example 5
Evaluation of Antibody Cytokine Engrafted Protein
Pharmacokinetics
[0281] rhIL10 has a short half-life, limiting its target tissue
exposure and requiring the patient to undergo multiple dosing. The
half-life of antibody cytokine engrafted proteins was assessed in
C57Bl/6 mice. Antibody cytokine engrafted proteins (e.g.
IgGIL10M13) were injected at 0.2 mg/kg subcutaneously and blood was
sampled beginning at 5 minutes post-injection up to 144 hours
post-injection. IgGIL10M13 had a significant half-life extension of
approximately 4.4 days (FIG. 5B) compared to rhIL10 which had a
half-life of approximately lhr (FIG. 5A).
Example 6
Evaluation of Antibody Cytokine Engrafted Protein
Pharmacodynamics
[0282] Consistent with extended half-life, antibody cytokine
engrafted proteins also demonstrated improved pharmacodynamics.
Phospho-STAT3 (pSTAT3), a marker of IL10 receptor activation and
signaling was monitored in mouse colon after subcutaneous dosing of
IgGIL10M13. Enhanced pSTAT3 signal was detected in colon at least
up to 72 hours post-dose, and absent by 144 hours post-dose. See
FIG. 5C. This profile is a dramatic improvement over rhIL10, whose
signal is absent by 24 hours post-dose. FIG. 5D depicts improved
duration of in vivo response of IgGIL10M13 as compared to rhIL10 as
measured by inhibition of TNF.alpha. in blood in response to LPS
challenge following antibody cytokine engrafted protein dosing.
Example 7
Efficacy of Antibody Cytokine Engrafted Proteins in a Mouse
Model
[0283] A direct comparison of efficacy for TNF.alpha. inhibition
after LPS challenge was performed. C57/BL6 mice were dosed
subcutaneously with vehicle, or equimolar levels of IL10 at 110
nmol/mouse, calculated for both recombinant IL10 and IgGIL10M13.
Mice were then challenged with LPS delivered intraperitoneally to
assess IL10 dependent inhibition of TNF.alpha. levels. IgGIL10M13
demonstrated comparable efficacy to rhIL10 at the initial
assessment time period of 0.5 hour, however, up to at least
forty-eight hours post dosing, IgGIL10M13 sustained superior
efficacy to rhIL10 as measured by TNF.alpha. production. This data
is shown in FIG. 6.
Example 8
Antibody Cytokine Engrafted Proteins have Improved Exposure
[0284] The peak serum concentration (Cmax) of antibody cytokine
engrafted proteins was assessed in C57Bl/6 mice. Antibody cytokine
engrafted proteins were injected at 0.2 mg/kg (10 ml/kg dose
volume) in 0.9% saline subcutaneously and blood was sampled
beginning at 1 hour post-injection and up to 144 hours
post-injection. Whole blood was collected into heparin-treated
tubes at each time point and centrifuged at 12,500 rpm for 10
minutes at 4.degree. C. Plasma supernatant was collected and stored
at -80.degree. C. until all time points were collected. Antibody
cytokine engrafted proteins levels in plasma were measured using
two different immunoassay methods to enable detection of both the
IL10 and antibody domains of the antibody cytokine engrafted
protein. As shown in FIG. 7, the antibody cytokine engrafted
protein (e.g. IgGIL10M13) maintained greater than 60% Cmax past 100
hours. In contrast, rhIL10 levels dropped below 20% Cmax within 3.5
hours.
Example 9
Antibody Cytokine Engrafted Proteins Act Only on Certain Cell Types
in Human Patients
[0285] CyTOF was run as previously described (see Example 3 and
Materials and Methods below) on immune cells from human healthy
donors and patients with Crohn's disease. As shown in the graphs in
FIG. 8, IgGIL10M13 stimulated only monocytes, and the stimulation
as measured by pSTAT3 levels is comparable to rhIL10. Monocytes are
the target cells for inflammatory related disorders such as Crohn's
disease and Ulcerative Colitis and express very high levels of IL10
receptor. However, FIG. 9 also shows the unwanted pro-inflammatory
effects of rhIL10, for example, the increased pSTAT3 signaling on
CD4 T cells, CD8 T cells and NK cells. It is noteworthy that
IgGIL10M13, does not display this unwanted pro-inflammatory effect
either on normal human cells or in cells taken Crohn's disease
patients. This demonstrates that IgGIL10M13 has a larger, safer
therapeutic index as administration of the antibody cytokine
engrafted protein will act only on the desired cell type and not on
other cell types such as CD8 T cells which would only worsen immune
related disorders such as Crohn's disease and Ulcerative
Colitis.
Example 10
IgGIL10M13 has Reduced Pro-Inflammatory Activity in PHA Stimulated
Human Whole Blood Compared to rhIL10
[0286] Despite extensive clinical data linking genetic IL 10
deficiency to IBD susceptibility, rhIL10 showed only mild efficacy
in IBD clinical trials (Herfarth et al., Gut 2002: 50(2):146-147).
Retrospective analyses of trial data suggest that rhIL10's efficacy
was limited by its intrinsic pro-inflammatory activity such as
enhanced production of IFN.gamma.. As discussed previously, in
human functional cell-based assays, rhIL10 signaling leads to
production of IFN.gamma. and Granzyme B from T cells and NK
cells.
[0287] Whole blood was taken from patients with Crohn's Disease and
the levels of IFN.gamma. were measured after stimulation with
rhIL10, IgGIL10M13 and PHA alone. This data is shown in FIGS.
9A-9C. Increasing doses of rhIL10 causes a sharp increase in
IFN.gamma. production, which then plateaus. In contrast, in
treatment of these cells with IgGIL10M13 little to no production of
IFN.gamma. was seen, indicating that IgGIL10M13 did not induce, or
induced only very low levels of IFN.gamma. production from T cells
or NK cells.
[0288] An additional titration experiment was performed with these
patient donor samples. In this experiment, IL10 levels from the
donor patient sera was measured and found to be in the range of 1.5
to 5 femtomolar (fM), although the scientific literature has
reported that patient IL10 levels could be as high as 20 fM
(Szkaradkiewicz et al., Arch. Immunol. Ther Exp 2009:
57(4):291-294). rhIL10 was administered to the donor patient cells
at the fixed concentrations of 2 femtomolar (fM), 2 pM, 2 nM and
200 nM. To these fixed concentrations of rhIL10, increasing
concentrations of the antibody cytokine engrafted protein
IgGIL10M13 was administered, and IFN.gamma. production assayed. The
data is shown in FIG. 9D. At the fixed concentrations of 2 fM and 2
pM, IgGIL10M13 competes with rhIL10 and reduced the production of
IFN.gamma. to baseline levels. At the fixed concentration of 2 nM,
IFN.gamma. production was reduced by nanomolar concentrations of
IgGIL10M13. Finally, at the fixed excess concentration of 200 nM
rhIL10, only very little reduction of IFN.gamma. production by
IgGIL10M13 was seen. This indicates that at physiological levels of
IL10, IgGIL10M13 competed out IL10, reducing the production of
IFN.gamma., and the unwanted pro-inflammatory effects.
Example 11
Aggregation Properties of Antibody Cytokine Engrafted Proteins
[0289] In clinical trials for IBD, rhIL10 was observed to have a
very short half-life; however simple Fc fusions to the IL10 dimer
to extend half-life were not pursued given aggregation properties
of such a molecule. FIG. 10A shows aggregation of both an IL10 wild
type linked to an Fc and IL10 monomer linked to an Fc. However, as
shown in FIG. 10B, the antibody structure of the antibody cytokine
engrafted protein prevents IL10 aggregation, thus promoting ease of
administration. In addition, reducing aggregation has the benefit
of reducing an immune reaction to the therapeutic, and the
generation of anti-drug antibodies.
Example 12
Retained Binding of Antibody Cytokine Engrafted Proteins
[0290] Palivizumab is an anti-RSV antibody, and was chosen as the
antibody structure for cytokine engrafting. This antibody had the
advantages of a known structure, and as its target was RSV, a
non-human target. The choice of a non-human target was to insure
that there would be no toxicity associated with the antibody
cytokine engrafted protein binding to an off target human antigen.
It was uncertain after engrafting IL10M into palivizumab, whether
the final IL10 antibody cytokine engrafted protein would still bind
the RSV target protein. As assayed by ELISA, the IL10 antibody
cytokine engrafted protein still bound to RSV target protein,
despite the presence of the IL10M. This data is shown in FIG.
11.
Example 13
Structural Conformation of the Antibody Cytokine Engrafted Protein
Results in Differential Activity Across Cell Types
[0291] Antibody cytokine engrafted proteins (e.g. IgGIL10M13)
incorporates monomeric IL10 (SEQ ID NO:209) into the Light Chain
CDR 1 of an antibody (FIG. 2). Insertion of a 6 amino acid
glycine-serine linker between helices D and E of IL10 renders the
normally heterodimeric molecule incapable of domain swapping
dimerization. As such, engrafting IL10M into an antibody results in
an antibody cytokine engrafted protein with 2 monomeric IL10
molecules. However, due to flexibility of the antibody Fab arms,
the angle and distance between the IL10 monomers is not fixed, as
in the wild-type IL10 dimer, thus affecting its interaction with
the IL10R1/R2 receptor complex. This is shown graphically in FIG.
12. Specifically, due to antibody engraftment, the angle of the
engrafted IL10 dimer is larger and variable, rendering signal
transduction less efficient on cells with lower expression levels
of IL10R1and R2 as found on the pro-inflammatory cell types such as
CD4 and CD8 T cells, B cells and NK cells. In contrast, antibody
cytokine engrafted proteins signal more efficiently on cells with
high IL-10R1 and R2 expression such as monocytes. A class average
negative stain EM study of IgGIL10M13 highlighted the additional
flexibility and wider angle between monomers, confirming that the
geometry is altered compared to rhIL10. The less restricted
geometry of the IL10 dimer in IgGIL10M13 alters its interaction
with IL10R complex. As a consequence, the structure of the
IgGIL10M13 antibody cytokine engrafted protein results in the
biological effect of only producing a productive signal on cell
types with high levels of IL10R1 and R2 expression.
Example 14
Crystal Structure of IgGIL10M13
[0292] The IgGIL10M13 Fab was concentrated to 16.2 mg/ml in 20 mM
HEPES pH 8.0, 150 mM NaCl and used directly in hanging drop vapor
diffusion crystallization trials. Crystallization screens were
setup by mixing 0.2 .mu.l of protein solution with 0.2 .mu.l of
reservoir solution and equilibrated against 50 .mu.l of the same
reservoir solutions. Crystals for data collection appeared after
3-4 weeks at 20.degree. C. from a reservoir solution of 20%
PEG3350, 200 mM magnesium acetate, pH 7.9. Prior to data
collection, the crystals were soaked in reservoir solution
supplemented with 20% ethylene glycol and flash cooled in liquid
nitrogen. Diffraction data were collected at the ALS beamline 5.0.3
with an ADSC Quantum 315R detector. Data was indexed and scaled
using the HKL2000 software package (Otwinowski and Minor. (1997)
Methods in Enzymology, Volume 276: Macromolecular Crystallography,
part A, p.30'7-326). The data for the IgGIL10M13 Fab was processed
to 2.40 .ANG. in space group P2.sub.1 with cell dimensions a=80.6
.ANG., b=104.7 .ANG., c=82.8 .ANG., alpha=90.degree.,
beta=115.3.degree., gamma=90.degree.. The structure was solved by
molecular replacement using PHASER (McCoy et al., (2007) J. Appl.
Cryst. 40:658-674) with the palivizumab Fab structure (PDB code:
2HWZ) and monomeric IL10 structure (PDB Code: 1LK3 chain A) as
search models. The top molecular replacement solution contained 2
molecules of the IgGIL10M13 Fab in the asymmetric unit. The final
model was built in COOT (Emsley & Cowtan (2004) Acta Cryst.
D60:2126-2132) and refined with PHENIX (Adams et al., (2010) Acta
Cryst. D66, 213-221). The R.sub.work and R.sub.free values are
18.8% and 23.9% respectively with root-mean-square (r.m.s)
deviation values from ideal bond lengths and bond angles were 0.005
.ANG. and 0.882.degree. respectively.
Overall Structure
[0293] The IgGIL10M13 Fab crystallized with 2 molecules in the
asymmetric unit, both with similar conformations. The electron
density maps were similar for both molecules. The overall structure
(FIG. 13A) shows that the Fab and grafted monomeric IL10 (IL10M)
can adopt a collinear arrangement (Fab light chain in white, Fab
heavy chain in black, IL10M in dark grey). FIG. 13B shows a closer
view of the grafting point in CDR-L1. The three flanking CDR
residues are show with dark grey sticks. Dashed lines illustrate
portions of the structure which could not be fit in the model due
to missing electron density, presumably due to structural
flexibility in these areas. The two areas include 6 residues at
N-terminus of IL10M just after the grafting point and 8 residues
between helices 4 and 5 in IL10M which encompass the inserted 6
residue linker (GGGSGG) (SEQ ID NO:251). There are also 3 pairs of
hydrogen bonding interactions between the grafted IL10M molecule
and portions of the Fab heavy chain (FIG. 13C). These include R138
and N104 (sidechain), R135 and D56 (sidechain), and N38 and K58
(backbone/sidechain).
Materials And Methods
Anti-Inflammatory Assays (LPS-Challenge Human Whole Blood)
[0294] IL10 antibody cytokine engrafted proteins were prepared at
10.times. at 1000 ng/ml in assay medium (RPMI 1640 with glutamine
(Hyclone), 10% heat inactivated FBS (Omega Scientific), 1%
Penicillin/Streptomycin (Gibco), 50 uM 2-mercaptoethonal (Gibco),
10 mM Hepes ph 7.4 (Hyclone), 0.1 mM Non-essential Amino Acids
(Hyclone), 1 mM Sodium Pyruvate (Hyclone) and 1.times. human
insulin/transferrin/selenium (Gibco). Each 10.times. stock of IL10
antibody cytokine engrafted protein in assay medium was serially
diluted 1:3 to create an 11-point dose titration.
[0295] Human whole blood was diluted to 90% in assay medium and
gently mixed. Unstimulated diluted whole blood was plated in n=3
wells (45 .mu.l/well), and assay medium added (5 ul/well) to bring
final well volume to 50 .mu.l. Lipopolysaccharide (LPS, Invivogen
stock 100 .mu.g/ml) was spiked into diluted whole blood at 220
ng/ml and gently mixed. LPS spiked whole blood was then plated,
IL10 proteins added to each respective well, then gently mixed and
incubated for .about.20 hours in a 37C incubator, 5% CO2. Plates
were then gently mixed and centrifuged at 1400 rpm for 5 minutes at
room temperature and supernatant harvested by transferring 10
.mu.l/well from assay plate to a proxy plate for TNF.alpha.
detection using the Human TNF-alpha HTRF kit (Cisbio.RTM.)
according to manufacturer's instructions, and results compared to a
standard curve generated using manufacturer provided control.
