U.S. patent application number 11/645287 was filed with the patent office on 2009-12-17 for human antibodies that bind human il-12 and methods for producing.
This patent application is currently assigned to Abbott GMBH & Co., KG. Invention is credited to Subhashis Banerjee, Sara Carmen, Elaine Joy Derbyshire, Sarah Leila Du Fou, Alexander Robert Duncan, John Gawain Elvin, Stuart Friedrich, Thor Las Holtet, Angela Kantor, Zehra Kaymakcalan, Boris Labkovsky, Michael Paskind, Michael Roguska, Paul Sakorafas, Jochen G. Salfeld, Stephen Smith, Daniel Edward Tracey, Geertruida M. Veldman, Amy Venturini, Nicholas W. Warne, Michael White, Angela Widom.
Application Number | 20090311241 11/645287 |
Document ID | / |
Family ID | 39563112 |
Filed Date | 2009-12-17 |
United States Patent
Application |
20090311241 |
Kind Code |
A9 |
Salfeld; Jochen G. ; et
al. |
December 17, 2009 |
Human antibodies that bind human IL-12 and methods for
producing
Abstract
Human antibodies, preferably recombinant human antibodies, that
specifically bind to human interleukin-12 (hIL-12) are disclosed.
Preferred antibodies have high affinity for hIL-12 and neutralize
hIL-12 activity in vitro and in vivo. An antibody of the invention
can be a full-length antibody or an antigen-binding portion
thereof. The antibodies, or antibody portions, of the invention are
useful for detecting hIL-12 and for inhibiting hIL-12 activity,
e.g., in a human subject suffering from a disorder in which hIL-12
activity is detrimental. Nucleic acids, vectors and host cells for
expressing the recombinant human antibodies of the invention, and
methods of synthesizing the recombinant human antibodies, are also
encompassed by the invention.
Inventors: |
Salfeld; Jochen G.; (North
Grafton, MA) ; Roguska; Michael; (Ashland, MA)
; Paskind; Michael; (Sterling, MA) ; Banerjee;
Subhashis; (Hamden, CT) ; Tracey; Daniel Edward;
(Harvard, MA) ; White; Michael; (Framingham,
MA) ; Kaymakcalan; Zehra; (Westborough, MA) ;
Labkovsky; Boris; (Marlborough, MA) ; Sakorafas;
Paul; (Newton Highlands, MA) ; Veldman; Geertruida
M.; (Sudbury, MA) ; Venturini; Amy;
(Lexington, MA) ; Widom; Angela; (Acton, MA)
; Friedrich; Stuart; (Cary, NC) ; Warne; Nicholas
W.; (Andover, MA) ; Kantor; Angela;
(Pepperell, MA) ; Elvin; John Gawain; (Meldreth,
GB) ; Duncan; Alexander Robert; (Carton, GB) ;
Derbyshire; Elaine Joy; (Crowthorne, GB) ; Carmen;
Sara; (Cambridge, GB) ; Smith; Stephen; (Ely,
GB) ; Holtet; Thor Las; (Ronde, DK) ; Du Fou;
Sarah Leila; (Hitchen, GB) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP/ABBOTT;FLOOR 30, SUITE 3000
ONE POST OFFICE SQUARE
BOSTON
MA
02109-2127
US
|
Assignee: |
Abbott GMBH & Co., KG
Wiesbaden
DE
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20080063634 A1 |
March 13, 2008 |
|
|
Family ID: |
39563112 |
Appl. No.: |
11/645287 |
Filed: |
December 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10884830 |
Jul 1, 2004 |
7504485 |
|
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11645287 |
Dec 22, 2006 |
|
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09534717 |
Mar 24, 2000 |
6914128 |
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10884830 |
Jul 1, 2004 |
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60126603 |
Mar 25, 1999 |
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Current U.S.
Class: |
424/130.1 ;
436/86; 530/389.2; 530/402 |
Current CPC
Class: |
A61P 17/00 20180101;
C07K 2317/92 20130101; A61P 25/00 20180101; A61P 25/14 20180101;
A61P 43/00 20180101; A61P 35/04 20180101; C07K 2317/30 20130101;
C07K 2317/33 20130101; C07K 2317/76 20130101; A61P 7/06 20180101;
A61P 9/04 20180101; A61P 3/10 20180101; A61P 31/14 20180101; A61P
35/00 20180101; C07K 16/244 20130101; A61P 21/04 20180101; A61P
25/28 20180101; A61P 31/00 20180101; A61P 33/00 20180101; A61P
37/00 20180101; C07K 2317/565 20130101; A61P 25/16 20180101; A61P
29/00 20180101; A61P 37/06 20180101; A61P 17/06 20180101; C07K
2317/56 20130101; A61P 27/02 20180101; A61P 1/04 20180101; A61P
13/12 20180101; A61P 1/00 20180101; A61P 1/16 20180101; A61P 5/14
20180101; A61K 2039/505 20130101; A61P 5/16 20180101; A61P 19/04
20180101; A61P 37/08 20180101; C07K 2317/21 20130101; A61P 7/02
20180101; A61P 31/04 20180101; A61P 9/10 20180101; A61P 19/02
20180101; A61P 11/00 20180101; A61P 11/06 20180101; C07K 2317/73
20130101; A61P 9/00 20180101 |
Class at
Publication: |
424/130.1 ;
436/086; 530/389.2; 530/402 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 37/00 20060101 A61P037/00; C07K 14/54 20060101
C07K014/54; C07K 16/24 20060101 C07K016/24; G01N 33/00 20060101
G01N033/00 |
Claims
1. An isolated antibody, or antigen binding portion thereof, that
is capable of binding to the p40 subunit of IL-12 and is capable of
altering the conformational structure of said p40 subunit of
IL-12.
2. The isolated antibody of claim 1, or antigen binding portion
thereof, which dissociates from the p40 subunit of human IL-12 with
a K.sub.d of 1.times.10.sup.-10 M or less or a k.sub.off rate
constant of 1.times.10.sup.-3 s.sup.-1 or less, as determined by
surface plasmon resonance.
3. The isolated antibody of claim 1, or antigen binding portion
thereof, which is a neutralizing antibody
4. The isolated antibody of claim 3, or antigen binding portion
thereof, which inhibits phytohemagglutinin blast proliferation in
an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less, or which inhibits human IFN.gamma. production with an
IC.sub.50 of 1.times.10.sup.-10 M or less.
5. The isolated antibody of claim 1, or antigen binding portion
thereof, which is a human antibody.
6. A pharmaceutical composition comprising the antibody or an
antigen binding portion thereof of claim 1, and a pharmaceutically
acceptable carrier.
7. A method for detecting the p40 subunit of an interleukin
comprising contacting the p40 subunit of IL-12 with the antibody,
or antigen-binding portion thereof, of claim 1 such that said p40
subunit is detected.
8. A method for detecting the p40 subunit of an interleukin
comprising contacting the p40 subunit of the interleukin with the
antibody, or antigen-binding portion thereof, of claim 1 such that
the p40 subunit of the interleukin is detected
9. The method of claim 7, wherein the p40 subunit of the
interleukin is detected in vitro.
10. The method of claim 7, wherein the p40 subunit of the
interleukin is detected in a biological sample for diagnostic
purposes.
11. A method for inhibiting an activity of an interleukin
comprising a p40 subunit, comprising contacting the interleukin
with the antibody, or antigen-binding portion thereof, of claim 1
such that the activity is inhibited.
12. A method for inhibiting an activity of an interleukin
comprising a p40 subunit in a human subject suffering from a
disorder in which the activity is detrimental, comprising
administering to the human subject the antibody, or antigen-binding
portion thereof, of claim 1 such that the activity in the human
subject is inhibited.
13. The method of claim 11, wherein said interleukin is IL-12.
14. The method of claim 11, wherein said interleukin comprises a
p40 subunit and a p19 subunit.
15. The method of claim 11, wherein said interleukin is IL-23.
16. The method of claim 12, wherein the disorder is selected from
the group consisting of rheumatoid arthritis, osteoarthritis,
juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis,
reactive arthritis, spondyoarthropathy, ankylosing spondylitis,
systemic lupus erythematosis, Crohn's disease, ulcerative colitis,
inflammatory bowel disease, multiple sclerosis, insulin dependent
diabetes mellitus, thyroiditis, asthma, allergic diseases,
psoriasis, dermatitisscleroderma, thyroiditis, graft versus host
disease, organ transplant rejection, acute or chronic immune
disease associated with organ transplantation, sarcoidosis,
atherosclerosis, disseminated intravascular coagulation, Kawasaki's
disease, Grave's disease, nephrotic syndrome, chronic fatigue
syndrome, polyarteritis nodosa, Wegener's granulomatosis,
Henoch-Schonlein purpura, microscopic vasculitis of the kidneys,
chronic active hepatitis, Sjogren's syndrome, uveitis, sepsis,
septic shock, sepsis syndrome, adult respiratory distress syndrome,
cachexia, infectious diseases, parasitic diseases, acquired
immunodeficiency syndrome, acute transverse myelitis, myasthenia
gravis, Huntington's chorea, Parkinson's disease, Alzheimer's
disease, stroke, primary biliary cirrhosis, fibrotic lung diseases,
hemolytic anemia, malignancies, heart failure and myocardial
infarction.
17. The method of claim 12, wherein the disorder is psoriasis.
18. The method of claim 12, wherein the disorder is rheumatoid
arthritis.
19. A method for altering the conformational structure of the p40
subunit of IL-12, the method comprising contacting said subunit
with an antibody, or antigen binding portion thereof, that is
capable of binding to said subunit and is capable of altering the
conformational structure of said subunit in an amount effective to
alter the conformational structure of said subunit, thereby
altering the conformational structure of said subunit.
20. A method for inhibiting the activity of an interleukin
comprising a p40 subunit, the method comprising contacting the
interleukin with an antibody, or antigen binding portion thereof,
that is capable of binding to the p40 subunit of IL-12 and is
capable of altering the conformational structure of the interleukin
in an amount effective to alter the conformational structure of the
interleukin, thereby inhibiting the activity of the interleukin.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/534,717, filed Mar. 24, 2000, which issued
on Jul. 5, 2005 as U.S. Pat. No. 6,914,128. This application also
claims priority to U.S. provisional application Ser. No.
60/126,603, filed Mar. 25, 1999. The entire contents of each of the
foregoing applications are hereby incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Human interleukin 12 (IL-12) has recently been characterized
as a cytokine with a unique structure and pleiotropic effects
(Kobayashi, et al. (1989) J. Exp Med. 170:827-845; Seder, et al.
(1993) Proc. Natl. Acad. Sci. 90:10188-10192; Ling, et al. (1995)
J. Exp Med. 154:116-127; Podlaski, et al. (1992) Arch. Biochem.
Biophys. 294:230-237). IL-12 plays a critical role in the pathology
associated with several diseases involving immune and inflammatory
responses. A review of IL-12, its biological activities, and its
role in disease can be found in Gately et al. (1998) Ann. Rev.
Immunol. 16: 495-521.
[0003] Structurally, IL-12 is a heterodimeric protein comprising a
35 kDa subunit (p35) and a 40 kDa subunit (p40) which are both
linked together by a disulfide bridge (referred to as the "p70
subunit"). The heterodimeric protein is produced primarily by
antigen-presenting cells such as monocytes, macrophages and
dendritic cells. These cell types also secrete an excess of the p40
subunit relative to p70 subunit. The p40 and p35 subunits are
genetically unrelated and neither has been reported to possess
biological activity, although the p40 homodimer may function as an
IL-12 antagonist.
[0004] Functionally, IL-12 plays a central role in regulating the
balance between antigen specific T helper type (Th1) and type 2
(Th2) lymphocytes. The Th1 and Th2 cells govern the initiation and
progression of autoimmune disorders, and IL-12 is critical in the
regulation of Th.sub.1-lymphocyte differentiation and maturation.
Cytokines released by the Th1 cells are inflammatory and include
interferon .gamma. (IFN.gamma.), IL-2 and lymphotoxin (LT). Th2
cells secrete IL-4, IL-5, IL-6, IL-10 and IL-13 to facilitate
humoral immunity, allergic reactions, and immunosuppression.
[0005] Consistent with the preponderance of Th1 responses in
autoimmune diseases and the proinflammatory activities of
IFN.gamma., IL-12 may play a major role in the pathology associated
with many autoimmune and inflammatory diseases such as rheumatoid
arthritis (RA), multiple sclerosis (MS), and Crohn's disease.
[0006] Human patients with MS have demonstrated an increase in
IL-12 expression as documented by p40 mRNA levels in acute MS
plaques. (Windhagen et al., (1995) J. Exp. Med. 182: 1985-1996). In
addition, ex vivo stimulation of antigen-presenting cells with
CD40L-expressing T cells from MS patients resulted in increased
IL-12 production compared with control T cells, consistent with the
observation that CD40/CD40L interactions are potent inducers of
IL-12.
[0007] Elevated levels of IL-12 p70 have been detected in the
synovia of RA patients compared with healthy controls (Morita et al
(1998) Arthritis and Rheumatism. 41: 306-314). Cytokine messenger
ribonucleic acid (mRNA) expression profile in the RA synovia
identified predominantly Th1 cytokines. (Bucht et al., (1996) Clin.
Exp. Immunol. 103: 347-367). IL-12 also appears to play a critical
role in the pathology associated with Crohn's disease (CD).
Increased expression of INF.gamma. and IL-12 has been observed in
the intestinal mucosa of patients with this disease (Fais et al.
(1994) J. Interferon Res. 14:235-238; Parronchi et al., (1997) Am.
J. Path. 150:823-832; Monteleone et al., (1997) Gastroenterology.
112:1169-1178, and Berrebi et al., (1998) Am. J. Path 152:667-672).
The cytokine secretion profile of T cells from the lamina propria
of CD patients is characteristic of a predominantly Th1 response,
including greatly elevated IFN.gamma. levels (Fuss, et al., (1996)
J. Immunol. 157:1261-1270). Moreover, colon tissue sections from CD
patients show an abundance of IL-12 expressing macrophages and
IFN.gamma. expressing T cells (Parronchi et al (1997) Am. J. Path.
150:823-832).
[0008] Due to the role of human IL-12 in a variety of human
disorders, therapeutic strategies have been designed to inhibit or
counteract IL-12 activity. In particular, antibodies that bind to,
and neutralize, IL-12 have been sought as a means to inhibit IL-12
activity. Some of the earliest antibodies were murine monoclonal
antibodies (mAbs), secreted by hybridomas prepared from lymphocytes
of mice immunized with IL-12 (see e.g., World Patent Application
Publication No. WO 97/15327 by Strober et al.; Neurath et al.
(1995) J. Exp. Med. 182:1281-1290; Duchmann et al. (1996) J.
Immunol. 26:934-938). These murine IL-12 antibodies are limited for
their use in vivo due to problems associated with administration of
mouse antibodies to humans, such as short serum half life, an
inability to trigger certain human effector functions and
elicitation of an unwanted immune response against the mouse
antibody in a human (the "human anti-mouse antibody" (HAMA)
reaction).
[0009] In general, attempts to overcome the problems associated
with use of fully-murine antibodies in humans, have involved
genetically engineering the antibodies to be more "human-like." For
example, chimeric antibodies, in which the variable regions of the
antibody chains are murine-derived and the constant regions of the
antibody chains are human-derived, have been prepared (Junghans, et
al. (1990) Cancer Res. 50:1495-1502; Brown et al. (1991) Proc.
Natl. Acad. Sci. 88:2663-2667; Kettleborough et al. (1991) Protein
Engineering. 4:773-783). However, because these chimeric and
humanized antibodies still retain some murine sequences, they still
may elicit an unwanted immune reaction, the human anti-chimeric
antibody (HACA) reaction, especially when administered for
prolonged periods.
[0010] A preferred IL-12 inhibitory agent to murine antibodies or
derivatives thereof (e.g., chimeric or humanized antibodies) would
be an entirely human anti-IL-12 antibody, since such an agent
should not elicit the HAMA reaction, even if used for prolonged
periods. However, such antibodies have not been described in the
art and, therefore are still needed.