Pro-Inflammatory Assays (Human Primary CD8 T-Cell Activation)
[0296] Peripheral blood mononuclear cells (PBMCs) were isolated
from buffy coats from the Blutspende Zentrum Basel. Eight Leucosep
tubes (Greiner, 227290) per buffy coat were each filled with 15 ml
Ficoll-Paque PLUS.RTM. (GE Healthcare, 17-1440-03) and centrifuged
(lminute, 1000 g, 20.degree. C.). Buffy coats were diluted 1:4 in
phosphate buffered saline (PBS) pH 7.4 (Gibco, 10010-015) and 35 ml
were overlaid onto each Ficoll gradient. After centrifugation (800
g, 20 min, 20.degree. C.) the lymphocytes separate into a white
cell layer. This cell layer was transferred to fresh tubes and
washed with PBS. Erythrocytes were lysed by resuspending cell
pellets in 2 ml Gey's red blood lysing buffer (155 mM NH.sub.4Cl,
10 mM KHCO.sub.3, 0.1 mM EDTA) per tube. After incubation for 5
min, the cells were washed twice with PBS. After the last wash, the
PBMCs were resuspended in T cell medium (TCM). TCM contains RPMI
1640 (Gibco, 21875-034), 10% fetal bovine serum (Gibco, 10082147),
1% Penicillin-Streptomycin (Gibco, 15070063) 2 mM GlutaMax (Gibco,
35050-038) and 50 U/ml penicillin, 50 .mu.g/ml streptomycin. PBMCs
were filtered through 40 .mu.m cell strainers (BD Falcon, 734-0002)
to obtain single cell suspensions, and counted. CD8.sup.+ T cells
were purified from PBMCs using the human CD8.sup.+ T cell
enrichment kit (StemCell, 19053) according to the manufacturer's
protocol for the Big Easy Magnet.RTM. (StemCell, 18001). After
isolation, the cells were washed, counted and resuspended in TCM at
a concentration of 1.8.times.10.sup.6 cells/ml. The anti-CD3/CD28
coated plate (see section 2.1.1) was washed once with 2 ml TCM per
well followed by the addition of 1.8.times.10.sup.6 CD8.sup.+ T
cells in 1 ml per well. Plates were centrifuged (5 min, 520 g,
20.degree. C.) and incubated for 3 days in a cell incubator
(Binder) at 37.degree. C., 5% CO.sub.2, 95% humidity
Stimulation with IL10 Antibody Cytokine Engrafted Proteins
[0297] After three days of incubation, activated CD8+ T cells were
pooled from the 24 well plates and washed once with TCM. The cells
were counted, resuspended in TCM at 3.times.10.sup.6 cells per ml
and 300,000 cells per well in 100 .mu.l were added into Nunclon
Delta Surface.RTM. 96 well round bottom plate (Thermo Scientific,
163320). IL10 antibody cytokine engrafted protein pre-dilutions at
double the final concentration were prepared in TCM in a separate
plate at the following concentrations: 40, 4, 0.4, 0.04, 0.004 and
0 nM. 100 .mu.l of these pre-dilutions were added in duplicates to
the 100 .mu.l cell suspension resulting in the final concentration
of the antibody cytokine engrafted proteins of 20, 2, 0.2, 0.02,
0.002 and 0 nM. Plates were incubated at 37.degree. C. for 48 h
PMA and Anti-CD3 Stimulation and Golgistop
[0298] After the incubation period, the plates were centrifuged (2
min, 970 g, 20.degree. C.) and the supernatant was discarded. Cell
pellets were resuspended in 200 .mu.l TCM containing 2 ng/ml
Phorbol-myristate-acetate (PMA, Sigma Aldrich, 79346), 2.5 .mu.g/ml
anti-CD3 (BD, 555336) and 1:1500 GolgiStop (BD, 554724). Plates
were incubated for 5 h at 37.degree. C. to restimulate the cells.
Afterwards, plates were centrifuged (2 minutes, 970 g, 20.degree.
C.), supernatants discarded and 200 .mu.l ice cold PBS was added to
wash the cell pellet. After centrifugation (2 minutes, 970 g,
20.degree. C.), PBS was removed and the cells were reuspended in
200 .mu.l ice cold 200 mM Tris-HCl buffer (pH 8.1) containing 2%
Triton X-100 (Serva, 39795) to lyse the cells. The plates were left
for 15 min on ice and centrifuged (10 min, 970 g, 4.degree. C.) to
eliminate debris. The supernatant was carefully transferred into a
new 96 well plate and frozen for storage. To detect
interferon-.gamma. (IFN-.gamma. in the supernatant, the human
IFN-.gamma. DuoSet.RTM. ELISA (R&D systems, DY285) was used
according to the manufacturer's protocol. The supernatants obtained
as described in section 2.1.5 were diluted 1:2 in the recommended
assay diluent. To detect Granzyme B (GrzB) in the supernatant, the
human Granzyme B.RTM. ELISA (Mabtech, 3485-1H-20) was used
according to the manufacturer's protocol. The supernatants were
diluted 1:50 in the recommended assay diluent. The optical density
(OD) of the ELISA plates were acquired by SpectraMax.RTM. 340PC
plate reader (Molecular Devices) and converted to concentrations by
SoftMax.RTM. Pro software according to the automatically generated
standard curve. The data were exported to Microsoft Excel where the
concentrations were back calculated regarding their dilution
factor.
IL10 Dependent Signaling
[0299] CyTOF is a FACS/Mass spectrometry technique to assess the
activation of multiple cell populations by a single agent
simultaneously. Antibody cytokine engrafted protein was incubated
with human whole blood 20 minutes. Post-stimulation, PBMCs were
treated with metal-conjugated antibodies against cell specific
surface receptors CD14, HLA-DR, CD4, CD8, CD19, CD56 and the
signaling marker pSTAT3 and analyzed by CyTOF. Results indicate
that IgGIL10M13 at all doses primarily activated monocyte and
macrophage cell populations with little activation of T cell, B
cell, NK cell and dendritic cell populations. Conversely, rhIL10
activated all cell populations tested to some extent.
Pharmacokinetics Evaluation
[0300] Half-life of the antibody cytokine engrafted proteins was
assessed in C57Bl/6 mice. Antibody cytokine engrafted proteins were
injected at 0.2 mg/kg (10 ml/kg dose volume) in 0.9% saline
subcutaneously and blood was sampled beginning at lhour
post-injection and up to 144 hours post-injection. Whole blood was
collected into heparin-treated tubes at each time point and
centrifuged at 12,500 rpm for 10 minutes at 4.degree. C. Plasma
supernatant was collected and stored at -80.degree. C. until all
time points were collected. Antibody cytokine engrafted proteins
levels in plasma were measured using two different immunoassay
methods to enable detection of both the IL10 and antibody domains
of the antibody cytokine engrafted protein. The first method
utilized a commercially available 1L-10 ELISA kit employed as
recommended (BD OptEIA.RTM. Human IL10 ELISA Set, Capture:
rhIL10/Detection: biotin-rhIL10). The second consisted of an IL10
based capture and Fc-based detect immunoassay run on a GyroLab.RTM.
xP Workstation (Gyros AB Uppsala, Sweden). Specifically, the
reagents employed consisted of Biotinylated Human IL10 capture
(R&D Systems BAF217) and Alexafluor 647 goat anti-human IgG,
Fc.gamma. specific detection (Jackson ImmunoResearch #109-605-098).
The assay was run on 200 nL CDs (Gyros #P0004180) using a
Gyros-approved wizard method. The buffers used were Rexxip A.RTM.
(Gyros #P0004820) for standard and sample dilution and Rexxip
F.RTM. (Gyros #P0004825) for detection preparation. Analysis of
results was done using the Gyrolab.RTM. data analysis software.
Pharmacodynamics Evaluation
[0301] Consistent with the extended half-life, antibody cytokine
engrafted proteins also demonstrated improved pharmacodynamics.
Phospho-stat3 (pSTAT3), a marker of IL10R activation was monitored
in target tissues (blood and colon) after subcutaneous dosing.
Antibody cytokine engrafted proteins were injected at 0.2 mg/kg (10
ml/kg dose volume) in 0.9% saline subcutaneously. Terminal whole
blood and colon tissue (2 cm at ileo-cecal junction) were harvested
beginning at 1 hour post-injection and up to 144 hours
post-injection. Whole blood was collected into heparin-treated
tubes containing 1.times. phosphatase inhibitors (Pierce Halt
Phosphatase Inhibitor Cocktail) and kept on ice until phospho-Stat3
assay. Colon tissue was collected in tubes containing cold PBS and
1.times. phosphatase inhibitors. Once all tissue was collected for
the time point, colon tissue was transferred into tubes containing
a steel bead and 700 .mu.l of Complete Cell Lysis Buffer containing
PBS with 10 .mu.M DTT, 1.times. protease inhibitor cocktail,
10.times. phosphatase inhibitor cocktail and 10.times. cell lysis
buffer (Active Motif Nuclear Extraction Kit). Colon tissue was
homogenized by tissue-lyser at 30 rps for 5 minutes at room
temperature. Lysed tissue was centrifuged for 10 minutes at
14,000.times.g at 4.degree. C. Supernatant was collected and stored
on ice until phospho-STAT3 assay.
[0302] A phospho-Stat3 assay plate (Meso Scale Discovery.RTM.
pSTAT3(Tyr705) Assay) was run on the same day as whole blood and
colon tissue collection and processing. Ice-cold whole blood was
lysed using the provided 1.times. MSD Lysis Buffer containing
1.times. Phosphatase Inhibitor 2, 1.times. Phosphatase Inhibitor 3
and 1.times. Protease Inhibitor. Each tube of whole blood was
centrifuged at 12,500 rpm for 10 minutes at 4.degree. C. Plasma was
collected and discarded. Pelleted whole blood was resuspended in
220 .mu.l of MSD Lysis Buffer+Inhibitors and vortexed thoroughly.
Lysed whole blood was plated into respective wells of the
phospho-STAT3 assay plate at 50 .mu.l/well. Colon tissue
supernatant protein detection was performed using the Bradford
Assay (Pierce). Colon protein was then plated on the phospho-STAT3
assay plate at 50 .mu.l/well. Plates were incubated at room
temperature for 2 hours, washed, and treated with phospho-STAT3 or
Total STAT3 antibody (Meso Scale Discovery). Plates were analyzed
for relative fluorescence units (RFU) on the MSD Sector Imager 2400
(Meso Scale Discovery). Whole blood phospho-STAT3 RFU was
normalized to total STAT3 RFU. Colon protein phospho-STAT3 RFU was
normalized to loaded protein concentration. Enhanced pSTAT3 signal
is detected in both tissues at least up to 72 hours post-dose, and
absent by 144 hours post-dose. See FIG. 5, not shown. This profile
is a dramatic improvement over rhIL10, whose signal is absent by 24
hours post-dose.
Ex Vivo Efficacy
[0303] A direct comparison of efficacy for TNF.alpha. inhibition
after LPS challenge was performed. In the assay, C57/B16 mice were
dosed subcutaneously with vehicle, 0.2 mg/kg of IgGIL10M13 (10
ml/kg dose volume), or equimolar levels of rhIL10. Whole blood was
collected prior to, at 1.5 hrs post-dose and up to 144 hrs
post-dose. Whole blood was collected into heparin-treated tubes.
Prior to blood collection, assay medium was prepared. Assay medium
contained RPMI 1640 with glutamine (Hyclone) with 10% heat
inactivated FBS (Omega Scientific), 1% Penicillin/Streptomycin
(Gibco), 50 .mu.M 2-mercaptoethonal (Gibco), 10 mM Hepes ph 7.4
(Hyclone), 0.1 mM Non-essential Amino Acids (Hyclone) and 1 mM
Sodium Pyruvate (Hyclone). Mouse whole blood was plated in
25W/well/replicate/mouse on a 384 well plate. Assay medium was
added to unstimulated control wells (25 .mu.l/well) to bring final
well volume to 50 .mu.l. For LPS challenge, LPS (Invivogen, stock
100 .mu.g/ml) was spiked into assay medium at 200 ng/ml [100 ng/ml
final in assay] and gently mixed. LPS spiked BCM was then plated at
25 .mu.l/well for each required well containing mouse whole blood.
The plate was gently mixed and incubated for 21 hours in a
37.degree. C. incubator, 5% CO2. The next day, the assay plate was
centrifuged at 1400 rpm for 5 minutes at room temperature.
Supernatants from each well were collected and frozen at
-80.degree. C. until all time points had been assayed. Once all
time points could be analysed, supernatants were plated onto an MSD
V-plex.RTM. Mouse Pro-Inflammatory Cytokine Assay Plate (Meso Scale
Discovery). Plates were analysed for TNF.alpha. (pg/ml) on the MSD
Sector Imager 2400.RTM. (Meso Scale Discovery)
Immunostimulatory Assays
MC/9 Cell Proliferation
[0304] The immunostimulatory activity of IL10 antibody cytokine
engrafted proteins were assessed by examining the ability to
stimulate MC/9 (a mast cell line derived from mouse fetal liver)
cell proliferation. First, IL10 antibody cytokine engrafted
proteins were diluted in a separate 384 well plate at 7.times. in
growth media with no T-STIM (7.times.=700 ng/ml). IL-4 was included
as a positive control (7.times.=1,000 ng/ml, [final]=143 ng/ml).
Second, 30 .mu.l of 1.67.times.10.sup.4cells/ml MC/9 cells were
plated in a white 384-well TC treated plate (500 cells/well) in
media with no T-STIM (washing cells prior to assay was not
necessary). Third, 5 .mu.l of diluted IL10 antibody cytokine
engrafted protein was added to the cells. Plate was briefly mixed
using a plate mixer, and then pulse spun at 1,000 rpm. Plate was
covered with custom porous lid to aerate cells. Fourth, plate was
incubated at 37.degree. C. for 72 hours. Fifth, 30 .mu.l of Promega
Cell Titer Glo.RTM. was added to the cells, incubated for 10 min at
room temp and read on Envision.RTM. (0.1 sec read).
B Cell Proliferation
[0305] PBMCs were isolated from human buffy coats as described
above. For the isolation of B cells, the human B cell enrichment
kit.RTM. (Stemcell, 19054) was used according to manufacturer's
protocol for the Big Easy Magnet.RTM. (Stemcell, 18001). After
isolation, the B cells were washed with B cell medium (BCM),
counted and resuspended at a concentration of 4.times.10.sup.5
cells/ml. BCM contains RPMI 1640, 10% fetal bovine serum (Gibco,
10082147), 1% Penicillin-Streptomycin (Gibco, 15070063) and 2 mM
GlutaMax (Gibco, 35050-038), 1% Non-essential amino acids (Gibco,
11140035) and 1 mM sodium pyruvate (Gibco, 11360-039), 50 U/ml
penicillin and 50 .mu.g/ml streptomycin. Isolated B cells were
counted and resuspended at a concentration of
4.times.10.sup.5cells/ml in BCM. To stimulate the B cells, 8.4
.mu.g/ml anti-CD40 and 40 U/ml recombinant human IL-2 (R&D
systems, 202-IL-50) were added to the suspension. B cells were then
added at a concentration of 4.times.10.sup.4 cells in 100 .mu.l
into wells of Nunclon Delta Surface.RTM. 96 well round bottom
plates (Thermo Scientific, 163320). IL10 antibody engrafted protein
pre-dilutions at the double the final concentration were prepared
in a separate plate. A titration of the compounds was performed
obtaining the following concentrations: 40, 4, 0.4, 0.04, 0.004 and
0 nM. 100 .mu.l of these pre-dilutions were added in duplicates to
the 100.mu.1 cell suspension resulting in the final concentration
of the compounds of 20, 2, 0.2, 0.02, 0.002 and 0 nM and a final
concentration of 4.2 mg/ml anti-CD40 and 20 U/ml recombinant IL-2.
The plate was incubated for 5 days in a cell culture incubator
(37.degree. C., 5% CO.sub.2, 95% humidity). After 5 days of
incubation, proliferation of B cells was determined by the
thymidine incorporation assay. Cells were pulsed with 0.5 .mu.Ci
3H-thymidine (ANAWA, ART-178) per well in 20 .mu.l BCM for the
final 16 h of the culture period at 37.degree. C. Using the TomTec
9600.RTM. harvester, cells were harvested onto a membrane according
to manufacturer's protocol. Membranes were sealed in a bag,
scintillation liquid was added and radioactivity was measured on
scintillation counter. For each of the antibody engrafted proteins,
the percentage of proliferation at 20 nM ("top") was calculated
relative to the proliferation at an equimolar concentration of
rhIL10, in relation to the respective background cytokine
production ("bottom"). The resulting % of max values were
calculated using the following formula:
% of
max=(top.sub.compound-bottom.sub.compound)/(top.sub.rhIL-10-bottom.-
sub.rHIL-10) * 100
Means of different experimental series were determined, and the
standard error of the mean (SEM) was calculated if the compound was
measured on more than two donors.
[0306] It is understood that the examples and embodiments described
herein are for illustrative purposes and that various modifications
or changes in light thereof will be suggested to persons skilled in
the art and are to be included within the spirit and purview of
this application and scope of the appended claims. All
publications, sequence accession numbers, patents, and patent
applications cited herein are hereby incorporated by reference in
their entirety for all purposes.
Sequence CWU 1
1
25317PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 1Gly Phe Thr Phe Ser Ser Tyr1
526PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 2Ser Gly Ser Gly Gly Ser1
536PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 3Thr Arg Thr Lys Arg Phe1
54174PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 4Ser Gln Ser Val Ser Pro Gly Gln
Gly Thr Gln Ser Glu Asn Ser Cys1 5 10 15Thr His Phe Pro Gly Asn Leu
Pro Asn Met Leu Arg Asp Leu Arg Asp 20 25 30Ala Phe Ser Arg Val Lys
Thr Phe Phe Gln Met Lys Asp Gln Leu Asp 35 40 45Asn Leu Leu Leu Lys
Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu 50 55 60Gly Cys Gln Ala
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val65 70 75 80Met Pro
Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn 85 90 95Ser
Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys 100 105
110His Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser
115 120 125Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln
Glu Lys 130 135 140Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe
Ile Asn Tyr Ile145 150 155 160Glu Ala Tyr Met Thr Met Lys Ile Arg
Asn Ser Ser Ser Tyr 165 17053PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 5Gly Ala Ser166PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 6Tyr Gly Ser Ser Pro Leu1 575PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 7Ser Tyr Ala Met Ser1 5817PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 8Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Gly Asp Ser
Val Lys1 5 10 15Gly96PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 9Thr Arg Thr Lys Arg Phe1
510178PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 10Arg Ala Ser Gln Ser Val Ser Pro
Gly Gln Gly Thr Gln Ser Glu Asn1 5 10 15Ser Cys Thr His Phe Pro Gly
Asn Leu Pro Asn Met Leu Arg Asp Leu 20 25 30Arg Asp Ala Phe Ser Arg
Val Lys Thr Phe Phe Gln Met Lys Asp Gln 35 40 45Leu Asp Asn Leu Leu
Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly 50 55 60Tyr Leu Gly Cys
Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu65 70 75 80Glu Val
Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His 85 90 95Val
Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg 100 105
110Arg Cys His Arg Phe Leu Pro Cys Glu Gly Gly Gly Ser Gly Gly Asn
115 120 125Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys
Leu Gln 130 135 140Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
Ile Phe Ile Asn145 150 155 160Tyr Ile Glu Ala Tyr Met Thr Met Lys
Ile Arg Asn Ser Ser Ser Tyr 165 170 175Leu Ala117PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 11Gly Ala Ser Ser Arg Ala Thr1 5129PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 12Gln Gln Tyr Gly Ser Ser Pro Leu Thr1 513115PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 13Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr
Tyr Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Arg Thr Lys
Arg Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser
11514274PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 14Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Pro Gly 20 25 30Gln Gly Thr
Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu 35 40 45Pro Asn
Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr 50 55 60Phe
Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser65 70 75
80Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu
85 90 95Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn
Gln 100 105 110Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
Asn Leu Lys 115 120 125Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg
Phe Leu Pro Cys Glu 130 135 140Asn Gly Gly Gly Ser Gly Gly Lys Ser
Lys Ala Val Glu Gln Val Lys145 150 155 160Asn Ala Phe Asn Lys Leu
Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser 165 170 175Glu Phe Asp Ile
Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys 180 185 190Ile Arg
Asn Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 195 200
205Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly
210 215 220Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu225 230 235 240Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln 245 250 255Gln Tyr Gly Ser Ser Pro Leu Thr Phe
Gly Gly Gly Thr Lys Val Glu 260 265 270Ile Lys15445PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 15Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr
Tyr Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Arg Thr Lys
Arg Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135
140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys 195 200 205Val Asp Lys Arg Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250
255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys 275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375
380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 435 440 44516381PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 16Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Pro Gly 20 25 30Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His
Phe Pro Gly Asn Leu 35 40 45Pro Asn Met Leu Arg Asp Leu Arg Asp Ala
Phe Ser Arg Val Lys Thr 50 55 60Phe Phe Gln Met Lys Asp Gln Leu Asp
Asn Leu Leu Leu Lys Glu Ser65 70 75 80Leu Leu Glu Asp Phe Lys Gly
Tyr Leu Gly Cys Gln Ala Leu Ser Glu 85 90 95Met Ile Gln Phe Tyr Leu
Glu Glu Val Met Pro Gln Ala Glu Asn Gln 100 105 110Asp Pro Asp Ile
Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys 115 120 125Thr Leu
Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu 130 135
140Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Val
Lys145 150 155 160Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr
Lys Ala Met Ser 165 170 175Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu
Ala Tyr Met Thr Met Lys 180 185 190Ile Arg Asn Ser Ser Ser Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly 195 200 205Gln Ala Pro Arg Leu Leu
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly 210 215 220Ile Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu225 230 235 240Thr
Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln 245 250
255Gln Tyr Gly Ser Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu
260 265 270Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser 275 280 285Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn 290 295 300Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala305 310 315 320Leu Gln Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys 325 330 335Asp Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 340 345 350Tyr Glu Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 355 360 365Ser
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 370 375
380177PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 17Gly Phe Thr Phe Ser Ser Tyr1
5186PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 18Ser Gly Ser Gly Gly Ser1
5196PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 19Thr Arg Thr Lys Arg Phe1
5208PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 20Ser Gln Ser Val Ser Ser Ser Tyr1
521170PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 21Gly Ala Ser Ser Ser Pro Gly Gln
Gly Thr Gln Ser Glu Asn Ser Cys1 5 10 15Thr His Phe Pro Gly Asn Leu
Pro Asn Met Leu Arg Asp Leu Arg Asp 20 25 30Ala Phe Ser Arg Val Lys
Thr Phe Phe Gln Met Lys Asp Gln Leu Asp 35 40 45Asn Leu Leu Leu Lys
Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu 50 55 60Gly Cys Gln Ala
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val65 70 75 80Met Pro
Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn 85 90 95Ser
Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys 100 105
110His Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser
115 120 125Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln
Glu Lys 130 135 140Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe
Ile Asn Tyr Ile145 150 155 160Glu Ala Tyr Met Thr Met Lys Ile Arg
Asn 165 170226PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 22Tyr Gly Ser Ser Pro Leu1
5235PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 23Ser Tyr Ala Met Ser1
52417PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 24Ala Ile Ser Gly Ser Gly Gly Ser Thr
Tyr Tyr Gly Asp Ser Val Lys1 5 10 15Gly256PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 25Thr Arg Thr Lys Arg Phe1 52612PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 26Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala1 5
1027173PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 27Gly Ala Ser Ser Ser Pro Gly Gln
Gly Thr Gln Ser Glu Asn Ser Cys1 5 10 15Thr His Phe Pro Gly Asn Leu
Pro Asn Met Leu Arg Asp Leu Arg Asp 20 25 30Ala Phe Ser Arg Val Lys
Thr Phe Phe Gln Met Lys Asp Gln Leu Asp 35 40 45Asn Leu Leu Leu Lys
Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu 50 55 60Gly Cys Gln Ala
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val65 70 75 80Met Pro
Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn 85 90 95Ser
Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys 100 105
110His Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser
115 120 125Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln
Glu Lys 130 135 140Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe
Ile Asn Tyr Ile145 150 155 160Glu Ala Tyr Met Thr Met Lys Ile Arg
Asn Arg Ala Thr 165 170289PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 28Gln Gln Tyr Gly Ser Ser Pro Leu Thr1 529115PRTArtificial
Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 29Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly
Ser Gly Gly Ser Thr Tyr Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Thr Arg Thr Lys Arg Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105
110Val Ser Ser 11530274PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 30Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Ser Pro Gly Gln
Gly Thr Gln Ser Glu Asn 50 55 60Ser Cys Thr His Phe Pro Gly Asn Leu
Pro Asn Met Leu Arg Asp Leu65 70 75 80Arg Asp Ala Phe Ser Arg Val
Lys Thr Phe Phe Gln Met Lys Asp Gln 85 90 95Leu Asp Asn Leu Leu Leu
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly 100 105 110Tyr Leu Gly Cys
Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu 115 120 125Glu Val
Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His 130 135
140Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu
Arg145 150 155 160Arg Cys His Arg Phe Leu Pro Cys Glu Asn Gly Gly
Gly Ser Gly Gly 165 170 175Lys Ser Lys Ala Val Glu Gln Val Lys Asn
Ala Phe Asn Lys Leu Gln 180 185 190Glu Lys Gly Ile Tyr Lys Ala Met
Ser Glu Phe Asp Ile Phe Ile Asn 195 200 205Tyr Ile Glu Ala Tyr Met
Thr Met Lys Ile Arg Asn Arg Ala Thr Gly 210 215 220Ile Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu225 230 235 240Thr
Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln 245 250
255Gln Tyr Gly Ser Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu
260 265 270Ile Lys31445PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 31Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr
Tyr Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Arg Thr Lys
Arg Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135
140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys 195 200 205Val Asp Lys Arg Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250
255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys 275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375
380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly385 390 395 400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 435 440 44532381PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 32Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Ser Pro Gly Gln
Gly Thr Gln Ser Glu Asn 50 55 60Ser Cys Thr His Phe Pro Gly Asn Leu
Pro Asn Met Leu Arg Asp Leu65 70 75 80Arg Asp Ala Phe Ser Arg Val
Lys Thr Phe Phe Gln Met Lys Asp Gln 85 90 95Leu Asp Asn Leu Leu Leu
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly 100 105 110Tyr Leu Gly Cys
Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu 115 120 125Glu Val
Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His 130 135
140Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu
Arg145 150 155 160Arg Cys His Arg Phe Leu Pro Cys Glu Asn Gly Gly
Gly Ser Gly Gly 165 170 175Lys Ser Lys Ala Val Glu Gln Val Lys Asn
Ala Phe Asn Lys Leu Gln 180 185 190Glu Lys Gly Ile Tyr Lys Ala Met
Ser Glu Phe Asp Ile Phe Ile Asn 195 200 205Tyr Ile Glu Ala Tyr Met
Thr Met Lys Ile Arg Asn Arg Ala Thr Gly 210 215 220Ile Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu225 230 235 240Thr
Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln 245 250
255Gln Tyr Gly Ser Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu
260 265 270Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser 275 280 285Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn 290 295 300Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala305 310 315 320Leu Gln Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys 325 330 335Asp Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 340 345 350Tyr Glu Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 355 360 365Ser
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 370 375
380337PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 33Gly Phe Thr Phe Ser Ser Tyr1
5346PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 34Ser Gly Ser Gly Gly Ser1
5356PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 35Thr Arg Thr Lys Arg Phe1
5368PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 36Ser Gln Ser Val Ser Ser Ser Tyr1
5373PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 37Gly Ala Ser138172PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 38Tyr Gly Ser Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn
Ser Cys Thr1 5 10 15His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp
Leu Arg Asp Ala 20 25 30Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys
Asp Gln Leu Asp Asn 35 40 45Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp
Phe Lys Gly Tyr Leu Gly 50 55 60Cys Gln Ala Leu Ser Glu Met Ile Gln
Phe Tyr Leu Glu Glu Val Met65 70 75 80Pro Gln Ala Glu Asn Gln Asp
Pro Asp Ile Lys Ala His Val Asn Ser 85 90 95Leu Gly Glu Asn Leu Lys
Thr Leu Arg Leu Arg Leu Arg Arg Cys His 100 105 110Arg Phe Leu Pro
Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys 115 120 125Ala Val
Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly 130 135
140Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile
Glu145 150 155 160Ala Tyr Met Thr Met Lys Ile Arg Asn Ser Pro Leu
165 170395PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 39Ser Tyr Ala Met Ser1
54017PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 40Ala Ile Ser Gly Ser Gly Gly Ser Thr
Tyr Tyr Gly Asp Ser Val Lys1 5 10 15Gly416PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 41Thr Arg Thr Lys Arg Phe1 54212PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 42Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala1 5
10437PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 43Gly Ala Ser Ser Arg Ala Thr1
544175PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 44Gln Gln Tyr Gly Ser Ser Pro Gly
Gln Gly Thr Gln Ser Glu Asn Ser1 5 10 15Cys Thr His Phe Pro Gly Asn
Leu Pro Asn Met Leu Arg Asp Leu Arg 20 25 30Asp Ala Phe Ser Arg Val
Lys Thr Phe Phe Gln Met Lys Asp Gln Leu 35 40 45Asp Asn Leu Leu Leu
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr 50 55 60Leu Gly Cys Gln
Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu65 70 75 80Val Met
Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val 85 90 95Asn
Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg 100 105
110Cys His Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys
115 120 125Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu
Gln Glu 130 135 140Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile
Phe Ile Asn Tyr145 150 155 160Ile Glu Ala Tyr Met Thr Met Lys Ile
Arg Asn Ser Pro Leu Thr 165 170 17545115PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 45Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr
Tyr Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Arg Thr Lys
Arg Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser
11546274PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 46Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
85 90 95Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly
Asn 100 105 110Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser
Arg Val Lys 115 120 125Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn
Leu Leu Leu Lys Glu 130 135 140Ser Leu Leu Glu Asp Phe Lys Gly Tyr
Leu Gly Cys Gln Ala Leu Ser145 150 155 160Glu Met Ile Gln Phe Tyr
Leu Glu Glu Val Met Pro Gln Ala Glu Asn 165 170 175Gln Asp Pro Asp
Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu 180 185 190Lys Thr
Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys 195 200
205Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Val
210 215 220Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
Ala Met225 230 235 240Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu
Ala Tyr Met Thr Met 245 250 255Lys Ile Arg Asn Ser Pro Leu Thr Phe
Gly Gly Gly Thr Lys Val Glu 260 265 270Ile Lys47445PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 47Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr
Tyr Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Arg Thr Lys
Arg Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135
140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly 165 170
175Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys 195 200 205Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295
300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys305 310 315 320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser 340 345 350Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410
415Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435
440 44548381PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 48Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
85 90 95Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly
Asn 100 105 110Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser
Arg Val Lys 115 120 125Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn
Leu Leu Leu Lys Glu 130 135 140Ser Leu Leu Glu Asp Phe Lys Gly Tyr
Leu Gly Cys Gln Ala Leu Ser145 150 155 160Glu Met Ile Gln Phe Tyr
Leu Glu Glu Val Met Pro Gln Ala Glu Asn 165 170 175Gln Asp Pro Asp
Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu 180 185 190Lys Thr
Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys 195 200
205Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Val
210 215 220Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
Ala Met225 230 235 240Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu
Ala Tyr Met Thr Met 245 250 255Lys Ile Arg Asn Ser Pro Leu Thr Phe
Gly Gly Gly Thr Lys Val Glu 260 265 270Ile Lys Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser 275 280 285Asp Glu Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 290 295 300Asn Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala305 310 315
320Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
325 330 335Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp 340 345 350Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu 355 360 365Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys 370 375 38049173PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 49Gly Phe Thr Phe Ser Pro Gly Gln Gly Thr Gln Ser Glu
Asn Ser Cys1 5 10 15Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg
Asp Leu Arg Asp 20 25 30Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met
Lys Asp Gln Leu Asp 35 40 45Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu
Asp Phe Lys Gly Tyr Leu 50 55 60Gly Cys Gln Ala Leu Ser Glu Met Ile
Gln Phe Tyr Leu Glu Glu Val65 70 75 80Met Pro Gln Ala Glu Asn Gln
Asp Pro Asp Ile Lys Ala His Val Asn 85 90 95Ser Leu Gly Glu Asn Leu
Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys 100 105 110His Arg Phe Leu
Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser 115 120 125Lys Ala
Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys 130 135
140Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr
Ile145 150 155 160Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Ser Ser
Tyr 165 170506PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 50Ser Gly Ser Gly Gly Ser1
5516PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 51Thr Arg Thr Lys Arg Phe1
5528PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 52Ser Gln Ser Val Ser Ser Ser Tyr1
5533PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 53Gly Ala Ser1546PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 54Tyr Gly Ser Ser Pro Leu1 555172PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 55Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr
His Phe Pro1 5 10 15Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp
Ala Phe Ser Arg 20 25 30Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu
Asp Asn Leu Leu Leu 35 40 45Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly
Tyr Leu Gly Cys Gln Ala 50 55 60Leu Ser Glu Met Ile Gln Phe Tyr Leu
Glu Glu Val Met Pro Gln Ala65 70 75 80Glu Asn Gln Asp Pro Asp Ile
Lys Ala His Val Asn Ser Leu Gly Glu 85 90 95Asn Leu Lys Thr Leu Arg
Leu Arg Leu Arg Arg Cys His Arg Phe Leu 100 105 110Pro Cys Glu Asn
Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu 115 120 125Gln Val
Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys 130 135
140Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr
Met145 150 155 160Thr Met Lys Ile Arg Asn Ser Ser Tyr Ala Met Ser
165 1705617PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 56Ala Ile Ser Gly Ser Gly
Gly Ser Thr Tyr Tyr Gly Asp Ser Val Lys1 5 10 15Gly576PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 57Thr Arg Thr Lys Arg Phe1 55812PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 58Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala1 5
10597PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 59Gly Ala Ser Ser Arg Ala Thr1
5609PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 60Gln Gln Tyr Gly Ser Ser Pro Leu Thr1
561281PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 61Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Pro Gly 20 25 30Gln Gly Thr Gln Ser Glu
Asn Ser Cys Thr His Phe Pro Gly Asn Leu 35 40 45Pro Asn Met Leu Arg
Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr 50 55 60Phe Phe Gln Met
Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser65 70 75 80Leu Leu
Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu 85 90 95Met
Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln 100 105
110Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys
115 120 125Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro
Cys Glu 130 135 140Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val
Glu Gln Val Lys145 150 155 160Asn Ala Phe Asn Lys Leu Gln Glu Lys
Gly Ile Tyr Lys Ala Met Ser 165 170 175Glu Phe Asp Ile Phe Ile Asn
Tyr Ile Glu Ala Tyr Met Thr Met Lys 180 185 190Ile Arg Asn Ser Ser
Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 195 200 205Lys Gly Leu
Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 210 215 220Tyr
Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn225 230
235 240Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp 245 250 255Thr Ala Val Tyr Tyr Cys Ala Arg Thr Arg Thr Lys Arg
Phe Trp Gly 260 265 270Gln Gly Thr Leu Val Thr Val Ser Ser 275
28062108PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 62Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
85 90 95Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10563611PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 63Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Gly 20 25 30Gln Gly Thr
Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu 35 40 45Pro Asn
Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr 50 55 60Phe
Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser65 70 75
80Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu
85 90 95Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn
Gln 100 105 110Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
Asn Leu Lys 115 120 125Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg
Phe Leu Pro Cys Glu 130 135 140Asn Gly Gly Gly Ser Gly Gly Lys Ser
Lys Ala Val Glu Gln Val Lys145 150 155 160Asn Ala Phe Asn Lys Leu
Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser 165 170 175Glu Phe Asp Ile
Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys 180 185 190Ile Arg
Asn Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 195 200
205Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr
210 215 220Tyr Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn225 230 235 240Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp 245 250 255Thr Ala Val Tyr Tyr Cys Ala Arg Thr
Arg Thr Lys Arg Phe Trp Gly 260 265 270Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 275 280 285Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 290 295 300Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val305 310 315
320Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
325 330 335Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 340 345 350Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 355 360 365Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys 370 375 380Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly385 390 395 400Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 405 410 415Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 420 425 430Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 435 440
445His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
450 455 460Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly465 470 475 480Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 485 490 495Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 500 505 510Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser 515 520 525Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 530 535 540Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro545 550 555
560Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
565 570 575Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 580 585 590His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 595 600 605Pro Gly Lys 61064215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 64Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly
Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95Leu Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135
140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn Arg Gly Glu
Cys 210 215657PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 65Gly Phe Thr Phe Ser Ser
Tyr1 566172PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 66Ser Gly Ser Gly Ser
Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys1 5 10 15Thr His Phe Pro
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp 20 25 30Ala Phe Ser
Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp 35 40 45Asn Leu
Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu 50 55 60Gly
Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val65 70 75
80Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn
85 90 95Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg
Cys 100 105 110His Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly
Gly Lys Ser 115 120 125Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn
Lys Leu Gln Glu Lys 130 135 140Gly Ile Tyr Lys Ala Met Ser Glu Phe
Asp Ile Phe Ile Asn Tyr Ile145 150 155 160Glu Ala Tyr Met Thr Met
Lys Ile Arg Asn Gly Ser 165 170676PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 67Thr Arg Thr Lys Arg Phe1 5688PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 68Ser Gln Ser Val Ser Ser Ser Tyr1 5693PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 69Gly Ala Ser1706PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 70Tyr Gly Ser Ser Pro Leu1 5715PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 71Ser Tyr Ala Met Ser1 572183PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 72Ala Ile Ser Gly Ser Gly Ser Pro Gly Gln Gly Thr Gln
Ser Glu Asn1 5 10 15Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met
Leu Arg Asp Leu 20 25 30Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe
Gln Met Lys Asp Gln 35 40 45Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu
Leu Glu Asp Phe Lys Gly 50 55 60Tyr Leu Gly Cys Gln Ala Leu Ser Glu
Met Ile Gln Phe Tyr Leu Glu65 70 75 80Glu Val Met Pro Gln Ala Glu
Asn Gln Asp Pro Asp Ile Lys Ala His 85 90 95Val Asn Ser Leu Gly Glu
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg 100 105 110Arg Cys His Arg
Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly 115 120 125Lys Ser
Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln 130 135
140Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile
Asn145 150 155 160Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
Gly Ser Thr Tyr 165 170 175Tyr Gly Asp Ser Val Lys Gly
180736PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 73Thr Arg Thr Lys Arg Phe1
57412PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 74Arg Ala Ser Gln Ser Val Ser Ser Ser
Tyr Leu Ala1 5 10757PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 75Gly Ala Ser Ser Arg Ala
Thr1 5769PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 76Gln Gln Tyr Gly Ser Ser
Pro Leu Thr1 577281PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 77Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Ser Gly Ser Pro Gly Gln Gly Thr Gln Ser Glu 50 55 60Asn
Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp65 70 75
80Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp
85 90 95Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe
Lys 100 105 110Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln
Phe Tyr Leu 115 120 125Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp
Pro Asp Ile Lys Ala 130 135 140His Val Asn Ser Leu Gly Glu Asn Leu
Lys Thr Leu Arg Leu Arg Leu145 150 155 160Arg Arg Cys His Arg Phe
Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly 165 170 175Gly Lys Ser Lys
Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu 180 185 190Gln Glu
Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile 195 200
205Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Gly Ser Thr
210 215 220Tyr Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn225 230 235 240Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp 245 250 255Thr Ala Val Tyr Tyr Cys Ala Arg Thr
Arg Thr Lys Arg Phe Trp Gly 260 265 270Gln Gly Thr Leu Val Thr Val
Ser Ser 275 28078108PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 78Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
85 90 95Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10579611PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 79Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Ser Gly Ser Pro Gly Gln Gly Thr Gln Ser Glu 50 55 60Asn
Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp65 70 75
80Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp
85 90 95Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe
Lys 100 105 110Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln
Phe Tyr Leu 115 120 125Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp
Pro Asp Ile Lys Ala 130 135 140His Val Asn Ser Leu Gly Glu Asn Leu
Lys Thr Leu Arg Leu Arg Leu145 150 155 160Arg Arg Cys His Arg Phe
Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly 165 170 175Gly Lys Ser Lys
Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu 180 185 190Gln Glu
Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile 195 200
205Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Gly Ser Thr
210 215 220Tyr Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn225 230 235 240Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp 245 250 255Thr Ala Val Tyr Tyr Cys Ala Arg Thr
Arg Thr Lys Arg Phe Trp Gly 260 265 270Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 275 280 285Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 290 295 300Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val305 310 315
320Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
325 330 335Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 340 345 350Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 355 360 365Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys 370 375 380Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly385 390 395 400Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 405 410 415Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 420 425 430Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 435 440
445His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
450 455 460Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly465 470 475 480Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 485 490 495Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 500 505 510Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser 515 520 525Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 530 535 540Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro545 550 555
560Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
565 570 575Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 580 585 590His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 595 600 605Pro Gly Lys 61080215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 80Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly
Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95Leu Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135
140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn Arg Gly Glu
Cys 210 215817PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 81Gly Phe Thr Phe Ser Ser
Tyr1 5826PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 82Ser Gly Ser Gly Gly Ser1
583172PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 83Thr Arg Thr Ser Pro Gly Gln Gly
Thr Gln Ser Glu Asn Ser Cys Thr1 5 10 15His Phe Pro Gly Asn Leu Pro
Asn Met Leu Arg Asp Leu Arg Asp Ala 20 25 30Phe Ser Arg Val Lys Thr
Phe Phe Gln Met Lys Asp Gln Leu Asp Asn 35 40 45Leu Leu Leu Lys Glu
Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly 50 55 60Cys Gln Ala Leu
Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met65 70 75 80Pro Gln
Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser 85 90 95Leu
Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His 100 105
110Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys
115 120 125Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu
Lys Gly 130 135 140Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile
Asn Tyr Ile Glu145 150 155 160Ala Tyr Met Thr Met Lys Ile Arg Asn
Lys Arg Phe 165 170848PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 84Ser Gln Ser Val Ser Ser Ser Tyr1 5853PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 85Gly Ala Ser1866PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 86Tyr Gly Ser Ser Pro Leu1 5875PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 87Ser Tyr Ala Met Ser1 58817PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 88Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Gly Asp Ser
Val Lys1 5 10 15Gly89172PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 89Thr Arg Thr Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn
Ser Cys Thr1 5 10 15His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp
Leu Arg Asp Ala 20 25 30Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys
Asp Gln Leu Asp Asn 35 40 45Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp
Phe Lys Gly Tyr Leu Gly 50 55 60Cys Gln Ala Leu Ser Glu Met Ile Gln
Phe Tyr Leu Glu Glu Val Met65 70 75 80Pro Gln Ala Glu Asn Gln Asp
Pro Asp Ile Lys Ala His Val Asn Ser 85 90 95Leu Gly Glu Asn Leu Lys
Thr Leu Arg Leu Arg Leu Arg Arg Cys His 100 105 110Arg Phe Leu Pro
Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys 115 120 125Ala Val
Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly 130 135
140Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile
Glu145 150 155 160Ala Tyr Met Thr Met Lys Ile Arg Asn Lys Arg Phe
165 1709012PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 90Arg Ala Ser Gln Ser Val
Ser Ser Ser
Tyr Leu Ala1 5 10917PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 91Gly Ala Ser Ser Arg Ala
Thr1 5929PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 92Gln Gln Tyr Gly Ser Ser
Pro Leu Thr1 593281PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 93Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Gly Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Thr Arg Thr Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn
Ser 100 105 110Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg
Asp Leu Arg 115 120 125Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln
Met Lys Asp Gln Leu 130 135 140Asp Asn Leu Leu Leu Lys Glu Ser Leu
Leu Glu Asp Phe Lys Gly Tyr145 150 155 160Leu Gly Cys Gln Ala Leu
Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu 165 170 175Val Met Pro Gln
Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val 180 185 190Asn Ser
Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg 195 200
205Cys His Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys
210 215 220Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu
Gln Glu225 230 235 240Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
Ile Phe Ile Asn Tyr 245 250 255Ile Glu Ala Tyr Met Thr Met Lys Ile
Arg Asn Lys Arg Phe Trp Gly 260 265 270Gln Gly Thr Leu Val Thr Val
Ser Ser 275 28094108PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 94Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