SUMMARY OF THE INVENTION
[0011] The present invention provides human antibodies that bind
human IL-12. The invention also relates to the treatment or
prevention of acute or chronic diseases or conditions whose
pathology involves IL-12, using the human anti-IL-12 antibodies of
the invention.
[0012] In one aspect, the invention provides an isolated human
antibody, or an antigen-binding portion thereof, that binds to
human IL-12.
[0013] In one embodiment, the invention provides a selectively
mutated human IL-12 antibody, comprising:
[0014] a human antibody or antigen-binding portion thereof,
selectively mutated at a preferred selective mutagenesis position,
contact or hypermutation position with an activity enhancing amino
acid residue such that it binds to human IL-12.
[0015] In a preferred embodiment, the invention provides a
selectively mutated human IL-12 antibody, comprising:
[0016] a human antibody or antigen-binding portion thereof,
selectively mutated at a preferred selective mutagenesis position
with an activity enhancing amino acid residue such that it binds to
human IL-12.
[0017] In another preferred embodiment, the selectively mutated
human IL-12 antibody or antigen-binding portion thereof is
selectively mutated at more than one preferred selective
mutagenesis position, contact or hypermutation positions with an
activity enhancing amino acid residue. In another preferred
embodiment, the selectively mutated human IL-12 antibody or
antigen-binding portion thereof is selectively mutated at no more
than three preferred selective mutagenesis positions, contact or
hypermutation positions. In another preferred embodiment, the
selectively mutated human IL-12 antibody or antigen-binding portion
thereof is selectively mutated at no more than two preferred
selective mutagenesis position, contact or hypermutation positions.
In yet another preferred embodiment, the selectively mutated human
IL-12 antibody or antigen-binding portion thereof, is selectively
mutated such that a target specificity affinity level is attained,
the target level being improved over that attainable when selecting
for an antibody against the same antigen using phage display
technology. In another preferred embodiment, the selectively
mutated human IL-12 antibody further retains at least one desirable
property or characteristic, e.g., preservation of non-cross
reactivity with other proteins or human tissues, preservation of
epitope recognition, production of an antibody with a close to a
germline immunoglobulin sequence.
[0018] In another embodiment, the invention provides an isolated
human antibody, or antigen-binding portion thereof, that binds to
human IL-12 and dissociates from human IL-12 with a K.sub.off rate
constant of 0.1 s.sup.-1 or less, as determined by surface plasmon
resonance, or which inhibits phytohemagglutinin blast proliferation
in an in vitro phytohemagglutinin blast proliferation assay (PHA
assay) with an IC.sub.50 of 1.times.10.sup.-6 M or less. More
preferably, the isolated human antibody or an antigen-binding
portion thereof, dissociates from human IL-12 with a K.sub.off rate
constant of 1.times.10.sup.-2 s.sup.-1 or less, or inhibits
phytohemagglutinin blast proliferation in an in vitro PHA assay
with an IC.sub.50 of 1.times.10.sup.-7 M or less. More preferably,
the isolated human antibody, or an antigen-binding portion thereof,
dissociates from human IL-12 with a K.sub.off rate constant of
1.times.10.sup.-3 s.sup.-1 or less, or inhibits phytohemagglutinin
blast proliferation in an in vitro PHA assay with an IC.sub.50 of
1.times.10.sup.-8 M or less. More preferably, the isolated human
antibody, or an antigen-binding portion thereof, dissociates from
human IL-12 with a K.sub.off rate constant of 1.times.10.sup.-4
s.sup.-1 or less, or inhibits phytohemagglutinin blast
proliferation in an in vitro PHA assay with an IC.sub.50 of
1.times.10.sup.-9 M or less. More preferably, the isolated human
antibody, or an antigen-binding portion thereof, dissociates from
human IL-12 with a K.sub.off rate constant of 1.times.10.sup.-5
s.sup.-1 or less, or inhibits phytohemagglutinin blast
proliferation in an in vitro PHA assay with an IC.sub.50 of
1.times.10.sup.-10 M or less. Even more preferably, the isolated
human antibody, or an antigen-binding portion thereof, dissociates
from human IL-12 with a K.sub.off rate constant of
1.times.10.sup.-5 s.sup.-1 or less, or inhibits phytohemagglutinin
blast proliferation in an in vitro PHA assay with an IC.sub.50 of
1.times.10.sup.-11 M or less.
[0019] In another embodiment, the invention provides an isolated
human antibody, or an antigen-binding portion thereof, which has
the following characteristics:
[0020] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-6 M or
less;
[0021] b) has a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 1; and
[0022] c) has a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 2.
[0023] In a preferred embodiment, the isolated human antibody, or
an antigen-binding portion thereof, has a heavy chain CDR2
comprising the amino acid sequence of SEQ ID NO: 3; and has a light
chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4. In a
preferred embodiment, the isolated human antibody, or an
antigen-binding portion thereof, has a heavy chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 5; and has a light chain CDR1
comprising the amino acid sequence of SEQ ID NO: 6. In a preferred
embodiment, the isolated human antibody, or antigen binding portion
thereof, has a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 7; and has a light chain variable
region comprising the amino acid sequence of SEQ ID NO: 8.
[0024] In another embodiment, the invention provides an isolated
human antibody, or an antigen-binding portion thereof, which has
the following characteristics:
[0025] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less;
[0026] b) has a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 9; and
[0027] c) has a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 10.
[0028] In a preferred embodiment, the isolated human antibody, or
an antigen-binding portion thereof, has a heavy chain CDR2
comprising the amino acid sequence of SEQ ID NO: 11; and has a
light chain CDR2 comprising the amino acid sequence of SEQ ID NO:
12. In a preferred embodiment, the isolated human antibody, or an
antigen-binding portion thereof, has a heavy chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 13; and has a light chain
CDR1 comprising the amino acid sequence of SEQ ID NO: 14. In a
preferred embodiment, the isolated human antibody has a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:
15; and has a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 16.
[0029] In another embodiment, the invention provides an isolated
human antibody, or an antigen-binding portion thereof, which
[0030] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less;
[0031] b) has a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 17; and
[0032] c) has a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 18.
[0033] In a preferred embodiment, the isolated human antibody, or
an antigen-binding portion thereof, has a heavy chain CDR2
comprising the amino acid sequence of SEQ ID NO: 19; and a light
chain CDR2 comprising the amino acid sequence of SEQ ID NO: 20. In
a preferred embodiment, the isolated human antibody, or an
antigen-binding portion thereof, has a heavy chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 21; and a light chain CDR1
comprising the amino acid sequence of SEQ ID NO: 22. In a preferred
embodiment, the isolated human antibody, or an antigen-binding
portion thereof, has the heavy chain variable region comprising the
amino acid sequence of SEQ ID NO: 23, and a light chain variable
region comprising the amino acid sequence of SEQ ID NO: 24. In a
preferred embodiment, the isolated human antibody comprises a heavy
chain constant region selected from the group consisting of IgG1,
IgG2, IgG3, IgG4, IgM, IgA and IgE constant regions or any allelic
variation thereof as discussed in Kabat et al. (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), included herein by reference. In a more
preferred embodiment, the antibody heavy chain constant region is
IgG1. In another preferred embodiment, the isolated human antibody
is a Fab fragment, or a F(ab').sub.2 fragment or a single chain Fv
fragment.
[0034] In another embodiment, the invention provides an isolated
human antibody, or an antigen-binding portion thereof, which
[0035] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less;
[0036] b) has a heavy chain CDR3 comprising the amino acid sequence
selected from the group consisting of SEQ ID NO: 404-SEQ ID NO:
469; and
[0037] c) has a light chain CDR3 comprising the amino acid sequence
selected from the group consisting of SEQ ID NO: 534-SEQ ID NO:
579.
[0038] In a preferred embodiment, the isolated human antibody, or
an antigen-binding portion thereof, has a heavy chain CDR2
comprising the amino acid sequence selected from the group
consisting of SEQ ID NO:335-SEQ ID NO: 403; and a light chain CDR2
comprising the amino acid sequence selected from the group
consisting of SEQ ID NO: 506-SEQ ID NO: 533. In a preferred
embodiment, the isolated human antibody, or an antigen-binding
portion thereof, has a heavy chain CDR1 comprising the amino acid
sequence selected from the group consisting of SEQ ID NO: 288-SEQ
ID NO: 334; and a light chain CDR1 comprising the amino acid
sequence selected from the group consisting of SEQ ID NO: 470-SEQ
ID NO: 505. In a preferred embodiment, the isolated human antibody,
or an antigen-binding portion thereof, comprising a the heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:
23, and a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 24. In a preferred embodiment, the isolated
human antibody comprises a heavy chain constant region, or an Fab
fragment or a F(ab').sub.2 fragment or a single chain Fv fragment
as described above.
[0039] In another embodiment, the invention provides an isolated
human antibody, or an antigen-binding portion thereof, which
[0040] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less;
[0041] b) has a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 25; and
[0042] c) has a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 26.
[0043] In a preferred embodiment, the isolated human antibody, or
an antigen-binding portion thereof, has a heavy chain CDR2
comprising the amino acid sequence of SEQ ID NO: 27; and a light
chain CDR2 comprising the amino acid sequence of SEQ ID NO: 28. In
a preferred embodiment, the isolated human antibody, or an
antigen-binding portion thereof, has a heavy chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 29; and a light chain CDR1
comprising the amino acid sequence of SEQ ID NO: 30. In a preferred
embodiment, the isolated human antibody, or an antigen-binding
portion thereof, which has a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO: 31, and a light chain
variable region comprising the amino acid sequence of SEQ ID NO:
32. In a preferred embodiment, the isolated human antibody
comprises a heavy chain constant region, or an Fab fragment, or a
F(ab').sub.2 fragment or a single chain Fv fragment as described
above.
[0044] In another embodiment, the invention provides an isolated
human antibody, or an antigen-binding portion thereof, which
[0045] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-6 M or
less;
[0046] b) comprises a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 3 and a heavy chain CDR1 comprising the
amino acid sequence of SEQ ID NO:5, or a mutant thereof having one
or more amino acid substitutions at a contact position or a
hypermutation position, wherein said mutant has a k.sub.off rate no
more than 10-fold higher than the antibody comprising a heavy chain
CDR3 comprising the amino acid sequence of SEQ ID NO: 1, a heavy
chain CDR2 comprising the amino acid sequence of SEQ ID NO: 3, and
a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:
5; and
[0047] c) comprises a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 2, a light chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 4, and a light chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 6, or a mutant thereof having
one or more amino acid substitutions at a contact position or a
hypermutation position, wherein said mutant has a k.sub.off rate no
more than 10-fold higher than the antibody comprising a light chain
CDR3 comprising the amino acid sequence of SEQ ID NO: 2, a light
chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and
a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:
6.
[0048] In another embodiment, the invention provides an isolated
human antibody, or an antigen-binding portion thereof, which
[0049] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less;
[0050] b) comprises a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 11 and a heavy chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 13, or a mutant thereof
having one or more amino acid substitutions at a contact position
or a hypermutation position, wherein said mutant has a k.sub.off
rate no more than 10-fold higher than the antibody comprising a
heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:
9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID
NO: 11, and a heavy chain CDR1 comprising the amino acid sequence
of SEQ ID NO: 13; and
[0051] c) comprises a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 10, a light chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 12, and a light chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 14, or a mutant thereof
having one or more amino acid substitutions at a preferred
selective mutagenesis position, contact position or a hypermutation
position, wherein said mutant has a k.sub.off rate no more than
10-fold higher than the antibody comprising a light chain CDR3
comprising the amino acid sequence of SEQ ID NO: 10, a light chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a
light chain CDR1 comprising the amino acid sequence of SEQ ID NO:
14.
[0052] In another embodiment, the invention provides an isolated
human antibody, or an antigen-binding portion thereof, which
[0053] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less;
[0054] b) comprises a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 19 and a heavy chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 21, or a mutant thereof
having one or more amino acid substitutions at a preferred
selective mutagenesis position, contact position or a hypermutation
position, wherein said mutant has a k.sub.off rate no more than
10-fold higher than the antibody comprising a heavy chain CDR3
comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a
heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:
21; and
[0055] c) comprises a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 18, a light chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 20, and a light chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 22, or a mutant thereof
having one or more amino acid substitutions at preferred selective
mutagenesis position, contact position or a hypermutation position,
wherein said mutant has a k.sub.off rate no more than 10-fold
higher than the antibody comprising a light chain CDR3 comprising
the amino acid sequence of SEQ ID NO: 18, a light chain CDR2
comprising the amino acid sequence of SEQ ID NO: 20, and a light
chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22.
[0056] The invention also provides nucleic acid molecules encoding
antibodies, or antigen binding portions thereof, of the invention.
A preferred isolated nucleic acid encodes the heavy chain CDR3
comprising the amino acid sequence of SEQ ID NO: 17. The isolated
nucleic acid encoding an antibody heavy chain variable region. In
another embodiment, the isolated nucleic acid encodes the CDR2 of
the antibody heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 19. In another embodiment, the isolated
nucleic acid encodes the CDR1 of the antibody heavy chain variable
region comprising the amino acid sequence of SEQ ID NO: 21. In
another embodiment, the isolated nucleic acid encodes an antibody
heavy chain variable region comprising the amino acid sequence of
SEQ ID NO: 23. In another embodiment, the isolated nucleic acid
encodes the light chain CDR3 comprising the amino acid sequence of
SEQ ID NO: 18. The isolated nucleic acid encoding an antibody light
chain variable region. In another embodiment, the isolated nucleic
acid encodes the CDR2 of the antibody light chain variable region
comprising the amino acid sequence of SEQ ID NO: 20. In another
embodiment, the isolated nucleic acid encodes the CDR1 of the
antibody light chain variable region comprising the amino acid
sequence of SEQ ID NO: 22. In another embodiment, the isolated
nucleic acid encodes an antibody light chain variable region
comprising the amino acid sequence of SEQ ID NO: 24.
[0057] In another embodiment, the invention provides an isolated
human antibody, or an antigen-binding portion thereof, which
[0058] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less;
[0059] b) comprises a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 27 and a heavy chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 29, or a mutant thereof
having one or more amino acid substitutions at a preferred
selective mutagenesis position, contact position or a hypermutation
position, wherein said mutant has a k.sub.off rate no more than
10-fold higher than the antibody comprising a heavy chain CDR3
comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a
heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:
29; and
[0060] c) comprises a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 28, and a light chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 30, or a mutant thereof
having one or more amino acid substitutions at a preferred
selective mutagenesis position, contact position or a hypermutation
position, wherein said mutant has a k.sub.off rate no more than
10-fold higher than the antibody comprising a light chain CDR3
comprising the amino acid sequence of SEQ ID NO: 26, a light chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a
light chain CDR1 comprising the amino acid sequence of SEQ ID NO:
30.
[0061] A preferred isolated nucleic acid encodes the heavy chain
CDR3 comprising the amino acid sequence of SEQ ID NO: 25. The
isolated nucleic acid encoding an antibody heavy chain variable
region. In another embodiment, the isolated nucleic acid encodes
the CDR2 of the antibody heavy chain variable region comprising the
amino acid sequence of SEQ ID NO: 27. In another embodiment, the
isolated nucleic acid encodes the CDR1 of the antibody heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:
29. In another embodiment, the isolated nucleic acid encodes an
antibody heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 31. In another embodiment, the isolated
nucleic acid encodes the light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 26. The isolated nucleic acid encoding an
antibody light chain variable region. In another embodiment, the
isolated nucleic acid encodes the CDR2 of the antibody light chain
variable region comprising the amino acid sequence of SEQ ID NO:
28. In another embodiment, the isolated nucleic acid encodes the
CDR1 of the antibody light chain variable region comprising the
amino acid sequence of SEQ ID NO: 30. In another embodiment, the
isolated nucleic acid encodes an antibody light chain variable
region comprising the amino acid sequence of SEQ ID NO: 32.
[0062] In another aspect, the invention provides an isolated human
antibody, or an antigen-binding portion thereof, which has the
following characteristics: [0063] a) that binds to human IL-12 and
dissociates from human IL-12 with a k.sub.off rate constant of 0.1
s.sup.-1 or less, as determined by surface plasmon resonance, or
which inhibits phytohemagglutinin blast proliferation in an in
vitro phytohemagglutinin blast proliferation assay (PHA assay) with
an IC.sub.50 of 1.times.10.sup.-6M or less. [0064] b) has a heavy
chain variable region comprising an amino acid sequence selected
from a member of the V.sub.H3 germline family, wherein the heavy
chain variable region has a mutation at a preferred selective
mutagenesis position, contact or hypermutation position with an
activity enhancing amino acid residue. [0065] c) has a light chain
variable region comprising an amino acid sequence selected from a
member of the V.sub..lamda.1 germline family, wherein the light
chain variable region has a mutation at a preferred selective
mutagenesis position, contact position or hypermutation position
with an activity enhancing amino acid residue.
[0066] In another embodiment, the invention provides an isolated
human antibody, or an antigen-binding portion thereof, which has
the following characteristics: [0067] a) that binds to human IL-12
and dissociates from human IL-12 with a k.sub.off rate constant of
0.1 s.sup.-1 or less, as determined by surface plasmon resonance,
or which inhibits phytohemagglutinin blast proliferation in an in
vitro phytohemagglutinin blast proliferation assay (PHA assay) with
an IC.sub.50 of 1.times.10.sup.-6M or less. [0068] b) has a heavy
chain variable region comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs: 595-667, wherein the heavy
chain variable region has a mutation at a preferred selective
mutagenesis position, contact position or hypermutation position
with an activity enhancing amino acid residue. [0069] c) has a
light chain variable region comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 669-675, wherein
the light chain variable region has a mutation at a preferred
selective mutagenesis position, contact or hypermutation position
with an activity enhancing amino acid residue.
[0070] In another embodiment, the invention provides an isolated
human antibody, or an antigen-binding portion thereof, which has
the following characteristics: [0071] a) that binds to human IL-12
and dissociates from human IL-12 with a k.sub.off rate constant of
0.1 s.sup.-1 or less, as determined by surface plasmon resonance,
or which inhibits phytohemagglutinin blast proliferation in an in
vitro phytohemagglutinin blast proliferation assay (PHA assay) with
an IC.sub.50 of 1.times.10.sup.-6M or less. [0072] b) has a heavy
chain variable region comprising the COS-3 germline amino acid
sequence, wherein the heavy chain variable region has a mutation at
a preferred selective mutagenesis position, contact or
hypermutation position with an activity enhancing amino acid
residue. [0073] c) has a light chain variable region comprising the
DPL8 germline amino acid sequence, wherein the light chain variable
region has a mutation at a preferred selective mutagenesis
position, contact or hypermutation position with an activity
enhancing amino acid residue.
[0074] In another embodiment, the invention provides an isolated
human antibody, or an antigen-binding portion thereof, which has
the following characteristics: [0075] a) that binds to human IL-12
and dissociates from human IL-12 with a k.sub.off rate constant of
0.1 s.sup.-1 or less, as determined by surface plasmon resonance,
or which inhibits phytohemagglutinin blast proliferation in an in
vitro phytohemagglutinin blast proliferation assay (PHA assay) with
an IC.sub.50 of 1.times.10.sup.-6M or less. [0076] b) has a heavy
chain variable region comprising an amino acid sequence selected
from a member of the V.sub.H3 germline family, wherein the heavy
chain variable region comprises a CDR2 that is structurally similar
to CDR2s from other V.sub.H3 germline family members, and a CDR1
that is structurally similar to CDR1s from other V.sub.H3 germline
family members, and wherein the heavy chain variable region has a
mutation at a preferred selective mutagenesis position, contact or
hypermutation position with an activity enhancing amino acid
residue; [0077] c) has a light chain variable region comprising an
amino acid sequence selected from a member of the V.sub..lamda.1
germline family, wherein the light chain variable region comprises
a CDR2 that is structurally similar to CDR2s from other
V.sub..lamda.1 germline family members, and a CDR1 that is
structurally similar to CDR1s from other V.sub..lamda.1 germline
family members, and wherein the light chain variable region has a
mutation at a preferred selective mutagenesis position, contact or
hypermutation position with an activity enhancing amino acid
residue.
[0078] In a preferred embodiment, the isolated human antibody, or
antigen binding portion thereof, has a mutation in the heavy chain
CDR3. In another preferred embodiment, the isolated human antibody,
or antigen binding portion thereof, has a mutation in the light
chain CDR3. In another embodiment, the isolated human antibody, or
antigen binding portion thereof, has a mutation in the heavy chain
CDR2. In another preferred embodiment, the isolated human antibody,
or antigen binding portion thereof, has a mutation in the light
chain CDR2. In another preferred embodiment, the isolated human
antibody, or antigen binding portion thereof, has a mutation in the
heavy chain CDR1. In another preferred embodiment, the isolated
human antibody, or antigen binding portion thereof, has a mutation
in the light chain CDR1.
[0079] In another aspect, the invention provides recombinant
expression vectors carrying the antibody-encoding nucleic acids of
the invention, and host cells into which such vectors have been
introduced, are also encompassed by the invention, as are methods
of making the antibodies of the invention by culturing the host
cells of the invention.
[0080] In another aspect, the invention provides an isolated human
antibody, or antigen-binding portion thereof, that neutralizes the
activity of human IL-12, and at least one additional primate IL-12
selected from the group consisting of baboon IL-12, marmoset IL-12,
chimpanzee IL-12, cynomolgus IL-12 and rhesus IL-12, but which does
not neutralize the activity of the mouse IL-12.
[0081] In another aspect, the invention provides a pharmaceutical
composition comprising the antibody or an antigen binding portion
thereof, of the invention and a pharmaceutically acceptable
carrier.
[0082] In another aspect, the invention provides a composition
comprising the antibody or an antigen binding portion thereof, and
an additional agent, for example, a therapeutic agent.
[0083] In another aspect, the invention provides a method for
inhibiting human IL-12 activity comprising contacting human IL-12
with the antibody of the invention, e.g., J695, such that human
IL-12 activity is inhibited.
[0084] In another aspect, the invention provides a method for
inhibiting human IL-12 activity in a human subject suffering from a
disorder in which IL-12 activity is detrimental, comprising
administering to the human subject the antibody of the invention,
e.g., J695, such that human IL-12 activity in the human subject is
inhibited. The disorder can be, for example, Crohn's disease,
multiple sclerosis or rheumatoid arthritis.
[0085] In another aspect, the invention features a method for
improving the activity of an antibody, or an antigen binding
portion thereof, to attain a predetermined target activity,
comprising:
[0086] a) providing a parent antibody a antigen-binding portion
thereof;
[0087] b) selecting a preferred selective mutagenesis position
selected from group consisting of H30, H31, H31B, H32, H33, H52,
H56, H58, L30, L31, L32, L50, L91, L92, L93, L94.
[0088] c) individually mutating the selected preferred selective
mutagenesis position to at least two other amino acid residues to
hereby create a first panel of mutated antibodies, or antigen
binding portions thereof;
[0089] d) evaluating the activity of the first panel of mutated
antibodies, or antigen binding portions thereof to determined if
mutation of a single selective mutagenesis position produces an
antibody or antigen binding portion thereof with the predetermined
target activity or a partial target activity;
[0090] e) combining in a stepwise fashion, in the parent antibody,
or antigen binding portion thereof, individual mutations shown to
have an improved activity, to form combination antibodies, or
antigen binding portions thereof.
[0091] f) evaluating the activity of the combination antibodies, or
antigen binding portions thereof to determined if the combination
antibodies, or antigen binding portions thereof have the
predetermined target activity or a partial target activity.
[0092] g) if steps d) or f) do not result in an antibody or antigen
binding portion thereof having the predetermined target activity,
or result an antibody with only a partial activity, additional
amino acid residues selected from the group consisting of H35, H50,
H53, H54, H95, H96, H97, H98, L30A and L96 are mutated to at least
two other amino acid residues to thereby create a second panel of
mutated antibodies or antigen-binding portions thereof;
[0093] h) evaluating the activity of the second panel of mutated
antibodies or antigen binding portions thereof, to determined if
mutation of a single amino acid residue selected from the group
consisting of H35, H50, H53, H54, H95, H96, H97, H98, L30A and L96
results an antibody or antigen binding portion thereof, having the
predetermined target activity or a partial activity;
[0094] i) combining in stepwise fashion in the parent antibody, or
antigen-binding portion thereof, individual mutations of step g)
shown to have an improved activity, to form combination antibodies,
or antigen binding portions thereof;
[0095] j) evaluating the activity of the combination antibodies or
antigen binding portions thereof, to determined if the combination
antibodies, or antigen binding portions thereof have the
predetermined target activity or a partial target activity;
[0096] k) if steps h) or j) do not result in an antibody or antigen
binding portion thereof having the predetermined target activity,
or result in an antibody with only a partial activity, additional
amino acid residues selected from the group consisting of H33B,
H52B and L31A are mutated to at least two other amino acid residues
to thereby create a third panel of mutated antibodies or antigen
binding portions thereof;
[0097] l) evaluating the activity of the third panel of mutated
antibodies or antigen binding portions thereof, to determine if a
mutation of a single amino acid residue selected from the group
consisting of H33B, H52B and L31A resulted in an antibody or
antigen binding portion thereof, having the predetermined target
activity or a partial activity;
[0098] m) combining in a stepwise fashion in the parent antibody,
or antigen binding portion thereof, individual mutation of step k)
shown to have an improved activity, to form combination antibodies,
or antigen binding portions, thereof;
[0099] n) evaluating the activity of the combination antibodies or
antigen-binding portions thereof, to determine if the combination
antibodies, or antigen binding portions thereof have the
predetermined target activity to thereby produce an antibody or
antigen binding portion thereof with a predetermined target
activity.
[0100] In another aspect, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0101] a) providing a parent antibody or antigen-binding portion
thereof;
[0102] b) selecting a preferred selective mutagenesis position,
contact or hypermutation position within a complementarity
determining region (CDR) for mutation, thereby identifying a
selected preferred selective mutagenesis position, contact or
hypermutation position;
[0103] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;
[0104] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof;
[0105] e) repeating steps b) through d) for at least one other
contact or hypermutation position;
[0106] f) combining, in the parent antibody, or antigen-binding
portion thereof, individual mutations shown to have improved
activity, to form combination antibodies, or antigen-binding
portions thereof; and
[0107] g) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof; until an antibody, or
antigen-binding portion thereof, with an improved activity,
relative to the parent antibody, or antigen-binding portion
thereof, is obtained.
[0108] In one embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0109] a) providing a recombinant parent antibody or
antigen-binding portion thereof; that was obtained by selection in
a phage-display system but whose activity is not further improved
by mutagenesis in said phage-display system;
[0110] b) selecting a preferred selective mutagenesis position,
contact or hypermutation position within a complementarity
determining region (CDR) for mutation, thereby identifying a
selected contact or hypermutation position;
[0111] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof, and expressing
said panel in a non-phage display system;
[0112] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof;
[0113] e) repeating steps b) through d) for at least one other
contact or hypermutation position;
[0114] f) combining, in the parent antibody, or antigen-binding
portion thereof, individual mutations shown to have improved
activity, to form combination antibodies, or antigen-binding
portions thereof; and
[0115] g) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof; until an antibody, or
antigen-binding portion thereof, with an improved activity,
relative to the parent antibody, or antigen-binding portion
thereof, is obtained.
[0116] In a preferred embodiment, the contact positions are
selected from the group consisting of H30, H31, H31B, H32, H33,
H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101,
L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96.
In another preferred embodiment, the hypermutation positions are
selected from the group consisting of H30, H31, H31B, H32, H52,
H56, H58, L30, L31, L32, L53 and L93. In a more preferred
embodiment the residues for selective mutagenesis are selected from
the preferred selective mutagenesis positions from the group
consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,
L32, L50, L91, L92, L93, L94. In a more preferred embodiment, the
contact positions are selected from the group consisting of L50 and
L94.
[0117] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0118] a) providing a recombinant parent antibody or
antigen-binding portion thereof;
[0119] b) selecting a preferred selective mutagenesis position,
contact or hypermutation position within a complementarity
determining region (CDR) for mutation, thereby identifying a
selected contact or hypermutation position;
[0120] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expressing said
panel in an appropriate expression system;
[0121] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0122] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof for at least one other property
or characteristics, wherein the property or characteristic is one
that needs to be retained in the antibody;
until an antibody, or antigen-binding portion thereof, with an
improved activity and at least one retained property or
characteristic, relative to the parent antibody, or antigen-binding
portion thereof, is obtained.
[0123] In a preferred embodiment, the contact positions are
selected from the group consisting of H30, H31, H31B, H32, H33,
H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101,
L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96
and the other characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence. In
another preferred embodiment, the hypermutation positions are
selected from the group consisting of H30, H31, H31B, H32, H52,
H56, H58, L30, L31, L32, L53 and L93 and the other characteristic
is selected from 1) preservation of non-crossreactivity with other
proteins or human tissues, 2) preservation of epitope recognition,
i.e. recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence. In a more preferred embodiment
the residues for selective mutagenesis are selected from the
preferred selective mutagenesis positions from the group consisting
of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50,
L91, L92, L93, L94 and the other characteristic is selected from 1)
preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing
p40 epitope preferably in the context of the p70 p40/p35
heterodimer preventing binding interference from free, soluble p40
and/or 3) to produce an antibody with a close to germline
immunoglobulin sequence. In a more preferred embodiment, the
contact positions are selected from the group consisting of L50 and
L94 and the other characteristic is selected from 1) preservation
of non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0124] In another embodiment of the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0125] a) providing a recombinant parent antibody or
antigen-binding portion thereof; that was obtained by selection in
a phage-display system but whose activity cannot be further
improved by mutagenesis in said phage-display system;
[0126] b) selecting a preferred selective mutagenesis position,
contact or hypermutation position within a complementarity
determining region (CDR) for mutation, thereby identifying a
selected preferred selective mutagenesis position, contact or
hypermutation position;
[0127] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof, and expressing
said panel in a non-phage display system;
[0128] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0129] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof for at least one other property
or characteristic, wherein the property or characteristic is one
that needs to be retained, until an antibody, or antigen-binding
portion thereof, with an improved activity and at least one
retained property or characteristic, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.
[0130] f) repeating steps a) through e) for at least one other
preferred selective mutagenesis position, contact or hypermutation
position;
[0131] g) combining, in the parent antibody, or antigen-binding
portion thereof, at least two individual activity enhancing amino
acid residues shown to have improved activity and at least on
retained property or characteristic, to form combination
antibodies, or antigen-binding portions thereof; and
[0132] h) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof, until an antibody, or
antigen-binding portion thereof, with an improved activity and at
least one retained property or characteristic, relative to the
parent antibody, or antigen-binding portion thereof, is
obtained.
[0133] In a preferred embodiment, the contact positions are
selected from the group consisting of H30, H31, H31B, H32, H33,
H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101,
L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96
and the other characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence. In
another preferred embodiment, the hypermutation positions are
selected from the group consisting of H30, H31, H31B, H32, H52,
H56, H58, L30, L31, L32, L53 and L93 and the other characteristic
is selected from 1) preservation of non-crossreactivity with other
proteins or human tissues, 2) preservation of epitope recognition,
i.e. recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence. In a more preferred embodiment
the residues for selective mutagenesis are selected from the
preferred selective mutagenesis positions from the group consisting
of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50,
L91, L92, L93, L94 and the other characteristic is selected from 1)
preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing
p40 epitope preferably in the context of the p70 p40/p35
heterodimer preventing binding interference from free, soluble p40
and/or 3) to produce an antibody with a close to germline
immunoglobulin sequence. In a more preferred embodiment, the
contact positions are selected from the group consisting of L50 and
L94 and the other characteristic is selected from 1) preservation
of non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0134] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0135] a) providing a recombinant parent antibody or
antigen-binding portion thereof; that was obtained by selection in
a phage-display system but whose activity cannot be further
improved by mutagenesis in said phage-display system;
[0136] b) selecting a contact or hypermutation position within a
complementarity determining region (CDR) for mutation, thereby
identifying a selected contact or hypermutation position;
[0137] c) individually mutating said selected contact or
hypermutation position to at least two other amino acid residues to
thereby create a panel of mutated antibodies, or antigen-binding
portions thereof, and expressing said panel in a non-phage display
system;
[0138] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0139] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof for at least one other property
or characteristics, wherein the property or characteristic is one
that needs to be retained;
until an antibody, or antigen-binding portion thereof, with an
improved activity and at least one retained property or
characteristic, relative to the parent antibody, or antigen-binding
portion thereof, is obtained.