85 90 95Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10595611PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 95Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Gly Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Thr Arg Thr Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn
Ser 100 105 110Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg
Asp Leu Arg 115 120 125Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln
Met Lys Asp Gln Leu 130 135 140Asp Asn Leu Leu Leu Lys Glu Ser Leu
Leu Glu Asp Phe Lys Gly Tyr145 150 155 160Leu Gly Cys Gln Ala Leu
Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu 165 170 175Val Met Pro Gln
Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val 180 185 190Asn Ser
Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg 195 200
205Cys His Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys
210 215 220Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu
Gln Glu225 230 235 240Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
Ile Phe Ile Asn Tyr 245 250 255Ile Glu Ala Tyr Met Thr Met Lys Ile
Arg Asn Lys Arg Phe Trp Gly 260 265 270Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 275 280 285Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 290 295 300Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val305 310 315
320Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
325 330 335Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 340 345 350Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 355 360 365Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys 370 375 380Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly385 390 395 400Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 405 410 415Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 420 425 430Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 435 440
445His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
450 455 460Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly465 470 475 480Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 485 490 495Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 500 505 510Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser 515 520 525Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 530 535 540Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro545 550 555
560Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
565 570 575Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 580 585 590His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 595 600 605Pro Gly Lys 61096215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 96Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly
Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95Leu Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135
140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn Arg Gly Glu
Cys 210 215979PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 97Gly Phe Ser Leu Ser Thr
Ser Gly Met1 5985PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 98Trp Trp Asp Asp Lys1
59910PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 99Ser Met Ile Thr Asn Trp Tyr Phe Asp
Val1 5 10100172PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 100Gln Leu Ser Ser Pro
Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr1 5 10 15His Phe Pro Gly
Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala 20 25 30Phe Ser Arg
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn 35 40 45Leu Leu
Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly 50 55 60Cys
Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met65 70 75
80Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser
85 90 95Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys
His 100 105 110Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly
Lys Ser Lys 115 120 125Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys
Leu Gln Glu Lys Gly 130 135 140Ile Tyr Lys Ala Met Ser Glu Phe Asp
Ile Phe Ile Asn Tyr Ile Glu145 150 155 160Ala Tyr Met Thr Met Lys
Ile Arg Asn Val Gly Tyr 165 1701013PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 101Asp Thr Ser11026PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 102Gly Ser Gly Tyr Pro Phe1 51037PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 103Thr Ser Gly Met Ser Val Gly1 510416PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 104Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu
Lys Ser1 5 10 1510510PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 105Ser Met Ile Thr Asn
Trp Tyr Phe Asp Val1 5 10106176PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 106Lys Ala Gln Leu Ser Ser Pro Gly Gln Gly Thr Gln Ser
Glu Asn Ser1 5 10 15Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu
Arg Asp Leu Arg 20 25 30Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln
Met Lys Asp Gln Leu 35 40 45Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu
Glu Asp Phe Lys Gly Tyr 50 55 60Leu Gly Cys Gln Ala Leu Ser Glu Met
Ile Gln Phe Tyr Leu Glu Glu65 70 75 80Val Met Pro Gln Ala Glu Asn
Gln Asp Pro Asp Ile Lys Ala His Val 85 90 95Asn Ser Leu Gly Glu Asn
Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg 100 105 110Cys His Arg Phe
Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys 115 120 125Ser Lys
Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu 130 135
140Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn
Tyr145 150 155 160Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Val
Gly Tyr Met His 165 170 1751077PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 107Asp Thr Ser Lys Leu Ala Ser1 51089PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 108Phe Gln Gly Ser Gly Tyr Pro Phe Thr1
5109120PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 109Gln Val Thr Leu Arg Glu Ser Gly
Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile
Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Ala Asn Gln Val65 70 75 80Val Leu
Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys
Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115 120110272PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 110Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Gln Leu Ser
Ser Pro Gly Gln 20 25 30Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe
Pro Gly Asn Leu Pro 35 40 45Asn Met Leu Arg Asp Leu Arg Asp Ala Phe
Ser Arg Val Lys Thr Phe 50 55 60Phe Gln Met Lys Asp Gln Leu Asp Asn
Leu Leu Leu Lys Glu Ser Leu65 70 75 80Leu Glu Asp Phe Lys Gly Tyr
Leu Gly Cys Gln Ala Leu Ser Glu Met 85 90 95Ile Gln Phe Tyr Leu Glu
Glu Val Met Pro Gln Ala Glu Asn Gln Asp 100 105 110Pro Asp Ile Lys
Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr 115 120 125Leu Arg
Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn 130 135
140Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Val Lys
Asn145 150 155 160Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
Ala Met Ser Glu 165 170 175Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala
Tyr Met Thr Met Lys Ile 180 185 190Arg Asn Val Gly Tyr Met His Trp
Tyr Gln Gln Lys Pro Gly Lys Ala 195 200 205Pro Lys Leu Leu Ile Tyr
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro 210 215 220Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Thr Ile225 230 235 240Ser
Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly 245 250
255Ser Gly Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
260 265 270111450PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 111Gln Val Thr Leu Arg
Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu
Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Ser
Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu
Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60Leu
Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ala Asn Gln Val65 70 75
80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr
85 90 95Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val
Trp Gly Ala 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310
315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425
430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445Gly Lys 450112379PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 112Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Gln Leu Ser
Ser Pro Gly Gln 20 25 30Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe
Pro Gly Asn Leu Pro 35 40 45Asn Met Leu Arg Asp Leu Arg Asp Ala Phe
Ser Arg Val Lys Thr Phe 50 55 60Phe Gln Met Lys Asp Gln Leu Asp Asn
Leu Leu Leu Lys Glu Ser Leu65 70 75 80Leu Glu Asp Phe Lys Gly Tyr
Leu Gly Cys Gln Ala Leu Ser Glu Met 85 90 95Ile Gln Phe Tyr Leu Glu
Glu Val Met Pro Gln Ala Glu Asn Gln Asp 100 105 110Pro Asp Ile Lys
Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr 115 120 125Leu Arg
Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn 130 135
140Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Val Lys
Asn145 150 155 160Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
Ala Met Ser Glu 165 170 175Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala
Tyr Met Thr Met Lys Ile 180 185 190Arg Asn Val Gly Tyr Met His Trp
Tyr Gln Gln Lys Pro Gly Lys Ala 195 200 205Pro Lys Leu Leu Ile Tyr
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro 210 215 220Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Thr Ile225 230 235 240Ser
Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly 245 250
255Ser Gly Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
260 265 270Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu 275 280 285Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe 290 295 300Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln305 310 315 320Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 325 330 335Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 340 345 350Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 355 360 365Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 370 3751139PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 113Gly Phe Ser Leu Ser Thr Ser Gly Met1 51145PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 114Trp Trp Asp Asp Lys1 511510PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 115Ser Met Ile Thr Asn Trp Tyr Phe Asp Val1 5
101166PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 116Gln Leu Ser Val Gly Tyr1
5117169PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 117Asp Thr Ser Pro Gly Gln Gly Thr
Gln Ser Glu Asn Ser Cys Thr His1 5 10 15Phe Pro Gly Asn Leu Pro Asn
Met Leu Arg Asp Leu Arg Asp Ala Phe 20 25 30Ser Arg Val Lys Thr Phe
Phe Gln Met Lys Asp Gln Leu Asp Asn Leu 35 40 45Leu Leu Lys Glu Ser
Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys 50 55 60Gln Ala Leu Ser
Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro65 70 75 80Gln Ala
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu 85 90 95Gly
Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg 100 105
110Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala
115 120 125Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys
Gly Ile 130 135 140Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn
Tyr Ile Glu Ala145 150 155 160Tyr Met Thr Met Lys Ile Arg Asn Ser
1651186PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 118Gly Ser Gly Tyr Pro Phe1
51197PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 119Thr Ser Gly Met Ser Val Gly1
512016PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 120Asp Ile Trp Trp Asp Asp Lys Lys Asp
Tyr Asn Pro Ser Leu Lys Ser1 5 10 1512110PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 121Ser Met Ile Thr Asn Trp Tyr Phe Asp Val1 5
1012210PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 122Lys Ala Gln Leu Ser Val Gly Tyr Met
His1 5 10123173PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 123Asp Thr Ser Pro Gly
Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His1 5 10 15Phe Pro Gly Asn
Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe 20 25 30Ser Arg Val
Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu 35 40 45Leu Leu
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys 50 55 60Gln
Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro65 70 75
80Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu
85 90 95Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His
Arg 100 105 110Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys
Ser Lys Ala 115 120 125Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu
Gln Glu Lys Gly Ile 130 135 140Tyr Lys Ala Met Ser Glu Phe Asp Ile
Phe Ile Asn Tyr Ile Glu Ala145 150 155 160Tyr Met Thr Met Lys Ile
Arg Asn Ser Lys Leu Ala Ser 165 1701249PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 124Phe Gln Gly Ser Gly Tyr Pro Phe Thr1
5125120PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 125Gln Val Thr Leu Arg Glu Ser Gly
Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile
Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Ala Asn Gln Val65 70 75 80Val Leu
Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys
Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115 120126272PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 126Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Gln Leu Ser
Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Pro Gly Gln Gly Thr Gln Ser
Glu Asn Ser Cys Thr His 50 55 60Phe Pro Gly Asn Leu Pro Asn Met Leu
Arg Asp Leu Arg Asp Ala Phe65 70 75 80Ser Arg Val Lys Thr Phe Phe
Gln Met Lys Asp Gln Leu Asp Asn Leu 85 90 95Leu Leu Lys Glu Ser Leu
Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys 100 105 110Gln Ala Leu Ser
Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro 115 120 125Gln Ala
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu 130 135
140Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His
Arg145 150 155 160Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly
Lys Ser Lys Ala 165 170 175Val Glu Gln Val Lys Asn Ala Phe Asn Lys
Leu Gln Glu Lys Gly Ile 180 185 190Tyr Lys Ala Met Ser Glu Phe Asp
Ile Phe Ile Asn Tyr Ile Glu Ala 195 200 205Tyr Met Thr Met Lys Ile
Arg Asn Ser Lys Leu Ala Ser Gly Val Pro 210 215 220Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Thr Ile225 230 235 240Ser
Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly 245 250
255Ser Gly Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
260 265 270127450PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 127Gln Val Thr Leu Arg
Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu
Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Ser
Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu
Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60Leu
Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ala Asn Gln Val65 70 75
80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr
85 90 95Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly
Ala 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly Lys 450128379PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 128Asp Ile Gln Met
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Lys Ala Gln Leu Ser Val Gly Tyr Met 20 25 30His Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp
Thr Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His 50 55
60Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe65
70 75 80Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn
Leu 85 90 95Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu
Gly Cys 100 105 110Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu
Glu Val Met Pro 115 120 125Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys
Ala His Val Asn Ser Leu 130 135 140Gly Glu Asn Leu Lys Thr Leu Arg
Leu Arg Leu Arg Arg Cys His Arg145 150 155 160Phe Leu Pro Cys Glu
Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala 165 170 175Val Glu Gln
Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile 180 185 190Tyr
Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala 195 200
205Tyr Met Thr Met Lys Ile Arg Asn Ser Lys Leu Ala Ser Gly Val Pro
210 215 220Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu
Thr Ile225 230 235 240Ser Ser Leu Gln
Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly 245 250 255Ser Gly
Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 260 265
270Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
275 280 285Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe 290 295 300Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln305 310 315 320Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser 325 330 335Thr Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu 340 345 350Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 355 360 365Pro Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 370 3751299PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 129Gly Phe Ser Leu Ser Thr Ser Gly Met1 51305PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 130Trp Trp Asp Asp Lys1 513110PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 131Ser Met Ile Thr Asn Trp Tyr Phe Asp Val1 5
101326PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 132Gln Leu Ser Val Gly Tyr1
51333PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 133Asp Thr Ser1134172PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 134Gly Ser Gly Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn
Ser Cys Thr1 5 10 15His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp
Leu Arg Asp Ala 20 25 30Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys
Asp Gln Leu Asp Asn 35 40 45Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp
Phe Lys Gly Tyr Leu Gly 50 55 60Cys Gln Ala Leu Ser Glu Met Ile Gln
Phe Tyr Leu Glu Glu Val Met65 70 75 80Pro Gln Ala Glu Asn Gln Asp
Pro Asp Ile Lys Ala His Val Asn Ser 85 90 95Leu Gly Glu Asn Leu Lys
Thr Leu Arg Leu Arg Leu Arg Arg Cys His 100 105 110Arg Phe Leu Pro
Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys 115 120 125Ala Val
Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly 130 135
140Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile
Glu145 150 155 160Ala Tyr Met Thr Met Lys Ile Arg Asn Tyr Pro Phe
165 1701357PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 135Thr Ser Gly Met Ser Val
Gly1 513616PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 136Asp Ile Trp Trp Asp Asp
Lys Lys Asp Tyr Asn Pro Ser Leu Lys Ser1 5 10 1513710PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 137Ser Met Ile Thr Asn Trp Tyr Phe Asp Val1 5
1013810PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 138Lys Ala Gln Leu Ser Val Gly Tyr Met
His1 5 101397PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 139Asp Thr Ser Lys Leu Ala
Ser1 5140175PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 140Phe Gln Gly Ser Gly
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser1 5 10 15Cys Thr His Phe
Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg 20 25 30Asp Ala Phe
Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu 35 40 45Asp Asn
Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr 50 55 60Leu
Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu65 70 75
80Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val
85 90 95Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg
Arg 100 105 110Cys His Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser
Gly Gly Lys 115 120 125Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
Asn Lys Leu Gln Glu 130 135 140Lys Gly Ile Tyr Lys Ala Met Ser Glu
Phe Asp Ile Phe Ile Asn Tyr145 150 155 160Ile Glu Ala Tyr Met Thr
Met Lys Ile Arg Asn Tyr Pro Phe Thr 165 170 175141120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 141Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys
Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser
Leu Ser Thr Ser 20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro
Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys
Lys Asp Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys
Asp Thr Ser Ala Asn Gln Val65 70 75 80Val Leu Lys Val Thr Asn Met
Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Met Ile
Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105 110Gly Thr Thr Val
Thr Val Ser Ser 115 120142272PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 142Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Gln Leu Ser
Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ala Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys
Phe Gln Gly Ser Gly Ser Pro Gly Gln 85 90 95Gly Thr Gln Ser Glu Asn
Ser Cys Thr His Phe Pro Gly Asn Leu Pro 100 105 110Asn Met Leu Arg
Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe 115 120 125Phe Gln
Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu 130 135
140Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu
Met145 150 155 160Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
Glu Asn Gln Asp 165 170 175Pro Asp Ile Lys Ala His Val Asn Ser Leu
Gly Glu Asn Leu Lys Thr 180 185 190Leu Arg Leu Arg Leu Arg Arg Cys
His Arg Phe Leu Pro Cys Glu Asn 195 200 205Gly Gly Gly Ser Gly Gly
Lys Ser Lys Ala Val Glu Gln Val Lys Asn 210 215 220Ala Phe Asn Lys
Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu225 230 235 240Phe
Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile 245 250
255Arg Asn Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
260 265 270143450PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 143Gln Val Thr Leu Arg
Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu
Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Ser
Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu
Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60Leu
Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ala Asn Gln Val65 70 75
80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr
85 90 95Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly
Ala 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly Lys 450144379PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 144Asp Ile Gln Met
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Lys Ala Gln Leu Ser Val Gly Tyr Met 20 25 30His Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp
Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55
60Gly Ser Gly Thr Ala Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65
70 75 80Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Ser Pro Gly
Gln 85 90 95Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn
Leu Pro 100 105 110Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
Val Lys Thr Phe 115 120 125Phe Gln Met Lys Asp Gln Leu Asp Asn Leu
Leu Leu Lys Glu Ser Leu 130 135 140Leu Glu Asp Phe Lys Gly Tyr Leu
Gly Cys Gln Ala Leu Ser Glu Met145 150 155 160Ile Gln Phe Tyr Leu
Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp 165 170 175Pro Asp Ile
Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr 180 185 190Leu
Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn 195 200
205Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Val Lys Asn
210 215 220Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met
Ser Glu225 230 235 240Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr
Met Thr Met Lys Ile 245 250 255Arg Asn Tyr Pro Phe Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 260 265 270Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 275 280 285Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 290 295 300Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln305 310 315
320Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
325 330 335Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu 340 345 350Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 355 360 365Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 370 375145175PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 145Gly Phe Ser Leu Ser
Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys1 5 10 15Thr His Phe Pro
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp 20 25 30Ala Phe Ser
Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp 35 40 45Asn Leu
Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu 50 55 60Gly
Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val65 70 75
80Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn
85 90 95Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg
Cys 100 105 110His Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly
Gly Lys Ser 115 120 125Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn
Lys Leu Gln Glu Lys 130 135 140Gly Ile Tyr Lys Ala Met Ser Glu Phe
Asp Ile Phe Ile Asn Tyr Ile145 150 155 160Glu Ala Tyr Met Thr Met
Lys Ile Arg Asn Ser Thr Ser Gly Met 165 170 1751465PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 146Trp Trp Asp Asp Lys1 514710PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 147Ser Met Ile Thr Asn Trp Tyr Phe Asp Val1 5
101486PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 148Gln Leu Ser Val Gly Tyr1
51493PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 149Asp Thr Ser11506PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 150Gly Ser Gly Tyr Pro Phe1 5151174PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 151Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr
His Phe Pro1 5 10 15Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp
Ala Phe Ser Arg 20 25 30Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu
Asp Asn Leu Leu Leu 35 40 45Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly
Tyr Leu Gly Cys Gln Ala 50 55 60Leu Ser Glu Met Ile Gln Phe Tyr Leu
Glu Glu Val Met Pro Gln Ala65 70 75 80Glu Asn Gln Asp Pro Asp Ile
Lys Ala His Val Asn Ser Leu Gly Glu 85 90 95Asn Leu Lys Thr Leu Arg
Leu Arg Leu Arg Arg Cys His Arg Phe Leu 100 105 110Pro Cys Glu Asn
Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu 115 120 125Gln Val
Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
130 135 140Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala
Tyr Met145 150 155 160Thr Met Lys Ile Arg Asn Ser Thr Ser Gly Met
Ser Val Gly 165 17015216PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 152Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu
Lys Ser1 5 10 1515310PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 153Ser Met Ile Thr Asn
Trp Tyr Phe Asp Val1 5 1015410PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 154Lys Ala Gln Leu Ser Val Gly Tyr Met His1 5
101557PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 155Asp Thr Ser Lys Leu Ala Ser1
51569PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 156Phe Gln Gly Ser Gly Tyr Pro Phe Thr1
5157286PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 157Gln Val Thr Leu Arg Glu Ser Gly
Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr
Phe Ser Gly Phe Ser Leu Ser Pro Gly 20 25 30Gln Gly Thr Gln Ser Glu
Asn Ser Cys Thr His Phe Pro Gly Asn Leu 35 40 45Pro Asn Met Leu Arg
Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr 50 55 60Phe Phe Gln Met
Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser65 70 75 80Leu Leu
Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu 85 90 95Met
Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln 100 105
110Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys
115 120 125Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro
Cys Glu 130 135 140Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val
Glu Gln Val Lys145 150 155 160Asn Ala Phe Asn Lys Leu Gln Glu Lys
Gly Ile Tyr Lys Ala Met Ser 165 170 175Glu Phe Asp Ile Phe Ile Asn
Tyr Ile Glu Ala Tyr Met Thr Met Lys 180 185 190Ile Arg Asn Ser Thr
Ser Gly Met Ser Val Gly Trp Ile Arg Gln Pro 195 200 205Pro Gly Lys
Ala Leu Glu Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys 210 215 220Lys
Asp Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp225 230
235 240Thr Ser Ala Asn Gln Val Val Leu Lys Val Thr Asn Met Asp Pro
Ala 245 250 255Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Ser Met Ile Thr
Asn Trp Tyr 260 265 270Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr
Val Ser Ser 275 280 285158106PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 158Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Gln Leu Ser
Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ala Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys
Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105159616PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 159Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys
Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser
Leu Ser Pro Gly 20 25 30Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His
Phe Pro Gly Asn Leu 35 40 45Pro Asn Met Leu Arg Asp Leu Arg Asp Ala
Phe Ser Arg Val Lys Thr 50 55 60Phe Phe Gln Met Lys Asp Gln Leu Asp
Asn Leu Leu Leu Lys Glu Ser65 70 75 80Leu Leu Glu Asp Phe Lys Gly
Tyr Leu Gly Cys Gln Ala Leu Ser Glu 85 90 95Met Ile Gln Phe Tyr Leu
Glu Glu Val Met Pro Gln Ala Glu Asn Gln 100 105 110Asp Pro Asp Ile
Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys 115 120 125Thr Leu
Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu 130 135
140Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Val
Lys145 150 155 160Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr
Lys Ala Met Ser 165 170 175Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu
Ala Tyr Met Thr Met Lys 180 185 190Ile Arg Asn Ser Thr Ser Gly Met
Ser Val Gly Trp Ile Arg Gln Pro 195 200 205Pro Gly Lys Ala Leu Glu
Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys 210 215 220Lys Asp Tyr Asn
Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp225 230 235 240Thr
Ser Ala Asn Gln Val Val Leu Lys Val Thr Asn Met Asp Pro Ala 245 250
255Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr
260 265 270Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
Ala Ser 275 280 285Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr 290 295 300Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro305 310 315 320Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val 325 330 335His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 340 345 350Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 355 360 365Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 370 375
380Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala385 390 395 400Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro 405 410 415Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val 420 425 430Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val 435 440 445Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 450 455 460Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln465 470 475 480Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 485 490
495Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
500 505 510Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr 515 520 525Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser 530 535 540Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr545 550 555 560Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr 565 570 575Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 580 585 590Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 595 600 605Ser
Leu Ser Leu Ser Pro Gly Lys 610 615160213PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 160Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Gln Leu Ser
Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ala Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys
Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135
140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys
2101619PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 161Gly Phe Ser Leu Ser Thr Ser Gly Met1
5162171PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 162Trp Trp Asp Ser Pro Gly Gln Gly
Thr Gln Ser Glu Asn Ser Cys Thr1 5 10 15His Phe Pro Gly Asn Leu Pro
Asn Met Leu Arg Asp Leu Arg Asp Ala 20 25 30Phe Ser Arg Val Lys Thr
Phe Phe Gln Met Lys Asp Gln Leu Asp Asn 35 40 45Leu Leu Leu Lys Glu
Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly 50 55 60Cys Gln Ala Leu
Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met65 70 75 80Pro Gln
Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser 85 90 95Leu
Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His 100 105
110Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys
115 120 125Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu
Lys Gly 130 135 140Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile
Asn Tyr Ile Glu145 150 155 160Ala Tyr Met Thr Met Lys Ile Arg Asn
Asp Lys 165 17016310PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 163Ser Met Ile Thr Asn
Trp Tyr Phe Asp Val1 5 101646PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 164Gln Leu Ser Val Gly Tyr1 51653PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 165Asp Thr Ser11666PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 166Gly Ser Gly Tyr Pro Phe1 51677PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 167Thr Ser Gly Met Ser Val Gly1 5168182PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 168Asp Ile Trp Trp Asp Ser Pro Gly Gln Gly Thr Gln Ser
Glu Asn Ser1 5 10 15Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu
Arg Asp Leu Arg 20 25 30Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln
Met Lys Asp Gln Leu 35 40 45Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu
Glu Asp Phe Lys Gly Tyr 50 55 60Leu Gly Cys Gln Ala Leu Ser Glu Met
Ile Gln Phe Tyr Leu Glu Glu65 70 75 80Val Met Pro Gln Ala Glu Asn
Gln Asp Pro Asp Ile Lys Ala His Val 85 90 95Asn Ser Leu Gly Glu Asn
Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg 100 105 110Cys His Arg Phe
Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys 115 120 125Ser Lys
Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu 130 135
140Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn
Tyr145 150 155 160Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Asp
Lys Lys Asp Tyr 165 170 175Asn Pro Ser Leu Lys Ser
18016910PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 169Ser Met Ile Thr Asn Trp
Tyr Phe Asp Val1 5 1017010PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 170Lys Ala Gln Leu Ser Val Gly Tyr Met His1 5
101717PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 171Asp Thr Ser Lys Leu Ala Ser1
51729PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 172Phe Gln Gly Ser Gly Tyr Pro Phe Thr1
5173286PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 173Gln Val Thr Leu Arg Glu Ser Gly
Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile
Trp Trp Asp Ser Pro Gly Gln Gly Thr Gln Ser 50 55 60Glu Asn Ser Cys
Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg65 70 75 80Asp Leu
Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys 85 90 95Asp
Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe 100 105
110Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr
115 120 125Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp
Ile Lys 130 135 140Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr
Leu Arg Leu Arg145 150 155 160Leu Arg Arg Cys His Arg Phe Leu Pro
Cys Glu Asn Gly Gly Gly Ser 165 170 175Gly Gly Lys Ser Lys Ala Val
Glu Gln Val Lys Asn Ala Phe Asn Lys 180 185 190Leu Gln Glu Lys Gly
Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe 195 200 205Ile Asn Tyr
Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Asp Lys 210 215 220Lys
Asp Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp225 230
235 240Thr Ser Ala Asn Gln Val Val Leu Lys Val Thr Asn Met Asp Pro
Ala 245 250 255Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Ser Met Ile Thr
Asn Trp Tyr 260 265 270Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr
Val Ser Ser 275 280 285174106PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 174Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Gln Leu Ser
Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ala Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys
Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys
100 105175616PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 175Gln Val Thr Leu Arg
Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu
Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Ser
Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu
Ala Asp Ile Trp Trp Asp Ser Pro Gly Gln Gly Thr Gln Ser 50 55 60Glu
Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg65 70 75
80Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys
85 90 95Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp
Phe 100 105 110Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile
Gln Phe Tyr 115 120 125Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln
Asp Pro Asp Ile Lys 130 135 140Ala His Val Asn Ser Leu Gly Glu Asn
Leu Lys Thr Leu Arg Leu Arg145 150 155 160Leu Arg Arg Cys His Arg
Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser 165 170 175Gly Gly Lys Ser
Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys 180 185 190Leu Gln
Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe 195 200
205Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Asp Lys
210 215 220Lys Asp Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser
Lys Asp225 230 235 240Thr Ser Ala Asn Gln Val Val Leu Lys Val Thr
Asn Met Asp Pro Ala 245 250 255Asp Thr Ala Thr Tyr Tyr Cys Ala Arg
Ser Met Ile Thr Asn Trp Tyr 260 265 270Phe Asp Val Trp Gly Ala Gly
Thr Thr Val Thr Val Ser Ser Ala Ser 275 280 285Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 290 295 300Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro305 310 315
320Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
325 330 335His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser 340 345 350Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile 355 360 365Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val 370 375 380Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala385 390 395 400Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 405 410 415Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 420 425 430Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 435 440
445Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
450 455 460Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln465 470 475 480Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala 485 490 495Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro 500 505 510Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr 515 520 525Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 530 535 540Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr545 550 555
560Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
565 570 575Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe 580 585 590Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys 595 600 605Ser Leu Ser Leu Ser Pro Gly Lys 610
615176213PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 176Asp Ile Gln Met Thr
Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Lys Ala Gln Leu Ser Val Gly Tyr Met 20 25 30His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr
Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly
Ser Gly Thr Ala Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr
85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
Pro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200
205Asn Arg Gly Glu Cys 2101779PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 177Gly Phe Ser Leu Ser Thr Ser Gly Met1 51785PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 178Trp Trp Asp Asp Lys1 5179176PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 179Ser Met Ile Thr Ser Pro Gly Gln Gly Thr Gln Ser Glu
Asn Ser Cys1 5 10 15Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg
Asp Leu Arg Asp 20 25 30Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met