[0140] In a preferred embodiment, the contact positions are
selected from the group consisting of H30, H31, H31B, H32, H33,
H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101,
L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96
and the other characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence. In
another preferred embodiment, the hypermutation positions are
selected from the group consisting of H30, H31, H31B, H32, H52,
H56, H58, L30, L31, L32, L53 and L93 and the other characteristic
is selected from 1) preservation of non-crossreactivity with other
proteins or human tissues, 2) preservation of epitope recognition,
i.e. recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence. In a more preferred embodiment
the residues for selective mutagenesis are selected from the
preferred selective mutagenesis positions from the group consisting
of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50,
L91, L92, L93, L94 and the other characteristic is selected from 1)
preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing
p40 epitope preferably in the context of the p70 p40/p35
heterodimer preventing binding interference from free, soluble p40
and/or 3) to produce an antibody with a close to germline
immunoglobulin sequence. In a more preferred embodiment, the
contact positions are selected from the group consisting of L50 and
L94 and the other characteristic is selected from 1) preservation
of non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0141] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0142] a) providing a recombinant parent antibody or
antigen-binding portion thereof; that was obtained by selection in
a phage-display system but whose activity cannot be further
improved by mutagenesis in said phage-display system;
[0143] b) selecting a preferred selective mutagenesis position,
contact or hypermutation position within a complementarity
determining region (CDR) for mutation, thereby identifying a
selected preferred selective mutagenesis position contact or
hypermutation position;
[0144] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof, and expressing
said panel in a non-phage display system;
[0145] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0146] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof for at least one other property
or characteristic, wherein the property or characteristic is one
that needs to be retained, until an antibody, or antigen-binding
portion thereof, with an improved activity and at least one
retained property or characteristic, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.
[0147] f) repeating steps a) through e) for at least one other
preferred selective mutagenesis position, contact or hypermutation
position;
[0148] g) combining, in the parent antibody, or antigen-binding
portion thereof, at least two individual activity enhancing amino
acid residues shown to have improved activity and at least on
retained other characteristic, to form combination antibodies, or
antigen-binding portions thereof; and
[0149] h) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof; until an antibody, or
antigen-binding portion thereof, with an improved activity and at
least one retained property or characteristic, relative to the
parent antibody, or antigen-binding portion thereof, is
obtained.
[0150] In a preferred embodiment, the contact positions are
selected from the group consisting of H30, H31, H31B, H32, H33,
H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101,
L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96
and the other characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence. In
another preferred embodiment, the hypermutation positions are
selected from the group consisting of H30, H31, H31B, H32, H52,
H56, H58, L30, L31, L32, L53 and L93 and the other characteristic
is selected from 1) preservation of non-crossreactivity with other
proteins or human tissues, 2) preservation of epitope recognition,
i.e. recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence. In a more preferred embodiment
the residues for selective mutagenesis are selected from the
preferred selective mutagenesis positions from the group consisting
of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50,
L91, L92, L93, L94 and the other characteristic is selected from 1)
preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing
p40 epitope preferably in the context of the p70 p40/p35
heterodimer preventing binding interference from free, soluble p40
and/or 3) to produce an antibody with a close to germline
immunoglobulin sequence. In a more preferred embodiment, the
contact positions are selected from the group consisting of L50 and
L94 and the other characteristic is selected from 1) preservation
of non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0151] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0152] a) providing a parent antibody or antigen-binding portion
thereof,
[0153] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and L96;
[0154] c) individually mutating said selected position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof,
[0155] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0156] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof, for changes in at least one
other property or characteristic;
until an antibody, or antigen-binding portion thereof, with an
improved activity, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.
[0157] Preferably, the other characteristic or property is selected
from 1) preservation of non-crossreactivity with other proteins or
human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence
[0158] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0159] a) providing a parent antibody or antigen-binding portion
thereof;
[0160] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and L96;
[0161] c) individually mutating said selected position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;
[0162] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof, thereby
identifying an activity enhancing amino acid residue;
[0163] e) repeating steps b) through d) for at least one other CDR
position which is neither the position selected under b) nor a
position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,
H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50,
L52, L53, L55, L91, L92, L93, L94 and L96;
[0164] f) combining, in the parent antibody, or antigen-binding
portion thereof, at least two individual activity enhancing amino
acid residues shown to have improved activity, to form combination
antibodies, or antigen-binding portions thereof; and
[0165] g) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino
acid residues, relative to the parent antibody or antigen-binding
portion thereof until an antibody, or antigen-binding portion
thereof, with an improved activity, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.
[0166] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0167] a) providing a recombinant parent antibody or
antigen-binding portion thereof; that was obtained by selection in
a phage-display system but whose activity cannot be further
improved by mutagenesis in said phage-display system;
[0168] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and;
[0169] c) individually mutating said selected contact or
hypermutation position to at least two other amino acid residues to
thereby create a panel of mutated antibodies, or antigen-binding
portions thereof, and expressing said panel in a non-phage display
system;
[0170] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0171] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof, for changes in at least one
other property or characteristic until an antibody, or
antigen-binding portion thereof, with an improved activity,
relative to the parent antibody, or antigen-binding portion
thereof, is obtained.
[0172] Preferably, the other characteristic or property is selected
from 1) preservation of non-crossreactivity with other proteins or
human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence.
[0173] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0174] a) providing a parent antibody or antigen-binding portion
thereof that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in
said phage-display system;
[0175] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and L96;
[0176] c) individually mutating said selected position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expression in a
non-phage display system;
[0177] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0178] e) repeating steps b) through d) for at least one other
position within the CDR which is neither the position selected
under b) nor a position at H30, H31, H31B, H32, H33, H35, H50, H52,
H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32,
L34, L50, L52, L53, L55, L91, L92, L93, L94;
[0179] f) combining, in the parent antibody, or antigen-binding
portion thereof, at least two individual activity enhancing amino
acid residues shown to have improved activity, to form combination
antibodies, or antigen-binding portions thereof; and
[0180] g) evaluating the activity and other property or
characteristic of the combination antibodies, or antigen-binding
portions thereof with two activity enhancing amino acid residues,
relative to the parent antibody or antigen-binding portion thereof;
until an antibody, or antigen-binding portion thereof, with an
improved activity, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.
[0181] Preferably, the other characteristic or property is selected
from 1) preservation of non-crossreactivity with other proteins or
human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence.
[0182] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0183] a) providing a parent antibody or antigen-binding portion
thereof;
[0184] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and L96;
[0185] c) individually mutating said selected position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;
[0186] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof, thereby
identifying an activity enhancing amino acid residue;
[0187] e) evaluating the panel of mutated antibodies or
antigen-binding portions thereof, relative to the parent antibody
or antigen-portion thereof, for changes in at least one other
property or characteristic;
[0188] f) repeating steps b) through e) for at least one other CDR
position which is neither the position selected under b) nor a
position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,
H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50,
L52, L53, L55, L91, L92, L93, L94 and L96;
[0189] g) combining, in the parent antibody, or antigen-binding
portion thereof, at least two individual activity enhancing amino
acid residues shown to have improved activity and not affecting at
least one other property or characteristic, to form combination
antibodies, or antigen-binding portions thereof; and
[0190] h) evaluating the activity and the retention of at least one
other characteristic or property of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino
acid residues, relative to the parent antibody or antigen-binding
portion thereof until an antibody, or antigen-binding portion
thereof, with an improved activity and at least one retained
property or characteristic, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.
[0191] In another embodiment the invention provides a method to
improve the affinity of an antibody or antigen-binding portion
thereof, comprising:
[0192] a) providing a parent antibody or antigen-binding portion
thereof that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in
said phage-display system;
[0193] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and L96;
[0194] c) individually mutating said selected position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expression in a
non-phage display system;
[0195] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0196] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof, for changes in at least one
other characteristic or property until an antibody, or
antigen-binding portion thereof, with an improved activity,
relative to the parent antibody, or antigen-binding portion
thereof, is obtained.
[0197] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0198] a) providing a parent antibody or antigen-binding portion
thereof;
[0199] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation at a position other than H30,
H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95,
H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91,
L92, L93, L94 and L96;
[0200] c) individually mutating said selected position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;
[0201] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof, thereby
identifying an activity enhancing amino acid residue;
[0202] e) evaluating the panel of mutated antibodies or
antigen-binding portions thereof, relative to the parent antibody
or antigen-portion thereof, for changes in at least one other
property or characteristic;
[0203] f) repeating steps b) through e) for at least one other CDR
position which is neither the position selected under b) nor a
position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,
H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50,
L52, L53, L55, L91, L92, L93, L94 and L96;
[0204] g) combining, in the parent antibody, or antigen-binding
portion thereof, at least two individual activity enhancing amino
acid residues shown to have improved activity but not affecting at
least one other property or characteristic, to form combination
antibodies, or antigen-binding portions thereof with at least one
retained property or characteristic; and
[0205] h) evaluating the activity and the retention of at least one
property of characteristic of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino
acid residues, relative to the parent antibody or antigen-binding
portion thereof until an antibody, or antigen-binding portion
thereof, with an improved activity and at least one retained
property or characteristic, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.
[0206] Preferably, the other characteristic or property is selected
from 1) preservation of non-crossreactivity with other proteins or
human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence
[0207] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, without affecting other characteristics, comprising:
[0208] a) providing a parent antibody or antigen-binding portion
thereof;
[0209] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and L96;
[0210] c) individually mutating said selected position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;
[0211] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0212] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof, for changes in at least one
other property or characteristic until an antibody, or
antigen-binding portion thereof, with an improved activity and
retained other characteristic or property, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.
[0213] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0214] a) providing a parent antibody or antigen-binding portion
thereof that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in
said phage-display system;
[0215] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and L96;
[0216] c) individually mutating said selected position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expression in a
non-phage display system;
[0217] d) evaluating the activity and retention of at least one
other characteristic or property of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof, thereby
identifying an activity enhancing amino acid residue;
[0218] e) repeating steps b) through d) for at least one other CDR
position which is neither the position selected under b nor other
than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56,
H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53,
L55, L91, L92, L93, L94 and L96;
[0219] f) combining, in the parent antibody, or antigen-binding
portion thereof, at least two individual activity enhancing amino
acid residues shown to have improved activity and not to affect at
least one other characteristic or property, to form combination
antibodies, or antigen-binding portions thereof; and
[0220] g) evaluating the activity and retention of at least one
other characteristic or property of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino
acid residues, relative to the parent antibody or antigen-binding
portion thereof until an antibody, or antigen-binding portion
thereof, with an improved activity and at least one other retained
characteristic or property, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.
[0221] Preferably, the other characteristic or property is selected
from 1) preservation of non-crossreactivity with other proteins or
human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence.
[0222] In yet another aspect, the invention provides an isolated
antibody (e.g., a human antibody) or antigen binding portion
thereof that is capable of binding to the p40 subunit of IL-12
(e.g., the p40 subunit of human IL-12) and is capable of altering
the conformational structure of said p40 subunit of IL-12.
[0223] In a related aspect, the invention provides an isolated
antibody (e.g., a human antibody) or antigen binding portion
thereof that is capable of binding to the p40 subunit of IL-12
(e.g., the p40 subunit of human IL-12) and is capable of altering
the conformational structure of an interleukin, e.g., the p40
subunit of an interleukin. In one embodiment, the interleukin is
IL-12. In another embodiment, the interleukin comprises a p40
subunit and a p19 subunit, e.g., the interleukin is IL-23. In yet
another embodiment, the interleukin is a human interleukin such as
human IL-12 or human IL-23.
[0224] In one embodiment, the antibody is Y61 or J695. In another
embodiment, the antibody is not Y61 or J695.
[0225] In another embodiment, the isolated antibody, or antigen
binding portion thereof, alters the conformational structure of the
interleukin, e.g., IL-12 or IL-23, such that binding by the
interleukin, e.g., IL-12 or IL-23, to an interleukin-interacting
molecule is modulated.
[0226] In yet another embodiment, the interleukin-interacting
molecule is an interleukin receptor, e.g., IL-12 or IL-23
receptor.
[0227] In a further embodiment, the isolated antibody, or antigen
binding portion thereof, alters the conformational structure of an
interleukin, e.g., IL-12 or IL-23, such that binding to the
interleukin, e.g., IL-12 or IL-23, by a second antibody is
inhibited.
[0228] In one embodiment, the interleukin is IL-12 and the second
antibody binds to an epitope of the p40 subunit of IL-12 to which
an antibody selected from the group consisting of 1D4.7, C8.6.2 and
C340 binds.
[0229] In another embodiment, the interleukin is IL-12 and the
second antibody is selected from the group consisting of 1D4.7,
C8.6.2 and C340.
[0230] In another aspect, the invention provides an isolated
antibody, e.g., a human antibody, or antigen binding portion
thereof, that binds to an epitope of the p40 subunit of IL-12 to
which an antibody selected from the group consisting of 1D4.7,
C8.6.2, C340 and 7G3 does not bind.
[0231] In one embodiment, the antibody is Y61 or J695. In another
embodiment, the antibody is not Y61 or J695.
[0232] In another embodiment, the isolated antibody, or antigen
binding portion thereof, binds to an epitope of the p40 subunit of
IL-12 to which an antibody selected from the group consisting of
Y61 and J695 binds.
[0233] In one embodiment, the antibody is not Y61 or J695.
[0234] In a further embodiment, the isolated antibody, or antigen
binding portion thereof, inhibits the binding to IL-12 by a second
antibody.
[0235] In yet a further embodiment, the second antibody binds to an
epitope of IL-12 to which an antibody selected from the group
consisting of 1D4.7, C8.6.2, C340 binds.
[0236] In one embodiment, the second antibody is selected from the
group consisting of 1D4.7, C8.6.2 and C340.
[0237] In one embodiment, the isolated antibody, or antigen binding
portion thereof, dissociates from the p40 subunit of human IL-12
with a K.sub.d of 1.times.10.sup.-10 M or less or a k.sub.off rate
constant of 1.times.10.sup.-3 s.sup.-1 or less, as determined by
surface plasmon resonance.
[0238] In another embodiment, the isolated antibody, or antigen
binding portion thereof, is a neutralizing antibody
[0239] In a further embodiment, the isolated antibody, or antigen
binding portion thereof, inhibits phytohemagglutinin blast
proliferation in an in vitro PHA assay with an IC.sub.50 of
1.times.10.sup.-9 M or less, or which inhibits human IFN.gamma.
production with an IC.sub.50 of 1.times.10.sup.-10 M or less.
[0240] In one embodiment, the isolated antibody, or antigen binding
portion thereof, is a human antibody.
[0241] In another aspect, the invention provides a pharmaceutical
composition comprising an antibody of the invention, or an antigen
binding portion thereof, and a pharmaceutically acceptable carrier.
The pharmaceutical composition may further comprise an additional
therapeutic agent, e.g., a therapeutic agent selected from the
group consisting of budenoside, epidermal growth factor,
corticosteroids, cyclosporin, sulfasalazine, aminosalicylates,
6-mercaptopurine, azathioprine, metronidazole, lipoxygenase
inhibitors, mesalamine, olsalazine, balsalazide, antioxidants,
thromboxane inhibitors, IL-1 receptor antagonists, anti-IL-1.beta.
monoclonal antibodies, anti-IL-6 monoclonal antibodies, growth
factors, elastase inhibitors, pyridinyl-imidazole compounds,
antibodies or agonists of TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8,
IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF, antibodies of
CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or
their ligands, methotrexate, cyclosporin, FK506, rapamycin,
mycophenolate mofetil, leflunomide, NSAIDs, ibuprofen,
corticosteroids, prednisolone, phosphodiesterase inhibitors,
adenosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, IRAK, NIK, IKK, p38, MAP kinase inhibitors,
IL-1.beta. converting enzyme inhibitors, TNF.alpha. converting
enzyme inhibitors, T-cell signalling inhibitors, metalloproteinase
inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines,
angiotensin converting enzyme inhibitors, soluble cytokine
receptors, soluble p55 TNF receptor, soluble p75 TNF receptor,
sIL-1RI, sIL-1RII, sIL-6R, antiinflammatory cytokines, IL-4, IL-10,
IL-11, IL-13 and TGF.beta..
[0242] In another embodiment, the therapeutic agent is selected
from the group consisting of anti-TNF antibodies and antibody
fragments thereof, TNFR-Ig constructs, TACE inhibitors, PDE4
inhibitors, corticosteroids, budenoside, dexamethasone,
sulfasalazine, 5-aminosalicylic acid, olsalazine, IL-1.beta.
converting enzyme inhibitors, IL-1ra, tyrosine kinase inhibitors,
6-mercaptopurines and IL-11.
[0243] In yet another embodiment, the therapeutic agent is selected
from the group consisting of corticosteroids, prednisolone,
methylprednisolone, azathioprine, cyclophosphamide, cyclosporine,
methotrexate, 4-aminopyridine, tizanidine, interferon-.beta.1a,
interferon-.beta.1b, Copolymer 1, hyperbaric oxygen, intravenous
immunoglobulin, clabribine, antibodies or agonists of TNF, LT,
IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF,
FGF, PDGF, antibodies to CD2, CD3, CD4, CD8, CD25, CD28, CD30,
CD40, CD45, CD69, CD80, CD86, CD90 or their ligands, methotrexate,
cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide,
NSAIDs, ibuprofen, corticosteroids, prednisolone, phosphodiesterase
inhibitors, adensosine agonists, antithrombotic agents, complement
inhibitors, adrenergic agents, IRAK, NIK, IKK, p38 or MAP kinase
inhibitors, IL-1.beta. converting enzyme inhibitors, TACE
inhibitors, T-cell signalling inhibitors, kinase inhibitors,
metalloproteinase inhibitors, sulfasalazine, azathioprine,
6-mercaptopurines, angiotensin converting enzyme inhibitors,
soluble cytokine receptors, soluble p55 TNF receptor, soluble p75
TNF receptor, sIL-1RI, sIL-1RII, sIL-6R, sIL-13R, anti-P7s,
p-selectin glycoprotein ligand (PSGL), antiinflammatory cytokines,
IL-4, IL-10, IL-13 and TGF.beta..
[0244] In another aspect, the invention provides a composition
comprising an antibody (e.g., a human antibody) or antigen binding
portion thereof, that is capable of binding to the p40 subunit of
IL-12 and is capable of altering the conformational structure of an
interleukin comprising a p40 subunit, e.g., IL-12 or IL-23, in an
amount effective for altering the conformational structure of the
interleukin. In one embodiment, the interleukin is IL-12. In
another embodiment, the interleukin comprises a p40 subunit and a
p19 subunit, e.g., the interleukin is IL-23.
[0245] In one embodiment, the amount of the antibody effective to
alter the conformational structure of the p40 subunit of an
interleukin, e.g., IL-12, is between about 0.1 and 2500 .mu.g/ml,
preferably between about 1.0 and 250 .mu.g/ml, more preferably
between about 2.0 and 125 .mu.g/ml, more preferably between about
4.0 and 65 .mu.g/ml, even more preferably between about 6.0 and 50
.mu.g/ml, preferably between about 8.0 and 40 .mu.g/ml, and most
preferably between about 10 and 25 .mu.g/ml. In various
embodiments, the amount of the antibody effective to alter the
conformational structure of the p40 subunit of an interleukin,
e.g., IL-12, is about 1, 2, 3, 4, 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, 45, 50, 55, 60 .mu.g/ml or
more. Ranges intermediate to the above recited amounts, e.g.,
between about 5.0 and 55 .mu.g/ml, between about 9.0 and 35
.mu.g/ml, and between about 15 and 20 .mu.g/ml, are also intended
to be part of this invention. For example, ranges of values using a
combination of any of the above recited values as upper and/or
lower limits are intended to be included. Further, discrete amounts
intermediate to any of the above recited amounts, e.g., 10.5, 11.5,
and 12.5 .mu.g/ml, are also intended to be part of this
invention.
[0246] In another aspect, the invention provides a composition
comprising an antibody (e.g., a human antibody) or antigen binding
portion thereof, that binds to the p40 subunit of IL-12 and is
capable of altering the conformational structure of said p40
subunit packaged or promoted with instructions for use in altering
the conformational structure of said p40 subunit.
[0247] In a related aspect, the invention provides a composition
comprising an antibody (e.g., a human antibody) or antigen binding
portion thereof, that binds to the p40 subunit of IL-12 and is
capable of altering the conformational structure of an interleukin
comprising a p40 subunit, e.g., IL-12 or IL-23, packaged or
promoted with instructions for use in altering the conformational
structure of the interleukin, e.g., IL-12 or IL-23.
[0248] In one embodiment, the composition further comprises a means
for determining whether the conformational structure of the
interleukin, e.g., IL-12 or IL-23, has been altered.
[0249] In one embodiment, the interleukin is IL-12 and the
antibody, or antigen binding portion thereof, binds to an epitope
of the p40 subunit of IL-12 to which an antibody selected from the
group consisting of 1D4.7, C8.6.2, C340 and 7G3 does not bind.
[0250] In another embodiment, the antibody, or antigen binding
portion thereof, binds an epitope to which an antibody selected
from the group consisting of Y61 and J695 binds.
[0251] In yet another embodiment, the antibody, or antigen binding
portion thereof, is not Y61 or J695.
[0252] In a further embodiment, the antibody, or antigen binding
portion thereof, dissociates from the p40 subunit of human IL-12
with a K.sub.d of 1.times.10-1M or less or a k.sub.off rate
constant of 1.times.10.sup.-3 s.sup.-1 or less, as determined by
surface plasmon resonance.
[0253] In one embodiment, the antibody, or antigen binding portion
thereof, is a neutralizing antibody.
[0254] In one embodiment, the antibody, or antigen binding portion
thereof, is a human antibody.
[0255] In yet another aspect, the invention provides a composition
comprising a plurality of antibodies, or antigen binding portions
thereof, wherein each antibody of the plurality of antibodies binds
to a different epitope of the p40 subunit of IL-12.
[0256] In one embodiment, at least one antibody, or antigen binding
portion thereof, binds to an epitope of the p40 subunit of IL-12 to
which an antibody selected from the group consisting of Y61 and
J695 binds.
[0257] In another embodiment, at least one antibody, or antigen
binding portion thereof, binds to an epitope of the p40 subunit of
IL-12 to which an antibody selected from the group consisting of
1D4.7, C8.6.2, C340 and 7G3 does not bind.
[0258] In one embodiment, at least one antibody, or antigen binding
portion thereof, dissociates from the p40 subunit of human IL-12
with a K.sub.d of 1.times.10.sup.-10 M or less or a k.sub.off rate
constant of 1.times.10.sup.-3 s.sup.-1 or less, as determined by
surface plasmon resonance.
[0259] In another embodiment, at least one antibody, or antigen
binding portion thereof, is a neutralizing antibody.
[0260] In yet another embodiment, at least one antibody, or antigen
binding portion thereof, inhibits phytohemagglutinin blast
proliferation in an in vitro PHA assay with an IC.sub.50 of
1.times.10.sup.-9 M or less, or which inhibits human IFN.gamma.
production with an IC.sub.50 of 1.times.10.sup.-10 M or less.
[0261] In a further embodiment, at least one antibody, or antigen
binding portion thereof, is a human antibody.
[0262] In one embodiment, at least one antibody, or antigen binding
portion thereof, has a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 25 and a light chain CDR3 comprising the
amino acid sequence of SEQ ID NO: 26.
[0263] In another embodiment, the at least one antibody, or antigen
binding portion thereof, further has a heavy chain CDR2 comprising
the amino acid sequence of SEQ ID NO: 27 and a light chain CDR2
comprising the amino acid sequence of SEQ ID NO: 28.
[0264] In yet another embodiment, the at least one antibody, or
antigen binding portion thereof, further has a heavy chain CDR1
comprising the amino acid sequence of SEQ ID NO: 29 and a light
chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30.
[0265] In one aspect, the invention provides a method for detecting
the p40 subunit of an interleukin, e.g., IL-12 or IL-23, comprising
contacting the p40 subunit of IL-12 with an antibody of the
invention, or antigen-binding portion thereof, such that the p40
subunit is detected.
[0266] In a related aspect, the invention provides a method for
detecting the p40 subunit of an interleukin, e.g., IL-12 or IL-23,
comprising contacting the p40 subunit of the interleukin, e.g.,
IL-12 or IL-23, with an antibody of the invention, or
antigen-binding portion thereof, such that the p40 subunit of the
interleukin, e.g., IL-12 or IL-23, is detected.
[0267] In one embodiment, the p40 subunit of the interleukin, e.g.,
IL-12 or IL-23, is detected in vitro. In another embodiment, the
p40 subunit of the interleukin, e.g., IL-12 or IL-23, is detected
in a biological sample for diagnostic purposes.
[0268] In another aspect, the invention provides a method for
inhibiting an activity of an interleukin comprising a p40 subunit,
e.g., IL-12 or IL-23, comprising contacting the interleukin, e.g.,
IL-12 or IL-23, with an antibody, or antigen-binding portion
thereof, of the invention such that the activity is inhibited.
[0269] In a related aspect, the invention provides a method for
inhibiting an activity of an interleukin comprising a p40 subunit,
e.g., IL-12 or IL-23, in a human subject suffering from a disorder
in which the activity is detrimental, comprising administering to
the human subject an antibody, or antigen-binding portion thereof,
of the invention such that the activity of the interleukin, e.g.,
IL-12 or IL-23, in the human subject is inhibited.
[0270] In one embodiment, the interleukin is IL-12. In another
embodiment, the interleukin comprises a p40 subunit and a p19
subunit, e.g., the interleukin is IL-23.
[0271] In one embodiment, the disorder is selected from the group
consisting of rheumatoid arthritis, osteoarthritis, juvenile
chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive
arthritis, spondyoarthropathy, ankylosing spondylitis, systemic
lupus erythematosis, Crohn's disease, ulcerative colitis,
inflammatory bowel disease, multiple sclerosis, insulin dependent
diabetes mellitus, thyroiditis, asthma, allergic diseases,
psoriasis, dermatitisscleroderma, thyroiditis, graft versus host
disease, organ transplant rejection, acute or chronic immune
disease associated with organ transplantation, sarcoidosis,
atherosclerosis, disseminated intravascular coagulation, Kawasaki's
disease, Grave's disease, nephrotic syndrome, chronic fatigue
syndrome, polyarteritis nodosa, Wegener's granulomatosis,
Henoch-Schonlein purpura, microscopic vasculitis of the kidneys,
chronic active hepatitis, Sjogren's syndrome, uveitis, sepsis,
septic shock, sepsis syndrome, adult respiratory distress syndrome,
cachexia, infectious diseases, parasitic diseases, acquired
immunodeficiency syndrome, acute transverse myelitis, myasthenia
gravis, Huntington's chorea, Parkinson's disease, Alzheimer's
disease, stroke, primary biliary cirrhosis, fibrotic lung diseases,
hemolytic anemia, malignancies, heart failure and myocardial
infarction.
[0272] In one embodiment, the disorder is rheumatoid arthritis.
[0273] In one embodiment, the disorder is Crohn's disease.
[0274] In one embodiment, the disorder is multiple sclerosis.
[0275] In one embodiment, the disorder is psoriasis.
[0276] In yet another aspect, the invention provides a method for
altering the conformational structure of the p40 subunit of IL-12,
the method comprising contacting said subunit with an antibody, or
antigen binding portion thereof, that is capable of binding said
subunit and is capable of altering the conformational structure of
said subunit, in an amount effective to alter the conformational
structure of said subunit, thereby altering the conformational
structure of said subunit.
[0277] In a related aspect, the invention provides a method for
altering the conformational structure of an interleukin comprising
a p40 subunit, e.g., IL-12 or IL-23, the method comprising
contacting the interleukin, e.g., IL-12 or IL-23, with an antibody,
or antigen binding portion thereof, that is capable of binding to
the p40 subunit of IL-12 and is capable of altering the
conformational structure of the interleukin, e.g., IL-12 or IL-23,
in an amount effective to alter the conformational structure of the
interleukin, e.g., IL-12 or IL-23, thereby altering the
conformational structure of the interleukin, e.g., IL-12 or
IL-23.
[0278] In another aspect, the invention provides a method for
inhibiting the activity of an interleukin comprising a p40 subunit,
e.g., IL-12 or IL-23, the method comprising contacting the
interleukin, e.g., IL-12 or IL-23, with an antibody, or antigen
binding portion thereof, that is capable of binding to the p40
subunit of IL-12 and is capable of altering the conformational
structure of the said subunit, in an amount effective to alter the
conformational structure of said subunit, thereby inhibiting the
activity of the interleukin, e.g., IL-12 or IL-23.
[0279] In a related aspect, the invention provides a method for
inhibiting the activity of an interleukin comprising a p40 subunit,
e.g., IL-12 or IL-23, the method comprising contacting the
interleukin, e.g., IL-12 or IL-23, with an antibody, or antigen
binding portion thereof, that is capable of binding to the p40
subunit of IL-12 and is capable of altering the conformational
structure of the interleukin, e.g., IL-12 or IL-23, in an amount
effective to alter the conformational structure of the interleukin,
e.g., IL-12 or IL-23, thereby inhibiting the activity of the
interleukin, e.g., IL-12 or IL-23.
[0280] In another aspect, the invention provides a method for
inhibiting the activity of an interleukin comprising a p40 subunit,
e.g., IL-12 or IL-23, in a human subject suffering from a disorder
in which the activity of the interleukin, e.g., IL-12 or IL-23, is
detrimental, the method comprising administering to the human
subject an antibody, or antigen binding portion thereof, that is
capable of binding to the p40 subunit of IL-12 and is capable of
altering the conformational structure of said subunit, in an amount
effective to alter the conformational structure of said subunit,
thereby inhibiting the activity of the interleukin, e.g., IL-12 or
IL-23, in the human subject.
[0281] In a related aspect, the invention provides a method for
inhibiting the activity of an interleukin comprising a p40 subunit,
e.g., IL-12 or IL-23, in a human subject suffering from a disorder
in which the activity of the interleukin, e.g., IL-12 or IL-23, is
detrimental, the method comprising administering to the human
subject an antibody, or antigen binding portion thereof, that is
capable of binding to the p40 subunit of IL-12 and is capable of
altering the conformational structure of the interleukin, e.g.,
IL-12 or IL-23, in an amount effective to alter the conformational
structure of the interleukin, e.g., IL-12 or IL-23, thereby
inhibiting the activity of the interleukin, e.g., IL-12 or IL-23,
in the human subject.
[0282] In one embodiment, the interleukin is IL-12. In another
embodiment, the interleukin comprises a p40 subunit and a p19
subunit, e.g., the interleukin is IL-23.
[0283] In one embodiment, the disorder is selected from the group
consisting of rheumatoid arthritis, osteoarthritis, juvenile
chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive
arthritis, spondyoarthropathy, ankylosing spondylitis, systemic
lupus erythematosis, Crohn's disease, ulcerative colitis,
inflammatory bowel disease, multiple sclerosis, insulin dependent
diabetes mellitus, thyroiditis, asthma, allergic diseases,
psoriasis, dermatitisscleroderma, thyroiditis, graft versus host
disease, organ transplant rejection, acute or chronic immune
disease associated with organ transplantation, sarcoidosis,
atherosclerosis, disseminated intravascular coagulation, Kawasaki's
disease, Grave's disease, nephrotic syndrome, chronic fatigue
syndrome, polyarteritis nodosa, Wegener's granulomatosis,
Henoch-Schonlein purpura, microscopic vasculitis of the kidneys,
chronic active hepatitis, Sjogren's syndrome, uveitis, sepsis,
septic shock, sepsis syndrome, adult respiratory distress syndrome,
cachexia, infectious diseases, parasitic diseases, acquired
immunodeficiency syndrome, acute transverse myelitis, myasthenia
gravis, Huntington's chorea, Parkinson's disease, Alzheimer's
disease, stroke, primary biliary cirrhosis, fibrotic lung diseases,
hemolytic anemia, malignancies, heart failure and myocardial
infarction.