Lys Asp Gln Leu Asp 35 40 45Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu
Asp Phe Lys Gly Tyr Leu 50 55 60Gly Cys Gln Ala Leu Ser Glu Met Ile
Gln Phe Tyr Leu Glu Glu Val65 70 75 80Met Pro Gln Ala Glu Asn Gln
Asp Pro Asp Ile Lys Ala His Val Asn 85 90 95Ser Leu Gly Glu Asn Leu
Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys 100 105 110His Arg Phe Leu
Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser 115 120 125Lys Ala
Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys 130 135
140Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr
Ile145 150 155 160Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Asn Trp
Tyr Phe Asp Val 165 170 1751806PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 180Gln Leu Ser Val Gly Tyr1 51813PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 181Asp Thr Ser11826PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 182Gly Ser Gly Tyr Pro Phe1 51837PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 183Thr Ser Gly Met Ser Val Gly1 518416PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 184Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu
Lys Ser1 5 10 15185176PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 185Ser Met Ile Thr Ser Pro Gly Gln Gly Thr Gln Ser Glu
Asn Ser Cys1 5 10 15Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg
Asp Leu Arg Asp 20 25 30Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met
Lys Asp Gln Leu Asp 35 40 45Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu
Asp Phe Lys Gly Tyr Leu 50 55 60Gly Cys Gln Ala Leu Ser Glu Met Ile
Gln Phe Tyr Leu Glu Glu Val65 70 75 80Met Pro Gln Ala Glu Asn Gln
Asp Pro Asp Ile Lys Ala His Val Asn 85 90 95Ser Leu Gly Glu Asn Leu
Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys 100 105 110His Arg Phe Leu
Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser 115 120 125Lys Ala
Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys 130 135
140Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr
Ile145 150 155 160Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Asn Trp
Tyr Phe Asp Val 165 170 17518610PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 186Lys Ala Gln Leu Ser Val Gly Tyr Met His1 5
101877PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 187Asp Thr Ser Lys Leu Ala Ser1
51889PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 188Phe Gln Gly Ser Gly Tyr Pro Phe Thr1
5189286PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 189Gln Val Thr Leu Arg Glu Ser Gly
Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile
Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Ala Asn Gln Val65 70 75 80Val Leu
Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys
Ala Arg Ser Met Ile Thr Ser Pro Gly Gln Gly Thr Gln Ser Glu 100 105
110Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp
115 120 125Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met
Lys Asp 130 135 140Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu
Glu Asp Phe Lys145 150 155 160Gly Tyr Leu Gly Cys Gln Ala Leu Ser
Glu Met Ile Gln Phe Tyr Leu 165 170 175Glu Glu Val Met Pro Gln Ala
Glu Asn Gln Asp Pro Asp Ile Lys Ala 180 185 190His Val Asn Ser Leu
Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu 195 200 205Arg Arg Cys
His Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly 210 215 220Gly
Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu225 230
235 240Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe
Ile 245 250 255Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
Asn Trp Tyr 260 265 270Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr
Val Ser Ser 275 280 285190106PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 190Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Gln Leu Ser
Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ala Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys
Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105191616PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 191Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys
Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser
Leu Ser Thr Ser 20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro
Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys
Lys Asp Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys
Asp Thr Ser Ala Asn Gln Val65 70 75 80Val Leu Lys Val Thr Asn Met
Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Met Ile
Thr Ser Pro Gly Gln Gly Thr Gln Ser Glu 100 105 110Asn Ser Cys Thr
His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp 115 120 125Leu Arg
Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp 130 135
140Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe
Lys145 150 155 160Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile
Gln Phe Tyr Leu 165 170 175Glu Glu Val Met Pro Gln Ala Glu Asn Gln
Asp Pro Asp Ile Lys Ala 180 185 190His Val Asn Ser Leu Gly Glu Asn
Leu Lys Thr Leu Arg Leu Arg Leu 195 200 205Arg Arg Cys His Arg Phe
Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly 210 215 220Gly Lys Ser Lys
Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu225 230 235 240Gln
Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile 245 250
255Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Asn Trp Tyr
260 265 270Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
Ala Ser 275 280 285Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr 290 295 300Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro305 310 315 320Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val 325 330 335His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 340 345 350Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 355 360 365Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 370 375
380Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala385 390 395 400Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro 405 410 415Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val 420 425 430Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val 435 440 445Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 450 455 460Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln465 470 475 480Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 485 490
495Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
500 505 510Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr 515 520 525Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser 530 535 540Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr545 550 555
560Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
565 570 575Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe 580 585 590Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys 595 600 605Ser Leu Ser Leu Ser Pro Gly Lys 610
615192213PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 192Asp Ile Gln Met Thr
Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Lys Ala Gln Leu Ser Val Gly Tyr Met 20 25 30His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr
Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly
Ser Gly Thr Ala Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr
85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
Pro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200
205Asn Arg Gly Glu Cys 2101939PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 193Gly Phe Ser Leu Ser Thr Ser Gly Met1 51945PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 194Trp Trp Asp Asp Lys1 519510PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 195Ser Met Ile Thr Asn Trp Tyr Phe Asp Val1 5
10196172PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 196Gln Leu Ser Ser Pro
Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr1 5 10 15His Phe Pro Gly
Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala 20 25 30Phe Ser Arg
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn 35 40 45Leu Leu
Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly 50 55 60Cys
Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met65 70 75
80Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser
85 90 95Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys
His 100 105 110Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly
Lys Ser Lys 115 120 125Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys
Leu Gln Glu Lys Gly 130 135 140Ile Tyr Lys Ala Met Ser Glu Phe Asp
Ile Phe Ile Asn Tyr Ile Glu145 150 155 160Ala Tyr Met Thr Met Lys
Ile Arg Asn Val Gly Tyr 165 1701973PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 197Asp Thr Ser11986PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 198Gly Ser Gly Tyr Pro Phe1 51997PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 199Thr Ser Gly Met Ser Val Gly1 520016PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 200Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu
Lys Ser1 5 10 1520110PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 201Ser Met Ile Thr Asn
Trp Tyr Phe Asp Val1 5 10202176PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 202Lys Ala Gln Leu Ser Ser Pro Gly Gln Gly Thr Gln Ser
Glu Asn Ser1 5 10 15Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu
Arg Asp Leu Arg 20 25 30Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln
Met Lys Asp Gln Leu 35 40 45Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu
Glu Asp Phe Lys Gly Tyr 50 55 60Leu Gly Cys Gln Ala Leu Ser Glu Met
Ile Gln Phe Tyr Leu Glu Glu65 70 75 80Val Met Pro Gln Ala Glu Asn
Gln Asp Pro Asp Ile Lys Ala His Val 85 90 95Asn Ser Leu Gly Glu Asn
Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg 100 105 110Cys His Arg Phe
Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys 115 120 125Ser Lys
Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu 130 135
140Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn
Tyr145 150 155 160Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Val
Gly Tyr Met His 165 170 1752037PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 203Asp Thr Ser Lys Leu Ala Ser1 52049PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 204Phe Gln Gly Ser Gly Tyr Pro Phe Thr1
5205120PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 205Gln Val Thr Leu Arg Glu Ser Gly
Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile
Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Ala Asn Gln Val65 70 75 80Val Leu
Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys
Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115 120206272PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 206Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Gln Leu Ser
Ser Pro Gly Gln 20 25 30Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe
Pro Gly Asn Leu Pro 35 40 45Asn Met Leu Arg Asp Leu Arg Asp Ala Phe
Ser Arg Val Lys Thr Phe 50 55 60Phe Gln Met Lys Asp Gln Leu Asp Asn
Leu Leu Leu Lys Glu Ser Leu65 70 75 80Leu Glu Asp Phe Lys Gly Tyr
Leu Gly Cys Gln Ala Leu Ser Glu Met 85 90 95Ile Gln Phe Tyr Leu Glu
Glu Val Met Pro Gln Ala Glu Asn Gln Asp 100 105 110Pro Asp Ile Lys
Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr 115 120 125Leu Arg
Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn 130 135
140Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Val Lys
Asn145 150 155 160Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
Ala Met Ser Glu 165 170 175Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala
Tyr Met Thr Met Lys Ile 180 185 190Arg Asn Val Gly Tyr Met His Trp
Tyr Gln Gln Lys Pro Gly Lys Ala 195 200 205Pro Lys Leu Leu Ile Tyr
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro 210 215 220Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Thr Ile225 230 235 240Ser
Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly 245 250
255Ser Gly Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
260 265 270207450PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 207Gln Val Thr Leu Arg
Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu
Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Ser
Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu
Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60Leu
Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ala Asn Gln Val65 70 75
80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr
85 90 95Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly
Ala 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Ala Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly Lys 450208379PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 208Asp Ile Gln Met
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Lys Ala Gln Leu Ser Ser Pro Gly Gln 20 25 30Gly Thr
Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro 35 40 45Asn
Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe 50 55
60Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu65
70 75 80Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu
Met 85 90 95Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn
Gln Asp 100 105 110Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
Asn Leu Lys Thr 115 120 125Leu Arg Leu Arg Leu Arg Arg Cys His Arg
Phe Leu Pro Cys Glu Asn 130 135 140Gly Gly Gly Ser Gly Gly Lys Ser
Lys Ala Val Glu Gln Val Lys Asn145 150 155 160Ala Phe Asn Lys Leu
Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu 165 170 175Phe Asp Ile
Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile 180 185 190Arg
Asn Val Gly Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 195 200
205Pro Lys Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
210 215 220Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu
Thr Ile225 230 235 240Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr
Tyr Cys Phe Gln Gly 245 250 255Ser Gly Tyr Pro Phe Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 260 265 270Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 275 280 285Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 290 295 300Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln305 310 315
320Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
325 330 335Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu 340 345 350Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 355 360 365Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 370 375209166PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 209Ser Pro Gly Gln Gly
Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro1 5 10 15Gly Asn Leu Pro
Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg 20 25 30Val Lys Thr
Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu 35 40 45Lys Glu
Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala 50 55 60Leu
Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala65 70 75
80Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe
Leu 100 105 110Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys
Ala Val Glu 115 120 125Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu
Lys Gly Ile Tyr Lys 130 135 140Ala Met Ser Glu Phe Asp Ile Phe Ile
Asn Tyr Ile Glu Ala Tyr Met145 150 155 160Thr Met Lys Ile Arg Asn
1652109PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 210Gly Phe Ser Leu Ser Thr Ser Gly Met1
52115PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 211Trp Trp Asp Asp Lys1
521210PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 212Ser Met Ile Thr Asn Trp Tyr Phe Asp
Val1 5 10213172PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 213Gln Leu Ser Ser Pro
Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr1 5 10 15His Phe Pro Gly
Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala 20 25 30Phe Ser Arg
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn 35 40
45Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly
50 55 60Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val
Met65 70 75 80Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His
Val Asn Ser 85 90 95Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu
Arg Arg Cys His 100 105 110Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly
Ser Gly Gly Lys Ser Lys 115 120 125Ala Val Glu Gln Val Lys Asn Ala
Phe Asn Lys Leu Gln Glu Lys Gly 130 135 140Ile Tyr Lys Ala Met Ser
Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu145 150 155 160Ala Tyr Met
Thr Met Lys Ile Arg Asn Val Gly Tyr 165 1702143PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 214Asp Thr Ser12156PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 215Gly Ser Gly Tyr Pro Phe1 52167PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 216Thr Ser Gly Met Ser Val Gly1 521716PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 217Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu
Lys Ser1 5 10 1521810PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 218Ser Met Ile Thr Asn
Trp Tyr Phe Asp Val1 5 10219176PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 219Lys Ala Gln Leu Ser Ser Pro Gly Gln Gly Thr Gln Ser
Glu Asn Ser1 5 10 15Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu
Arg Asp Leu Arg 20 25 30Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln
Met Lys Asp Gln Leu 35 40 45Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu
Glu Asp Phe Lys Gly Tyr 50 55 60Leu Gly Cys Gln Ala Leu Ser Glu Met
Ile Gln Phe Tyr Leu Glu Glu65 70 75 80Val Met Pro Gln Ala Glu Asn
Gln Asp Pro Asp Ile Lys Ala His Val 85 90 95Asn Ser Leu Gly Glu Asn
Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg 100 105 110Cys His Arg Phe
Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys 115 120 125Ser Lys
Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu 130 135
140Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn
Tyr145 150 155 160Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Val
Gly Tyr Met His 165 170 1752207PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 220Asp Thr Ser Lys Leu Ala Ser1 52219PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 221Phe Gln Gly Ser Gly Tyr Pro Phe Thr1
5222120PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 222Gln Val Thr Leu Arg Glu Ser Gly
Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile
Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Ala Asn Gln Val65 70 75 80Val Leu
Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys
Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115 120223272PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 223Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Gln Leu Ser
Ser Pro Gly Gln 20 25 30Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe
Pro Gly Asn Leu Pro 35 40 45Asn Met Leu Arg Asp Leu Arg Asp Ala Phe
Ser Arg Val Lys Thr Phe 50 55 60Phe Gln Met Lys Asp Gln Leu Asp Asn
Leu Leu Leu Lys Glu Ser Leu65 70 75 80Leu Glu Asp Phe Lys Gly Tyr
Leu Gly Cys Gln Ala Leu Ser Glu Met 85 90 95Ile Gln Phe Tyr Leu Glu
Glu Val Met Pro Gln Ala Glu Asn Gln Asp 100 105 110Pro Asp Ile Lys
Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr 115 120 125Leu Arg
Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn 130 135
140Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Val Lys
Asn145 150 155 160Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
Ala Met Ser Glu 165 170 175Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala
Tyr Met Thr Met Lys Ile 180 185 190Arg Asn Val Gly Tyr Met His Trp
Tyr Gln Gln Lys Pro Gly Lys Ala 195 200 205Pro Lys Leu Leu Ile Tyr
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro 210 215 220Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Thr Ile225 230 235 240Ser
Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly 245 250
255Ser Gly Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
260 265 270224450PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 224Gln Val Thr Leu Arg
Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu
Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Ser
Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu
Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60Leu
Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ala Asn Gln Val65 70 75
80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr
85 90 95Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly
Ala 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly Lys 450225379PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 225Asp Ile Gln Met
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Lys Ala Gln Leu Ser Ser Pro Gly Gln 20 25 30Gly Thr
Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro 35 40 45Asn
Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe 50 55
60Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu65
70 75 80Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu
Met 85 90 95Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn
Gln Asp 100 105 110Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
Asn Leu Lys Thr 115 120 125Leu Arg Leu Arg Leu Arg Arg Cys His Arg
Phe Leu Pro Cys Glu Asn 130 135 140Gly Gly Gly Ser Gly Gly Lys Ser
Lys Ala Val Glu Gln Val Lys Asn145 150 155 160Ala Phe Asn Lys Leu
Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu 165 170 175Phe Asp Ile
Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile 180 185 190Arg
Asn Val Gly Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 195 200
205Pro Lys Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
210 215 220Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu
Thr Ile225 230 235 240Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr
Tyr Cys Phe Gln Gly 245 250 255Ser Gly Tyr Pro Phe Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 260 265 270Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 275 280 285Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 290 295 300Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln305 310 315
320Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
325 330 335Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu 340 345 350Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 355 360 365Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 370 3752269PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 226Gly Phe Ser Leu Ser Thr
Ser Gly Met1 52275PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 227Trp Trp Asp Asp Lys1
522810PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 228Ser Met Ile Thr Asn Trp Tyr Phe Asp
Val1 5 10229172PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 229Gln Leu Ser Ser Pro
Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr1 5 10 15His Phe Pro Gly
Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala 20 25 30Phe Ser Arg
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn 35 40 45Leu Leu
Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly 50 55 60Cys
Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met65 70 75
80Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser
85 90 95Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys
His 100 105 110Arg Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly
Lys Ser Lys 115 120 125Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys
Leu Gln Glu Lys Gly 130 135 140Ile Tyr Lys Ala Met Ser Glu Phe Asp
Ile Phe Ile Asn Tyr Ile Glu145 150 155 160Ala Tyr Met Thr Met Lys
Ile Arg Asn Val Gly Tyr 165 1702303PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 230Asp Thr Ser12316PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 231Gly Ser Gly Tyr Pro Phe1 52327PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 232Thr Ser Gly Met Ser Val Gly1 523316PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 233Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu
Lys Ser1 5 10 1523410PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 234Ser Met Ile Thr Asn
Trp Tyr Phe Asp Val1 5 10235176PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 235Lys Ala Gln Leu Ser Ser Pro Gly Gln Gly Thr Gln Ser
Glu Asn Ser1 5 10 15Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu
Arg Asp Leu Arg 20 25 30Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln
Met Lys Asp Gln Leu 35 40 45Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu
Glu Asp Phe Lys Gly Tyr 50 55 60Leu Gly Cys Gln Ala Leu Ser Glu Met
Ile Gln Phe Tyr Leu Glu Glu65 70 75 80Val Met Pro Gln Ala Glu Asn
Gln Asp Pro Asp Ile Lys Ala His Val 85 90 95Asn Ser Leu Gly Glu Asn
Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg 100 105 110Cys His Arg Phe
Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys 115 120 125Ser Lys
Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu 130 135
140Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn
Tyr145 150 155 160Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Val
Gly Tyr Met His 165 170 1752367PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 236Asp Thr Ser Lys Leu Ala Ser1 52379PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 237Phe Gln Gly Ser Gly Tyr Pro Phe Thr1
5238120PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 238Gln Val Thr Leu Arg Glu Ser Gly
Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Ser Val Gly Trp
Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile
Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Ala Asn Gln Val65 70 75 80Val Leu
Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys
Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe
Asp Val Trp Gly Ala 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
120239272PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 239Asp Ile Gln Met Thr
Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Lys Ala Gln Leu Ser Ser Pro Gly Gln 20 25 30Gly Thr Gln
Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro 35 40 45Asn Met
Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe 50 55 60Phe
Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu65 70 75
80Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met
85 90 95Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln
Asp 100 105 110Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn
Leu Lys Thr 115 120 125Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe
Leu Pro Cys Glu Asn 130 135 140Gly Gly Gly Ser Gly Gly Lys Ser Lys
Ala Val Glu Gln Val Lys Asn145 150 155 160Ala Phe Asn Lys Leu Gln
Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu 165 170 175Phe Asp Ile Phe
Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile 180 185 190Arg Asn
Val Gly Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 195 200
205Pro Lys Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
210 215 220Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu
Thr Ile225 230 235 240Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr
Tyr Cys Phe Gln Gly 245 250 255Ser Gly Tyr Pro Phe Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 260 265 270240450PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 240Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys
Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser
Leu Ser Thr Ser 20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro
Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys
Lys Asp Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys
Asp Thr Ser Ala Asn Gln Val65 70 75 80Val Leu Lys Val Thr Asn Met
Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Met Ile
Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105 110Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135
140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly225 230 235 240Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250
255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala
Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375
380Val Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Ile His 420 425 430Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly Lys
450241379PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 241Asp Ile Gln Met Thr
Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Lys Ala Gln Leu Ser Ser Pro Gly Gln 20 25 30Gly Thr Gln
Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro 35 40 45Asn Met
Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe 50 55 60Phe
Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu65 70 75
80Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met
85 90 95Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln
Asp 100 105 110Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn
Leu Lys Thr 115 120 125Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe
Leu Pro Cys Glu Asn 130 135 140Gly Gly Gly Ser Gly Gly Lys Ser Lys
Ala Val Glu Gln Val Lys Asn145 150 155 160Ala Phe Asn Lys Leu Gln
Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu 165 170 175Phe Asp Ile Phe
Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile 180 185 190Arg Asn
Val Gly Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 195 200
205Pro Lys Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
210 215 220Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu
Thr Ile225 230 235 240Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr
Tyr Cys Phe Gln Gly 245 250 255Ser Gly Tyr Pro Phe Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 260 265 270Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 275 280 285Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 290 295 300Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln305 310 315
320Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
325 330 335Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu 340 345 350Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 355 360 365Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 370 375242360DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 242caggtcacac
tgagagagtc aggccctgcc ctggtcaagc ctactcagac cctgaccctg 60acctgcacct
ttagcggctt tagcctgagc actagcggaa tgagcgtggg ctggattaga
120cagccccctg gtaaagccct ggagtggctg gccgatattt ggtgggacga
taagaaggac 180tataacccta gcctgaagtc taggctgact atctctaagg
acactagcgc taatcaggtg 240gtgctgaaag tgactaatat ggaccccgcc
gacaccgcta cctactactg cgctagatct 300atgatcacta actggtactt
cgacgtgtgg ggcgctggca ctaccgtgac cgtgtctagc 360243816DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 243gatattcaga tgactcagtc acctagcacc ctgagcgcta
gtgtgggcga tagagtgact 60atcacctgta aagctcagct gtctagccca ggtcagggaa
ctcagtcaga gaatagctgc 120actcacttcc ccggtaacct gcctaatatg
ctgagagatc tgagggacgc cttctctagg 180gtcaagacct tctttcagat
gaaggatcag ctggataacc tgctgctgaa agagtcactg 240ctggaggact
ttaagggcta cctgggctgt caggccctga gcgagatgat tcagttctac
300ctggaagaag tgatgcccca ggccgagaat caggaccccg atattaaggc
tcacgtgaac 360tcactgggcg agaacctgaa aaccctgaga ctgaggctga
ggcggtgtca ccggtttctg 420ccctgcgaga acggcggagg tagcggcggt
aaatctaagg ccgtggaaca ggtcaaaaac 480gcctttaaca agctgcagga
aaagggaatc tataaggcta tgagcgagtt cgacatcttt 540attaactata
tcgaggccta tatgactatg aagattagga acgtgggcta tatgcactgg
600tatcagcaga agcccggtaa agcccctaag ctgctgatct acgacacctc
taagctggct 660agtggcgtgc cctctaggtt tagcggtagc ggtagtggca
ccgccttcac cctgactatc 720tctagcctgc agcccgacga cttcgctacc
tactactgtt ttcagggtag cggctacccc 780ttcaccttcg gcggaggcac
taagctggag attaag 8162441350DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 244caggtcacac tgagagagtc aggccctgcc ctggtcaagc
ctactcagac cctgaccctg 60acctgcacct ttagcggctt tagcctgagc actagcggaa
tgagcgtggg ctggattaga 120cagccccctg gtaaagccct ggagtggctg
gccgatattt ggtgggacga taagaaggac 180tataacccta gcctgaagtc
taggctgact atctctaagg acactagcgc taatcaggtg 240gtgctgaaag
tgactaatat ggaccccgcc gacaccgcta cctactactg cgctagatct
300atgatcacta actggtactt cgacgtgtgg ggcgctggca ctaccgtgac
cgtgtctagc 360gctagcacta agggcccaag tgtgtttccc ctggccccca
gcagcaagtc tacttccggc 420ggaactgctg ccctgggttg cctggtgaag
gactacttcc ccgagcccgt gacagtgtcc 480tggaactctg gggctctgac
ttccggcgtg cacaccttcc ccgccgtgct gcagagcagc 540ggcctgtaca
gcctgagcag cgtggtgaca gtgccctcca gctctctggg aacccagacc
600tatatctgca acgtgaacca caagcccagc aacaccaagg tggacaagag
agtggagccc 660aagagctgcg acaagaccca cacctgcccc ccctgcccag
ctccagaact gctgggaggg 720ccttccgtgt tcctgttccc ccccaagccc
aaggacaccc tgatgatcag caggaccccc 780gaggtgacct gcgtggtggt
ggacgtgtcc cacgaggacc cagaggtgaa gttcaactgg 840tacgtggacg
gcgtggaggt gcacaacgcc aagaccaagc ccagagagga gcagtacaac
900agcacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct
gaacggcaaa 960gaatacaagt gcaaagtctc caacaaggcc ctgccagccc
caatcgaaaa gacaatcagc 1020aaggccaagg gccagccacg ggagccccag
gtgtacaccc tgccccccag ccgggaggag 1080atgaccaaga accaggtgtc
cctgacctgt ctggtgaagg gcttctaccc cagcgatatc 1140gccgtggagt
gggagagcaa cggccagccc gagaacaact acaagaccac ccccccagtg
1200ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa
gtccaggtgg 1260cagcagggca acgtgttcag ctgcagcgtg atgcacgagg
ccctgcacaa ccactacacc 1320cagaagtccc tgagcctgag ccccggcaag
13502451137DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 245gatattcaga
tgactcagtc acctagcacc ctgagcgcta gtgtgggcga tagagtgact 60atcacctgta
aagctcagct gtctagccca ggtcagggaa ctcagtcaga gaatagctgc
120actcacttcc ccggtaacct gcctaatatg ctgagagatc tgagggacgc
cttctctagg 180gtcaagacct tctttcagat gaaggatcag ctggataacc
tgctgctgaa agagtcactg 240ctggaggact ttaagggcta cctgggctgt
caggccctga gcgagatgat tcagttctac 300ctggaagaag tgatgcccca
ggccgagaat caggaccccg atattaaggc tcacgtgaac 360tcactgggcg
agaacctgaa aaccctgaga ctgaggctga ggcggtgtca ccggtttctg
420ccctgcgaga acggcggagg tagcggcggt aaatctaagg ccgtggaaca
ggtcaaaaac 480gcctttaaca agctgcagga aaagggaatc tataaggcta
tgagcgagtt cgacatcttt 540attaactata tcgaggccta tatgactatg
aagattagga acgtgggcta tatgcactgg 600tatcagcaga agcccggtaa
agcccctaag ctgctgatct acgacacctc taagctggct 660agtggcgtgc
cctctaggtt tagcggtagc ggtagtggca ccgccttcac cctgactatc
720tctagcctgc agcccgacga cttcgctacc tactactgtt ttcagggtag
cggctacccc 780ttcaccttcg gcggaggcac taagctggag attaagcgta
cggtggccgc tcccagcgtg 840ttcatcttcc cccccagcga cgagcagctg
aagagcggca ccgccagcgt ggtgtgcctg 900ctgaacaact tctacccccg
ggaggccaag gtgcagtgga aggtggacaa cgccctgcag 960agcggcaaca
gccaggagag cgtcaccgag caggacagca aggactccac ctacagcctg
1020agcagcaccc tgaccctgag caaggccgac tacgagaagc ataaggtgta
cgcctgcgag 1080gtgacccacc agggcctgtc cagccccgtg accaagagct
tcaacagggg cgagtgc 1137246360DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 246caggtcacac tgagagagtc aggccctgcc ctggtcaagc
ctactcagac cctgaccctg 60acctgcacct ttagcggctt tagcctgagc actagcggaa
tgagcgtggg ctggattaga 120cagccccctg gtaaagccct ggagtggctg
gccgatattt ggtgggacga taagaaggac 180tataacccta gcctgaagtc
taggctgact atctctaagg acactagcgc taatcaggtg 240gtgctgaaag
tgactaatat ggaccccgcc gacaccgcta cctactactg cgctagatct
300atgatcacta actggtactt cgacgtgtgg ggcgctggca ctaccgtgac
cgtgtctagc 360247816DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polynucleotide" 247gatattcaga
tgactcagtc acctagcacc ctgagcgcta gtgtgggcga tagagtgact 60atcacctgta
aagctcagct gtctagccca ggtcagggaa ctcagtcaga gaatagctgc
120actcacttcc ccggtaacct gcctaatatg ctgagagatc tgagggacgc
cttctctagg 180gtcaagacct tctttcagat gaaggatcag ctggataacc
tgctgctgaa agagtcactg 240ctggaggact ttaagggcta cctgggctgt
caggccctga gcgagatgat tcagttctac 300ctggaagaag tgatgcccca
ggccgagaat caggaccccg atattaaggc tcacgtgaac 360tcactgggcg
agaacctgaa aaccctgaga ctgaggctga ggcggtgtca ccggtttctg
420ccctgcgaga acggcggagg tagcggcggt aaatctaagg ccgtggaaca
ggtcaaaaac 480gcctttaaca agctgcagga aaagggaatc tataaggcta
tgagcgagtt cgacatcttt 540attaactata tcgaggccta tatgactatg
aagattagga acgtgggcta tatgcactgg 600tatcagcaga agcccggtaa
agcccctaag ctgctgatct acgacacctc taagctggct 660agtggcgtgc
cctctaggtt tagcggtagc ggtagtggca ccgccttcac cctgactatc
720tctagcctgc agcccgacga cttcgctacc tactactgtt ttcagggtag
cggctacccc 780ttcaccttcg gcggaggcac taagctggag attaag
8162481350DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 248caggtcacac
tgagagagtc aggccctgcc ctggtcaagc ctactcagac cctgaccctg 60acctgcacct
ttagcggctt tagcctgagc actagcggaa tgagcgtggg ctggattaga
120cagccccctg gtaaagccct ggagtggctg gccgatattt ggtgggacga
taagaaggac 180tataacccta gcctgaagtc taggctgact atctctaagg
acactagcgc taatcaggtg 240gtgctgaaag tgactaatat ggaccccgcc
gacaccgcta cctactactg cgctagatct 300atgatcacta actggtactt
cgacgtgtgg ggcgctggca ctaccgtgac cgtgtctagc 360gctagcacta
agggcccctc cgtgttccct ctggcccctt ccagcaagtc tacctccggc
420ggcacagctg ctctgggctg cctggtcaag gactacttcc ctgagcctgt
gacagtgtcc 480tggaactctg gcgccctgac ctctggcgtg cacaccttcc
ctgccgtgct gcagtcctcc 540ggcctgtact ccctgtcctc cgtggtcaca
gtgccttcaa gcagcctggg cacccagacc 600tatatctgca acgtgaacca
caagccttcc aacaccaagg tggacaagcg ggtggagcct 660aagtcctgcg
acaagaccca cacctgtcct ccctgccctg ctcctgaact gctgggcggc
720ccttctgtgt tcctgttccc tccaaagccc aaggacaccc tgatgatctc
ccggacccct 780gaagtgacct gcgtggtggt ggccgtgtcc cacgaggatc
ctgaagtgaa gttcaattgg 840tacgtggacg gcgtggaggt gcacaacgcc
aagaccaagc ctcgggagga acagtacaac 900tccacctacc gggtggtgtc
cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 960gagtacaagt
gcaaagtctc caacaaggcc ctggccgccc ctatcgaaaa gacaatctcc
1020aaggccaagg gccagcctag ggaaccccag gtgtacaccc tgccacccag
ccgggaggaa 1080atgaccaaga accaggtgtc cctgacctgt ctggtcaagg
gcttctaccc ttccgatatc 1140gccgtggagt gggagtctaa cggccagcct
gagaacaact acaagaccac ccctcctgtg 1200ctggactccg acggctcctt
cttcctgtac tccaaactga ccgtggacaa gtcccggtgg 1260cagcagggca
acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc
1320cagaagtccc tgtccctgtc tcccggcaag 13502491137DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 249gatattcaga tgactcagtc acctagcacc ctgagcgcta
gtgtgggcga tagagtgact 60atcacctgta aagctcagct gtctagccca ggtcagggaa
ctcagtcaga gaatagctgc 120actcacttcc ccggtaacct gcctaatatg
ctgagagatc tgagggacgc cttctctagg 180gtcaagacct tctttcagat
gaaggatcag ctggataacc tgctgctgaa agagtcactg 240ctggaggact
ttaagggcta cctgggctgt caggccctga gcgagatgat tcagttctac
300ctggaagaag tgatgcccca ggccgagaat caggaccccg atattaaggc
tcacgtgaac 360tcactgggcg agaacctgaa aaccctgaga ctgaggctga
ggcggtgtca ccggtttctg 420ccctgcgaga acggcggagg tagcggcggt
aaatctaagg ccgtggaaca ggtcaaaaac 480gcctttaaca agctgcagga
aaagggaatc tataaggcta tgagcgagtt cgacatcttt 540attaactata
tcgaggccta tatgactatg aagattagga acgtgggcta tatgcactgg
600tatcagcaga agcccggtaa agcccctaag ctgctgatct acgacacctc
taagctggct 660agtggcgtgc cctctaggtt tagcggtagc ggtagtggca
ccgccttcac cctgactatc 720tctagcctgc agcccgacga cttcgctacc
tactactgtt ttcagggtag cggctacccc 780ttcaccttcg gcggaggcac
taagctggag attaagcgta cggtggccgc tcccagcgtg 840ttcatcttcc
cccccagcga cgagcagctg aagagcggca ccgccagcgt ggtgtgcctg
900ctgaacaact
tctacccccg ggaggccaag gtgcagtgga aggtggacaa cgccctgcag
960agcggcaaca gccaggagag cgtcaccgag caggacagca aggactccac
ctacagcctg 1020agcagcaccc tgaccctgag caaggccgac tacgagaagc
ataaggtgta cgcctgcgag 1080gtgacccacc agggcctgtc cagccccgtg
accaagagct tcaacagggg cgagtgc 1137250498DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 250agtcccggtc agggaactca gtcagagaat agctgcactc
acttccccgg taacctgcct 60aatatgctga gagatctgag ggacgccttc tctagggtca
agaccttctt tcagatgaag 120gatcagctgg ataacctgct gctgaaagag
tcactgctgg aggactttaa gggctacctg 180ggctgtcagg ccctgagcga
gatgattcag ttctacctgg aagaagtgat gccccaggcc 240gagaatcagg
accccgatat taaggctcac gtcaactcac tgggcgagaa cctgaaaacc
300ctgagactga ggctgaggcg gtgtcaccgg tttctgccct gcgagaacgg
cggaggtagc 360ggcggtaaat ctaaggccgt ggaacaggtc aaaaacgcct
ttaacaagct gcaggaaaag 420ggaatctata aggctatgag cgagttcgac
atctttatta actatatcga ggcctatatg 480actatgaaga ttaggaac
4982516PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 251Gly Gly Gly Ser Gly Gly1
52525PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 252Gly Gly Gly Gly Ser1
52535PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 253Gly Gly Gly Gly Ala1 5
* * * * *