[0284] In one embodiment, the disorder is rheumatoid arthritis.
[0285] In one embodiment, the disorder is Crohn's disease.
[0286] In one embodiment, the disorder is multiple sclerosis.
[0287] In one embodiment, the disorder is psoriasis.
[0288] In one embodiment, the antibody is not Y61 or J695.
[0289] In another embodiment, the amount of the antibody effective
to alter the conformational structure of the p40 subunit of an
interleukin, e.g., IL-12, is between about 0.1 and 2500 .mu.g/ml,
preferably between about 1.0 and 250 .mu.g/ml, more preferably
between about 2.0 and 125 .mu.g/ml, more preferably between about
4.0 and 65 .mu.g/ml, even more preferably between about 6.0 and 50
.mu.g/ml, preferably between about 8.0 and 40 .mu.g/ml, and most
preferably between about 10 and 25 .mu.g/ml. In various
embodiments, the amount of the antibody effective to alter the
conformational structure of the p40 subunit of an interleukin,
e.g., IL-12, is about 1, 2, 3, 4, 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, 45, 50, 55, 60 .mu.g/ml or
more. Ranges intermediate to the above recited amounts, e.g.,
between about 5.0 and 55 .mu.g/ml, between about 9.0 and 35
.mu.g/ml, and between about 15 and 20 .mu.g/ml, are also intended
to be part of this invention. For example, ranges of values using a
combination of any of the above recited values as upper and/or
lower limits are intended to be included. Further, discrete amounts
intermediate to any of the above recited amounts, e.g., 10.5, 11.5,
and 12.5 .mu.g/ml, are also intended to be part of this
invention.
[0290] In one embodiment, the p40 subunit of an interleukin, e.g.,
IL-12 is contacted with the antibody, or antigen binding portion
thereof, for a period of approximately 10 seconds, 30 seconds, 1
minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7
minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes,
13 minutes, 14 minutes, 15 minutes, 20 minutes, 25 minutes, 30
minutes, 1 hour, 2 hours, 5 hours or longer. In a preferred
embodiment, the p40 subunit of an interleukin is contacted with the
antibody, or antigen binding portion thereof, for a period of
between 5 and 10 minutes, e.g., 5, 6, 7, 8, 9 or 10 minutes. Times
intermediate to the above recited times, e.g., 2.5 minutes, 3.5
minutes, 4.5 minutes, 5.5 minutes, are also intended to be part of
this invention.
[0291] In another aspect, the invention provides a method for
identifying an antibody, or antigen binding portion thereof,
suitable for inhibiting the activity of an interleukin comprising a
p40 subunit, e.g., IL-12 or IL-23, the method comprising contacting
the p40 subunit of an interleukin, e.g., IL-12 or IL-23, or a
portion thereof, with an antibody, or antigen binding portion
thereof, and determining whether the antibody, or antigen binding
portion thereof, alters the conformational structure of said p40
subunit, thereby identifying an antibody, or antigen binding
portion thereof, suitable for inhibiting the activity of an
interleukin comprising a p40 subunit, e.g., IL-12 or IL-23.
[0292] In a related aspect, the invention provides a method for
identifying an antibody, or antigen binding portion thereof,
suitable for inhibiting the activity of an interleukin comprising a
p40 subunit, e.g., IL-12 or IL-23, the method comprising contacting
the p40 subunit of an interleukin, e.g., IL-12 or IL-23, or a
portion thereof, with an antibody, or antigen binding portion
thereof, and determining whether the antibody, or antigen binding
portion thereof, alters the conformational structure of the
interleukin, e.g., IL-12 or IL-23, thereby identifying an antibody,
or antigen binding portion thereof, suitable for inhibiting the
activity of an interleukin comprising a p40 subunit, e.g., IL-12 or
IL-23.
[0293] In one embodiment, the antibody, or antigen binding portion
thereof, is suitable for inhibiting the activity of the
interleukin, e.g., IL-12 or IL-23, in a human subject suffering
from a disorder in which the activity of the interleukin, e.g.,
IL-12 or IL-23; is detrimental.
[0294] In another embodiment, the method further comprises
determining whether binding by the interleukin, e.g., IL-12 or
IL-23, to an interleukin-interacting molecule is modulated.
[0295] In one embodiment, the molecule that interacts with the
interleukin, e.g., IL-12 or IL-23, is an interleukin receptor,
e.g., IL-12 receptor or IL-23 receptor.
[0296] In one embodiment, the method further comprises determining
whether binding to the interleukin, e.g., IL-12 or IL-23, by a
second antibody is inhibited.
[0297] In one embodiment, the interleukin is IL-12 and the second
antibody binds to an epitope of the p40 subunit of IL-12 to which
an antibody selected from the group consisting of 1D4.7, C8.6.2 and
C340 binds.
[0298] In one embodiment, the second antibody is selected from the
group consisting of 1D4.7, C8.6.2 and C340.
BRIEF DESCRIPTION OF THE DRAWINGS
[0299] FIGS. 1A-1B show the heavy chain variable region amino acid
sequence alignments of a series of human antibodies that bind human
IL-12 compared to germline sequences Cos-3/JH3 and Dpl18 Lv1042.
Kabat numbering is used to identify amino acid positions. For the
Joe 9 wild type, the full sequence is shown. For the other
antibodies, only those amino acids positions that differ from Joe 9
wild type are shown.
[0300] FIGS. 1C-1D show the light chain variable region amino acid
sequence alignments of a series of human antibodies that bind human
IL-12. Kabat numbering is used to identify amino acid positions.
For the Joe 9 wild type, the full sequence is shown. For the other
antibodies, only those amino acids positions that differ from Joe 9
wild type are shown.
[0301] FIGS. 2A-2E show the CDR positions in the heavy chain of the
Y61 antibody that were mutated by site-directed mutagenesis and the
respective amino acid substitutions at each position. The graphs at
the right of the figures show the off-rates for the substituted
antibodies (black bars) as compared to unmutated Y61 (open
bar).
[0302] FIGS. 2F-2H show the CDR positions in the light chain of the
Y61 antibody; that were mutated by site-directed mutagenesis and
the respective amino acid substitutions at each position. The
graphs at the right of the figures show the off-rates for the
substituted antibodies (black bars) as compared to unmutated Y61
(open bar).
[0303] FIG. 3 demonstrates the in vivo efficacy of the human
anti-IL-12 antibody J695, on plasma neopterin levels in cynomolgus
monkeys.
[0304] FIG. 4 shows a graph of mean arthritic score versus days
after immunization of mice with collagen, demonstrating that
treatment with C17.15 significantly decreases arthritis-related
symptoms as compared to treatment with rat IgG.
DETAILED DESCRIPTION OF THE INVENTION
[0305] In order that the present invention may be more readily
understood, certain terms are first defined.
[0306] The term "activity enhancing amino acid residue" includes an
amino acid residue which improves the activity of the antibody. It
should be understood that the activity enhancing amino acid residue
may replace an amino acid residue at a contact, hypermutation or
preferred selective mutagenesis position and, further, more than
one activity enhancing amino acid residue can be present within one
or more CDRs. An activity enhancing amino acid residue include, an
amino acid residue that improves the binding specificity/affinity
of an antibody, for example anti-human IL-12 antibody binding to
human IL-12. The activity enhancing amino acid residue is also
intended to include an amino acid residue that improves the
neutralization potency of an antibody, for example, the human IL-12
antibody which inhibits human IL-12.
[0307] The term "antibody" includes an immunoglobulin molecule
comprised of four polypeptide chains, two heavy (H) chains and two
light (L) chains inter-connected by disulfide bonds. Each heavy
chain is comprised of a heavy chain variable region (abbreviated
herein as HCVR or VH) and a heavy chain constant region. The heavy
chain constant region is comprised of three domains, CH1, CH2 and
CH3. Each light chain is comprised of a light chain variable region
(abbreviated herein as LCVR or VL) and a light chain constant
region. The light chain constant region is comprised of one domain,
CL. The VH and VL regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions
(CDRs), interspersed with regions that are more conserved, termed
framework regions (FR). Each VH and VL is composed of three CDRs
and four FRs, arranged from amino-terminus to carboxy-terminus in
the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
[0308] The term "antigen-binding portion" of an antibody (or
"antibody portion") includes fragments of an antibody that retain
the ability to specifically bind to an antigen (e.g., hIL-12). It
has been shown that the antigen-binding function of an antibody can
be performed by fragments of a full-length antibody. Examples of
binding fragments encompassed within the term "antigen-binding
portion" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL and CH1 domains; (ii) a
F(ab').sub.2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the VH and CH1 domains; (iv) a Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which consists of a VH domain; and (vi) an isolated
complementarity determining region (CDR). Furthermore, although the
two domains of the Fv fragment, VL and VH, are coded for by
separate genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the VL and VH regions pair to form monovalent
molecules (known as single chain Fv (scFv); see e.g., Bird et al.
(1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also
intended to be encompassed within the term "antigen-binding
portion" of an antibody. Other forms of single chain antibodies,
such as diabodies are also encompassed. Diabodies are bivalent,
bispecific antibodies in which VH and VL domains are expressed on a
single polypeptide chain, but using a linker that is too short to
allow for pairing between the two domains on the same chain,
thereby forcing the domains to pair with complementary domains of
another chain and creating two antigen binding sites (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).
Still further, an antibody or antigen-binding portion thereof may
be part of a larger immunoadhesion molecules, formed by covalent or
non-covalent association of the antibody or antibody portion with
one or more other proteins or peptides. Examples of such
immunoadhesion molecules include use of the streptavidin core
region to make a tetrameric scFv molecule (Kipriyanov, S. M., et
al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a
cysteine residue, a marker peptide and a C-terminal polyhistidine
tag to make bivalent and biotinylated scFv molecules (Kipriyanov,
S. M., et al. (1994) Mol. Immunol. 31:1047-1058). Antibody
portions, such as Fab and F(ab').sub.2 fragments, can be prepared
from whole antibodies using conventional techniques, such as papain
or pepsin digestion, respectively, of whole antibodies. Moreover,
antibodies, antibody portions and immunoadhesion molecules can be
obtained using standard recombinant DNA techniques, as described
herein. Preferred antigen binding portions are complete domains or
pairs of complete domains.
[0309] The term "backmutation" refers to a process in which some or
all of the somatically mutated amino acids of a human antibody are
replaced with the corresponding germline residues from a homologous
germline antibody sequence. The heavy and light chain sequences of
the human antibody of the invention are aligned separately with the
germline sequences in the VBASE database to identify the sequences
with the highest homology. Differences in the human antibody of the
invention are returned to the germline sequence by mutating defined
nucleotide positions encoding such different amino acid. The role
of each amino acid thus identified as candidate for backmutation
should be investigated for a direct or indirect role in antigen
binding and any amino acid found after mutation to affect any
desirable characteristic of the human antibody should not be
included in the final human antibody; as an example, activity
enhancing amino acids identified by the selective mutagenesis
approach will not be subject to backmutation. To minimize the
number of amino acids subject to backmutation those amino acid
positions found to be different from the closest germline sequence
but identical to the corresponding amino acid in a second germline
sequence can remain, provided that the second germline sequence is
identical and colinear to the sequence of the human antibody of the
invention for at least 10, preferably 12 amino acids, on both sides
of the amino acid in question. Backmutation may occur at any stage
of antibody optimization; preferably, backmutation occurs directly
before or after the selective mutagenesis approach. More
preferably, backmutation occurs directly before the selective
mutagenesis approach.
[0310] The phrase "human interleukin 12" (abbreviated herein as
hIL-12, or IL-12), as used herein, includes a human cytokine that
is secreted primarily by macrophages and dendritic cells. The term
includes a heterodimeric protein comprising a 35 kD subunit (p35)
and a 40 kD subunit (p40) which are both linked together with a
disulfide bridge. The heterodimeric protein is referred to as a
"p70 subunit". The structure of human IL-12 is described further
in, for example, Kobayashi, et al. (1989) J. Exp Med. 170:827-845;
Seder, et al. (1993) Proc. Natl. Acad. Sci. 90:10188-10192; Ling,
et al. (1995) J. Exp Med. 154:116-127; Podlaski, et al. (1992)
Arch. Biochem. Biophys. 294:230-237. The term human IL-12 is
intended to include recombinant human IL-12 (rh IL-12), which can
be prepared by standard recombinant expression methods.
[0311] The phrase "interleukin comprising a p40 subunit" includes
any interleukin, e.g., any human interleukin, that comprises a p40
subunit. Such interleukins are well known in the art and include
IL-12, e.g., human IL-12, and IL-23, e.g., human IL-23.
[0312] The terms "Kabat numbering", "Kabat definitions" and "Kabat
labeling" are used interchangeably herein. These terms, which are
recognized in the art, refer to a system of numbering amino acid
residues which are more variable (i.e. hypervariable) than other
amino acid residues in the heavy and light chain variable regions
of an antibody, or an antigen binding portion thereof (Kabat et al.
(1971) Ann. NY Acad, Sci. 190:382-391 and, 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). For the heavy chain variable region, the
hypervariable region ranges from amino acid positions 31 to 35 for
CDR1, amino acid positions 50 to 65 for CDR2, and amino acid
positions 95 to 102 for CDR3. For the light chain variable region,
the hypervariable region ranges from amino acid positions 24 to 34
for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid
positions 89 to 97 for CDR3.
[0313] The Kabat numbering is used herein to indicate the positions
of amino acid modifications made in antibodies of the invention.
For example, the Y61 anti-IL-12 antibody can be mutated from serine
(S) to glutamic acid (E) at position 31 of the heavy chain CDR1
(H31S.fwdarw.E), or glycine (G) can be mutated to tyrosine (Y) at
position 94 of the light chain CDR3 (L94G.fwdarw.Y).
[0314] The term "human antibody" includes antibodies having
variable and constant regions corresponding to human germline
immunoglobulin sequences as described by Kabat et al. (See Kabat,
et al. (1991) Sequences of proteins of Immunological Interest,
Fifth Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242). The human antibodies of the invention may
include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or
site-specific mutagenesis in vitro or by somatic mutation in vivo),
for example in the CDRs and in particular CDR3. The mutations
preferably are introduced using the "selective mutagenesis
approach" described herein. The human antibody can have at least
one position replaced with an amino acid residue, e.g., an activity
enhancing amino acid residue which is not encoded by the human
germline immunoglobulin sequence. The human antibody can have up to
twenty positions replaced with amino acid residues which are not
part of the human germline immunoglobulin sequence. In other
embodiments, up to ten, up to five, up to three or up to two
positions are replaced. In a preferred embodiment, these
replacements are within the CDR regions as described in detail
below. However, the term "human antibody", as used herein, is not
intended to include antibodies in which CDR sequences derived from
the germline of another mammalian species, such as a mouse, have
been grafted onto human framework sequences.
[0315] The phrase "recombinant human antibody" includes human
antibodies that are prepared, expressed, created or isolated by
recombinant means, such as antibodies expressed using a recombinant
expression vector transfected into a host cell (described further
in Section II, below), antibodies isolated from a recombinant,
combinatorial human antibody library (described further in Section
III, below), antibodies isolated from an animal (e.g., a mouse)
that is transgenic for human immunoglobulin genes (see e.g.,
Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295) or
antibodies prepared, expressed, created or isolated by any other
means that involves splicing of human immunoglobulin gene sequences
to other DNA sequences. Such recombinant human antibodies have
variable and constant regions derived from human germline
immunoglobulin sequences (See 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). In certain embodiments, however, such recombinant human
antibodies are subjected to in vitro mutagenesis (or, when an
animal transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the VH and VL
regions of the recombinant antibodies are sequences that, while
derived from and related to human germline VH and VL sequences, may
not naturally exist within the human antibody germline repertoire
in vivo. In certain embodiments, however, such recombinant
antibodies are the result of selective mutagenesis approach or
backmutation or both.
[0316] An "isolated antibody" includes an antibody that is
substantially free of other antibodies having different antigenic
specificities (e.g., an isolated antibody that specifically binds
hIL-12 is substantially free of antibodies that specifically bind
antigens other than hIL-12). An isolated antibody that specifically
binds hIL-12 may bind IL-12 molecules from other species (discussed
in further detail below). Moreover, an isolated antibody may be
substantially free of other cellular material and/or chemicals.
[0317] A "neutralizing antibody" (or an "antibody that neutralized
hIL-12 activity") includes an antibody whose binding to hIL-12
results in inhibition of the biological activity of hIL-12. This
inhibition of the biological activity of hIL-12 can be assessed by
measuring one or more indicators of hIL-12 biological activity,
such as inhibition of human phytohemagglutinin blast proliferation
in a phytohemagglutinin blast proliferation assay (PHA), or
inhibition of receptor binding in a human IL-12 receptor binding
assay (see Example 3-Interferon-gamma Induction Assay). These
indicators of hIL-12 biological activity can be assessed by one or
more of several standard in vitro or in vivo assays known in the
art (see Example 3).
[0318] The term "activity" includes activities such as the binding
specificity/affinity of an antibody for an antigen, for example, an
anti-hIL-12 antibody that binds to an IL-12 antigen and/or the
neutralizing potency of an antibody, for example, an anti-hIL-12
antibody whose binding to hIL-12 inhibits the biological activity
of hIL-12, e.g. inhibition of PHA blast proliferation or inhibition
of receptor binding in a human IL-12 receptor binding assay (see
Example 3).
[0319] The phrase "surface plasmon resonance" includes an optical
phenomenon that allows for the analysis of real-time biospecific
interactions by detection of alterations in protein concentrations
within a biosensor matrix, for example using the BIAcore system
(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For
further descriptions, see Example 5 and Jonsson, U., et al. (1993)
Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques
11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit.
8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem.
198:268-277.
[0320] The term "K.sub.off", as used herein, is intended to refer
to the off rate constant for dissociation of an antibody from the
antibody/antigen complex.
[0321] The term "K.sub.d", as used herein, is intended to refer to
the dissociation constant of a particular antibody-antigen
interaction.
[0322] The phrase "nucleic acid molecule" includes DNA molecules
and RNA molecules. A nucleic acid molecule may be single-stranded
or double-stranded, but preferably is double-stranded DNA.
[0323] The phrase "isolated nucleic acid molecule", as used herein
in reference to nucleic acids encoding antibodies or antibody
portions (e.g., VH, VL, CDR3) that bind hIL-12 including "isolated
antibodies"), includes a nucleic acid molecule in which the
nucleotide sequences encoding the antibody or antibody portion are
free of other nucleotide sequences encoding antibodies or antibody
portions that bind antigens other than hIL-12, which other
sequences may naturally flank the nucleic acid in human genomic
DNA. Thus, for example, an isolated nucleic acid of the invention
encoding a VH region of an anti-IL-12 antibody contains no other
sequences encoding other VH regions that bind antigens other than
IL-12. The phrase "isolated nucleic acid molecule" is also intended
to include sequences encoding bivalent, bispecific antibodies, such
as diabodies in which VH and VL regions contain no other sequences
other than the sequences of the diabody.
[0324] The term "vector" includes a nucleic acid molecule capable
of transporting another nucleic acid to which it has been linked.
One type of vector is a "plasmid", which refers to a circular
double stranded DNA loop into which additional DNA segments may be
ligated. Another type of vector is a viral vector, wherein
additional DNA segments may be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) can be
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors"
(or simply, "expression vectors"). In general, expression vectors
of utility in recombinant DNA techniques are often in the form of
plasmids. In the present specification, "plasmid" and "vector" may
be used interchangeably as the plasmid is the most commonly used
form of vector. However, the invention is intended to include such
other forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0325] The phrase "recombinant host cell" (or simply "host cell")
includes a cell into which a recombinant expression vector has been
introduced. It should be understood that such terms are intended to
refer not only to the particular subject cell but to the progeny of
such a cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term "host cell" as used
herein.
[0326] The term "modifying", as used herein, is intended to refer
to changing one or more amino acids in the antibodies or
antigen-binding portions thereof. The change can be produced by
adding, substituting or deleting an amino acid at one or more
positions. The change can be produced using known techniques, such
as PCR mutagenesis.
[0327] The phrase "contact position" includes an amino acid
position of in the CDR1, CDR2 or CDR3 of the heavy chain variable
region or the light chain variable region of an antibody which is
occupied by an amino acid that contacts antigen in one of the
twenty-six known antibody-antigen structures. If a CDR amino acid
in any of the 26 known solved structures of antibody-antigen
complexes contacts the antigen, then that amino acid can be
considered to occupy a contact position. Contact positions have a
higher probability of being occupied by an amino acid which contact
antigen than non-contact positions. Preferably a contact position
is a CDR position which contains an amino acid that contacts
antigen in greater than 3 of the 26 structures (>11.5%). Most
preferably a contact position is a CDR position which contains an
amino acid that contacts antigen in greater than 8 of the 25
structures (>32%).
[0328] The term "hypermutation position" includes an amino acid
residue that occupies position in the CDR1, CDR2 or CDR3 region of
the heavy chain variable region or the light chain variable region
of an antibody that is considered to have a high frequency or
probability for somatic hypermutation during in vivo affinity
maturation of the antibody. "High frequency or probability for
somatic hypermutation" includes frequencies or probabilities of a 5
to about 40% chance that the residue will undergo somatic
hypermutation during in vivo affinity maturation of the antibody.
It should be understood that all ranges within this stated range
are also intended to be part of this invention, e.g., 5 to about
30%, e.g., 5 to about 15%, e.g., 15 to about 30%.
[0329] The term "preferred selective mutagenesis position" includes
an amino acid residue that occupies a position in the CDR1, CDR2 or
CDR3 region of the heavy chain variable region or the light chain
variable region which can be considered to be both a contact and a
hypermutation position.
[0330] The phrase "selective mutagenesis approach" includes a
method of improving the activity of an antibody by selecting and
individually mutating CDR amino acids at least one preferred
selective mutagenesis position, hypermutation, and/or contact
position. A "selectively mutated" human antibody is an antibody
which contains a mutation at a position selected using a selective
mutagenesis approach. In another embodiment, the selective
mutagenesis approach is intended to provide a method of
preferentially mutating selected individual amino acid residues in
the CDR1, CDR2 or CDR3 of the heavy chain variable region
(hereinafter H1, H2, and H3, respectively), or the CDR1, CDR2 or
CDR3 of the light chain variable region (hereinafter referred to as
L1, L2, and L3, respectively) of an antibody. Amino acid residues
may be selected from preferred selective mutagenesis positions,
contact positions, or hypermutation positions. Individual amino
acids are selected based on their position in the light or heavy
chain variable region. It should be understood that a hypermutation
position can also be a contact position. In an embodiment, the
selective mutagenesis approach is a "targeted approach". The
language "targeted approach" is intended to include a method of
preferentially mutating selected individual amino acid residues in
the CDR1, CDR2 or CDR3 of the heavy chain variable region or the
CDR1, CDR2 or CDR3 of the light chain variable region of an
antibody in a targeted manner, e.g., a "Group-wise targeted
approach" or "CDR-wise targeted approach". In the "Group-wise
targeted approach", individual amino acid residues in particular
groups are targeted for selective mutations including groups I
(including L3 and H3), II (including H2 and L1) and III (including
L2 and H1), the groups being listed in order of preference for
targeting. In the "CDR-wise targeted approach", individual amino
acid residues in particular CDRs are targeted for selective
mutations with the order of preference for targeting as follows:
H3, L3, H2, L1, H1 and L2. The selected amino acid residue is
mutated, e.g., to at least two other amino acid residues, and the
effect of the mutation on the activity of the antibody is
determined. Activity is measured as a change in the binding
specificity/affinity of the antibody, and/or neutralization potency
of the antibody. It should be understood that the selective
mutagenesis approach can be used for the optimization of any
antibody derived from any source including phage display,
transgenic animals with human IgG germline genes, human antibodies
isolated from human B-cells. Preferably, the selective mutagenesis
approach is used on antibodies which can not be optimized further
using phage display technology. It should be understood that
antibodies from any source including phage display, transgenic
animals with human IgG germline genes, human antibodies isolated
from human B-cells can be subject to backmutation prior to or after
the selective mutagenesis approach.
[0331] The term "activity enhancing amino acid residue" includes an
amino acid residue which improves the activity of the antibody. It
should be understood that the activity enhancing amino acid residue
may replace an amino acid residue at a preferred selective
mutagenesis position, contact position, or a hypermutation position
and, further, more than one activity enhancing amino acid residue
can be present within one or more CDRs. An activity enchancing
amino acid residue include, an amino acid residue that improves the
binding specificity/affinity of an antibody, for example anti-human
IL-12 antibody binding to human IL-12. The activity enhancing amino
acid residue is also intended to include an amino acid residue that
improves the neutralization potency of an antibody, for example,
the human IL-12 antibody which inhibits human IL-12.
[0332] Various aspects of the invention are described in further
detail in the following subsections.
I. Human Antibodies that Bind Human IL-12
[0333] This invention provides isolated human antibodies, or
antigen-binding portions thereof, that bind to human IL-12.
Preferably, the human antibodies of the invention are recombinant,
neutralizing human anti-hIL-12 antibodies. Antibodies of the
invention that bind to human IL-12 can be selected, for example, by
screening one or more human VL and VH cDNA libraries with hIL-12,
such as by phage display techniques as described in Example 1.
Screening of human VL and VH cDNA libraries initially identified a
series of anti-IL-12 antibodies of which one antibody, referred to
herein as "Joe 9" (or "Joe 9 wild type"), was selected for further
development. Joe 9 is a relatively low affinity human IL-12
antibody (e.g., a K.sub.off of about 0.1 sec.sup.-1), yet is useful
for specifically binding and detecting hIL-12. The affinity of the
Joe 9 antibody was improved by conducting mutagenesis of the heavy
and light chain CDRs, producing a panel of light and heavy chain
variable regions that were "mixed and matched" and further mutated,
leading to numerous additional anti-hIL-12 antibodies with
increased affinity for hIL-12 (see Example 1, Table 2 (see Appendix
A) and the sequence alignments of FIGS. 1A-D).
[0334] Of these antibodies, the human anti-hIL-12 antibody referred
to herein as Y61 demonstrated a significant improvement in binding
affinity (e.g., a K.sub.off of about 2.times.10.sup.-4 sec.sup.-1).
The Y61 anti-hIL-12 antibody was selected for further affinity
maturation by individually mutating specific amino acids residues
within the heavy and light chain CDRs. Amino acids residues of Y61
were selected for site-specific mutation (selective mutagenesis
approach) based on the amino acid residue occupying a preferred
selective mutagenesis position, contact and/or a hypermutation
position. A summary of the substitutions at selected positions in
the heavy and light chain CDRs is shown in FIGS. 2A-2H. A preferred
recombinant neutralizing antibody of the invention, referred to
herein as J695, resulted from a Gly to Tyr substitution at position
50 of the light chain CDR2 of Y61, and a Gly to Tyr substitution at
position 94 of the light chain CDR3 of Y61.
[0335] Amino acid sequence alignments of the heavy and light chain
variable regions of a panel of anti-IL-12 antibodies of the
invention, on the lineage from Joe 9 wild type to J695, are shown
in FIGS. 1A-1D. These sequence alignments allowed for the
identification of consensus sequences for preferred heavy and light
chain variable regions of antibodies of the invention that bind
hIL-12, as well as consensus sequences for the CDR3, CDR2, and
CDR1, on the lineage from Joe 9 to J695. Moreover, the Y61
mutagenesis analysis summarized in FIGS. 2A-2H allowed for the
identification of consensus sequences for heavy and light chain
variable regions that bind hIL-12, as well as consensus sequences
for the CDR3, CDR2, and CDR1 that bind hIL-12 on the lineage from
Y61 to J695 that encompasses sequences with modifications from Y61
yet that retain good hIL-12 binding characteristics. Preferred CDR,
VH and VL sequences of the invention (including consensus
sequences) as identified by sequence identifiers in the attached
Sequence Listing, are summarized below. TABLE-US-00001 SEQ ANTIBODY
ID NO: CHAIN REGION SEQUENCE 1 Consensus CDR H3
(H/S)-G-S-(H/Y)-D-(N/T/Y) Joe 9 to J695 2 Consensus CDR L3
Q-(S/T)-Y-(D/E)-(S/R/K)-(S/G/Y)- Joe 9 to J695
(L/F/T/S)-(R/S/T/W/H)-(G/P)- (S/T/A/L)-(R/S/M/T/L)-(V/I/T/M/L) 3
Consensus CDR H2 F-I-R-Y-D-G-S-N-K-Y-Y-A-D-S-V-K-G Joe 9 to J695 4
Consensus CDR L2 (G/Y)-N-(D/S)-(Q/N)-R-P-S Joe 9 to J695 5
Consensus CDR H1 F-T-F-S-(S/E)-Y-G-M-H Joe 9 to J695 6 Consensus
CDR L1 (S/T)-G-(G/S)-(R/S)-S-N-I-(G/V)- Joe 9 to J695
(S/A)-(N/G/Y)-(T/D)-V-(K/H) 7 Consensus VH (full VH sequence; see
sequence listing) Joe 9 to J695 8 Consensus VL (full VL sequence;
see sequence listing) Joe 9 to J695 9 Consensus CDR H3
H-(G/V/C/H)-(S/T)-(H/T/V/R/I)- Y61 to J695 (D/S)-N/K/A/T/S/F/W/H)
10 Consensus CDR L3 Q-S-Y-(D/S)-(Xaa)- Y61 to J695
(G/D/Q/L/F/R/H/N/Y)-T-H-P-A-L-L 11 Consensus CDR H2
(F/T/Y)-I-(R/A)-Y-(D/S/E/A)-(G/R)- Y61 to J695
S-(Xaa)-K-(Y/E)-Y-A-D-S-V-K-G 12 Consensus CDR L2
(G/Y/S/T/N/Q)-N-D-Q-R-P-S Y61 to J695 13 Consensus CDR H1
F-T-F-(Xaa)-(Xaa)-(Y/H)- Y61 to J695 (G/M/A/N/S)-M-H 14 Consensus
CDR L1 S-G-G-R-S-N-I-G-(S/C/R/N/D/T)- Y61 to J695
(N/M/I)-(T/Y/D/H/K/P)-V-K 15 Consensus VH (full VH sequence; see
sequence listing) Y61 to J695 16 Consensus VL (full VL sequence;
see sequence listing) Y61 to J695 17 Y61 CDR H3 H-G-S-H-D-N 18 Y61
CDR L3 Q-S-Y-D-R-G-T-H-P-A-L-L 19 Y61 CDR H2
F-I-R-Y-D-G-S-N-K-Y-Y-A-D-S-V-K-G 20 Y61 CDR L2 G-N-D-Q-R-P-S 21
Y61 CDR H1 F-T-F-S-S-Y-G-M-H 22 Y61 CDR L1
S-G-G-R-S-N-I-G-S-N-T-V-K 23 Y61 VH (full VH sequence; see sequence
listing) 24 Y61 VL (full VL sequence; see sequence listing) 25 J695
CDR H3 H-G-S-H-D-N 26 J695 CDR L3 Q-S-Y-D-R-Y-T-H-P-A-L-L 27 J695
CDR H2 F-I-R-Y-D-G-S-N-K-Y-Y-A-D-S-V-K-G 28 J695 CDR L2
Y-N-D-Q-R-P-S 29 J695 CDR H1 F-T-F-S-S-Y-G-M-H 30 J695 CDR L1
S-G-S-R-S-N-I-G-S-N-T-V-K 31 J695 VH (full VH sequence; see
sequence listing) 32 J695 VL (full VL sequence; see sequence
listing)
[0336] Antibodies produced from affinity maturation of Joe 9 wild
type were functionally characterized by surface plasmon resonance
analysis to determine the K.sub.d and K.sub.off rate. A series of
antibodies were produced having a K.sub.off rate within the range
of about 0.1 s.sup.-1 to about 1.times.10.sup.-5 s.sup.-1, and more
preferably a K.sub.off of about 1.times.10.sup.-4 s.sup.-1 to
1.times.10.sup.-5 s.sup.-1 or less. Antibodies were also
characterized in vitro for their ability to inhibit
phytohemagglutinin (PHA) blast proliferation, as described in
Example 3. A series of antibodies were produced having an IC.sub.50
value in the range of about 1.times.10.sup.-6 M to about
1.times.10.sup.-11 M, more preferably about 1.times.10.sup.-10 M to
1.times.10.sup.-11 M or less.
[0337] Accordingly, in one aspect, the invention provides an
isolated human antibody, or antigen-binding portion thereof, that
binds to human IL-12 and dissociates from human IL-12 with a
K.sub.off rate constant of 0.1 s.sup.-1 or less, as determined by
surface plasmon resonance, or which inhibits phytohemagglutinin
blast proliferation in an in vitro phytohemagglutinin blast
proliferation assay (PHA assay) with an IC.sub.50 of
1.times.10.sup.-6 M or less. In preferred embodiments, the isolated
human IL-12 antibody, or an antigen-binding portion thereof,
dissociates from human IL-12 with a K.sub.off rate constant of
1.times.10.sup.-2 s.sup.-1 or less, or inhibits phytohemagglutinin
blast proliferation in an in vitro PHA assay with an IC.sub.50 of
1.times.10.sup.-7 M or less. In more preferred embodiments, the
isolated human IL-12 antibody, or an antigen-binding portion
thereof, dissociates from human IL-12 with a K.sub.off rate
constant of 1.times.10.sup.-3 s.sup.-1 or less, or inhibits
phytohemagglutinin blast proliferation in an in vitro PHA assay
with an IC.sub.50 of 1.times.10.sup.-8 M or less. In more preferred
embodiments, the isolated human IL-12 antibody, or an
antigen-binding portion thereof, dissociates from human IL-12 with
a K.sub.off rate constant of 1.times.10.sup.-4 s.sup.-1 or less, or
inhibits phytohemagglutinin blast proliferation in an in vitro PHA
assay with an IC.sub.50 of 1.times.10.sup.-9 M or less. In more
preferred embodiments, the isolated human IL-12 antibody, or an
antigen-binding portion thereof, dissociates from human IL-12 with
a K.sub.off rate constant of 1.times.10.sup.-5 s.sup.-1 or less, or
inhibits phytohemagglutinin blast proliferation in an in vitro PHA
assay with an IC.sub.50 of 1.times.10.sup.-10 M or less. In even
more preferred embodiments, the isolated human IL-12 antibody, or
an antigen-binding portion thereof, dissociates from human IL-12
with a K.sub.off rate constant of 1.times.10.sup.-5 s.sup.-1 or
less, or inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-11 M or
less.
[0338] The dissociation rate constant (K.sub.off) of an IL-12
antibody can be determined by surface plasmon resonance (see
Example 5). Generally, surface plasmon resonance analysis measures
real-time binding interactions between ligand (recombinant human
IL-12 immobilized on a biosensor matrix) and analyte (antibodies in
solution) by surface plasmon resonance (SPR) using the BIAcore
system (Pharmacia Biosensor, Piscataway, N.J.). Surface plasmon
analysis can also be performed by immobilizing the analyte
(antibodies on a biosensor matrix) and presenting the ligand
(recombinant IL-12 in solution). Neutralization activity of IL-12
antibodies, or antigen binding portions thereof, can be assessed
using one or more of several suitable in vitro assays (see Example
3).
[0339] It is well known in the art that antibody heavy and light
chain CDRs play an important role in the binding
specificity/affinity of an antibody for an antigen. Accordingly,
the invention encompasses human antibodies having light and heavy
chain CDRs of Joe 9, as well as other antibodies having CDRs that
have been modified to improve the binding specificity/affinity of
the antibody. As demonstrated in Example 1, a series of
modifications to the light and heavy chain CDRs results in affinity
maturation of human anti-hIL-12 antibodies. The heavy and light
chain variable region amino acid sequence alignments of a series of
human antibodies ranging from Joe 9 wild type to J695 that bind
human IL-12 is shown in FIGS. 1A-1D. Consensus sequence motifs for
the CDRs of antibodies can be determined from the sequence
alignment (as summarized in the table above). For example, a
consensus motif for the VH CDR3 of the lineage from Joe 9 to J695
comprises the amino acid sequence: (H/S)-G-S-(H/Y)-D-(N/T/Y) (SEQ
ID NO: 1), which encompasses amino acids from position 95 to 102 of
the consensus HCVR shown in SEQ ID NO: 7. A consensus motif for the
VL CDR3 comprises the amino acid sequence:
Q-(S/T)-Y-(D/E)-(S/R/K)-(S/G/Y)-(L/F/T/S)-(R/S/T/W/H)-(G/P)-(S/T/A/L)-(R/-
S/M/T/L-V/I/T/M/L) (SEQ ID NO: 2), which encompasses amino acids
from position 89 to 97 of the consensus LCVR shown in SEQ ID NO:
8.
[0340] Accordingly, in another aspect, the invention provides an
isolated human antibody, or an antigen-binding portion thereof,
which has the following characteristics:
[0341] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-6 M or
less;
[0342] b) has a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 1; and
[0343] c) has a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 2.
[0344] In a preferred embodiment, the antibody further comprises a
VH CDR2 comprising the amino acid sequence:
F-I-R-Y-D-G-S-N-K-Y-Y-A-D-S-V-K-G (SEQ ID NO: 3) (which encompasses
amino acids from position 50 to 65 of the consensus HCVR comprising
the amino acid sequence SEQ ID NO: 7) and further comprises a VL
CDR2 comprising the amino acid sequence: (G/Y)-N-(D/S)-(Q/N)-R-P-S
(SEQ ID NO: 4) (which encompasses amino acids from position 50 to
56 of the consensus LCVR comprising the amino acid sequence SEQ ID
NO: 8).
[0345] In another preferred embodiment, the antibody further
comprises a VH CDR1 comprising the amino acid sequence:
F-T-F-S-(S/E)-Y-G-M-H (SEQ ID NO: 5) (which encompasses amino acids
from position 27 to 35 of the consensus HCVR comprising the amino
acid sequence SEQ ID NO: 7) and further comprises a VL CDR1
comprising the amino acid sequence:
(S/T)-G-(G/S)-(R/S)-S-N-I-(G/V)-(S/A)-(N/G/Y)-(T/D)-V-(K/H) (SEQ ID
NO: 6) (which encompasses amino acids from position 24 to 34 of the
consensus LCVR comprising the amino acid sequence SEQ ID NO:
8).
[0346] In yet another preferred embodiment, the antibody of the
invention comprises a HCVR comprising the amino acid sequence of
SEQ ID NO: 7 and a LCVR comprising the amino acid sequence of SEQ
ID NO: 8.
[0347] Additional consensus motifs can be determined based on the
mutational analysis performed on Y61 that led to the J695 antibody
(summarized in FIGS. 2A-2H). As demonstrated by the graphs shown in
FIGS. 2A-2H, certain residues of the heavy and light chain CDRs of
Y61 were amenable to substitution without significantly impairing
the hIL-12 binding properties of the antibody. For example,
individual substitutions at position 30 in CDR H1 with twelve
different amino acid residues did not significantly reduce the
K.sub.off rate of the antibody, indicating that is position is
amenable to substitution with a variety of different amino acid
residues. Thus, based on the mutational analysis (i.e., positions
within Y61 that were amenable to substitution by other amino acid
residues) consensus motifs were determined. The consensus motifs
for the heavy and light chain CDR3s are shown in SEQ ID NOs: 9 and
10, respectively, consensus motifs for the heavy and light chain
CDR2s are shown in SEQ ID NOs: 11 and 12, respectively, and
consensus motifs for the heavy and light chain CDR1s are shown in
SEQ ID NOs: 13 and 14, respectively. Consensus motifs for the VH
and VL regions are shown in SEQ ID NOs: 15 and 16,
respectively.
[0348] Accordingly, in one aspect, the invention features an
isolated human antibody, or an antigen-binding portion thereof,
which has the following characteristics:
[0349] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less;
[0350] b) has a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 9; and
[0351] c) has a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 10.
[0352] In a preferred embodiment, the antibody further comprises a
VH CDR2 comprising the amino acid sequence of SEQ ID NO: 11 and
further comprises a VL CDR2 comprising the amino acid sequence of
SEQ ID NO: 12.
[0353] In another preferred embodiment, the antibody further
comprises a VH CDR1 comprising the amino acid sequence of SEQ ID
NO: 13 and further comprises a VL CDR1 comprising the amino acid
sequence of SEQ ID NO: 14.
[0354] In yet another preferred embodiment, the antibody of the
invention comprises a HCVR comprising the amino acid sequence of
SEQ ID NO: 15 and a LCVR comprising the amino acid sequence of SEQ
ID NO: 16.
[0355] A preferred antibody of the invention, the human anti-hIL-12
antibody Y61, was produced by affinity maturation of Joe 9 wild
type by PCR mutagenesis of the CDR3 (as described in Example 1).
Y61 had an improved specificity/binding affinity determined by
surface plasmon resonance and by in vitro neutralization assays.
The heavy and light chain CDR3s of Y61 are shown in SEQ ID NOs: 17
and 18, respectively, the heavy and light chain CDR2s of Y61 are
shown in SEQ ID NOs: 19 and 20, respectively, and the heavy and
light chain CDR1s of Y61 are shown in SEQ ID NOs: 21 and 22,
respectively. The VH of Y61 has the amino acid sequence of SEQ ID
NO: 23 and the VL of Y61 has the amino acid sequence of SEQ ID NO:
24 (these sequences are also shown in FIGS. 1A-1D, aligned with
Joe9).
[0356] Accordingly, in another aspect, the invention features an
isolated human antibody, or an antigen-binding portion thereof,
which
[0357] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less;
[0358] b) has a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 17; and
[0359] c) has a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 18.
[0360] In a preferred embodiment, the isolated human antibody, or
an antigen-binding portion thereof, has a heavy chain CDR2
comprising the amino acid sequence of SEQ ID NO: 19 and a light
chain CDR2 comprising the amino acid sequence of SEQ ID NO: 20.
[0361] In another preferred embodiment, the isolated human
antibody, or an antigen-binding portion thereof has a heavy chain
CDR1 comprising the amino acid sequence of SEQ ID NO: 21 and a
light chain CDR1 comprising the amino acid sequence of SEQ ID NO:
22.
[0362] In yet another preferred embodiment, the isolated human
antibody, or an antigen-binding portion thereof, comprising a the
heavy chain variable region comprising the amino acid sequence of
SEQ ID NO: 23, and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 24.
[0363] In certain embodiments, the full length antibody comprises a
heavy chain constant region, such as IgG1, IgG2, IgG3, IgG4, IgM,
IgA and IgE constant regions, and any allotypic variant therein as
described in Kabat (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).
Preferably, the antibody heavy chain constant region is an IgG1
heavy chain constant region. Alternatively, the antibody portion
can be an Fab fragment, an F(ab'.sub.2) fragment or a single chain
Fv fragment.
[0364] Modifications of individual residues of Y61 led to the
production of a panel of antibodies shown in FIGS. 2A-2H. The
specificity/binding affinity of each antibody was determined by
surface plasmon resonance and/or by in vitro neutralization
assays.
[0365] Accordingly, in another aspect, the invention features an
isolated human antibody, or an antigen-binding portion thereof,
which
[0366] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less;
[0367] b) has a heavy chain CDR3 comprising the amino acid sequence
selected from the group consisting of SEQ ID NO: 404-SEQ ID NO:
469; and
[0368] c) has a light chain CDR3 comprising the amino acid sequence
selected from the group consisting of SEQ ID NO: 534-SEQ ID NO:
579.
[0369] In preferred embodiment, the isolated human antibody, or an
antigen-binding portion thereof, has a heavy chain CDR2 comprising
the amino acid sequence selected from the group consisting of SEQ
ID NO:335-SEQ ID NO: 403; and a light chain CDR2 comprising the
amino acid sequence selected from the group consisting of SEQ ID
NO: 506-SEQ ID NO: 533.
[0370] In another preferred embodiment, the isolated human
antibody, or an antigen-binding portion thereof, has a heavy chain
CDR1 comprising the amino acid sequence selected from the group
consisting of SEQ ID NO: 288-SEQ ID NO: 334; and a light chain CDR1
comprising the amino acid sequence selected from the group
consisting of SEQ ID NO: 470-SEQ ID NO: 505.
[0371] In yet another preferred embodiment, the isolated human
antibody, or an antigen-binding portion thereof, comprising a the
heavy chain variable region comprising the amino acid sequence of
SEQ ID NO: 23, and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 24.
[0372] In certain embodiments, the full length antibody comprising
a heavy chain constant region such as IgG1, IgG2, IgG3, IgG4, IgM,
IgA and IgE constant regions and any allotypic variant therein as
described in Kabat (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).
Preferably, the antibody heavy chain constant region is an IgG1
heavy chain constant region. Alternatively, the antibody portion
can be a Fab fragment, an F(ab'.sub.2) fragment or a single chain
Fv fragment.
[0373] A particularly preferred recombinant, neutralizing antibody
of the invention, J695, was produced by site-directed mutagenesis
of contact and hypermutation amino acids residues of antibody Y61
(see Example 2 and section III below). J695 differs from Y61 by a
Gly to Tyr substitution in Y61 at position 50 of the light chain
CDR2 and by a Gly to Tyr substitution at position 94 of the light
chain CDR3. The heavy and light chain CDR3s of J695 are shown in
SEQ ID NOs: 25 and 26, respectively, the heavy and light chain
CDR2s of J695 are shown in SEQ ID NOs: 27 and 28, respectively, and
the heavy and light chain CDR1s of J695 are shown in SEQ ID NOs: 29
and 30, respectively. The VH of J695 has the amino acid sequence of
SEQ ID NO: 31 and the VL of J695 has the amino acid sequence of SEQ
ID NO: 32 (these sequences are also shown in FIGS. 1A-1D, aligned
with Joe9).
[0374] Accordingly, in another aspect, the invention features an
isolated human antibody, or an antigen-binding portion thereof,
which
[0375] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less;
[0376] b) has a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 25; and
[0377] c) has a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 26.
[0378] In preferred embodiment, the isolated human antibody, or an
antigen-binding portion thereof, has a heavy chain CDR2 comprising
the amino acid sequence of SEQ ID NO: 27, and a light chain CDR2
comprising the amino acid sequence of SEQ ID NO: 28.
[0379] In another preferred embodiment, the isolated human
antibody, or an antigen-binding portion thereof, has a heavy chain
CDR1 comprising the amino acid sequence of SEQ ID NO: 29, and a
light chain CDR1 comprising the amino acid sequence of SEQ ID NO:
30.
[0380] In yet another preferred embodiment, the isolated human
antibody, or an antigen-binding portion thereof, has a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:
31, and a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 32.
[0381] In certain embodiments, the full length antibody comprises a
heavy chain constant region, such as IgG1, IgG2, IgG3, IgG4, IgM,
IgA and IgE constant regions and any allotypic variant therein as
described in Kabat (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).
Preferably, the antibody heavy chain constant region is an IgG1
heavy chain constant region. Alternatively, the antibody portion
can be an Fab fragment, an F(ab'.sub.2) fragment or a single chain
Fv fragment.
[0382] Additional mutations in the preferred consensus sequences
for CDR3, CDR2, and CDR1 of antibodies on the lineage from Joe 9 to
J695, or from the lineage Y61 to J695, can be made to provide
additional anti-IL-12 antibodies of the invention. Such methods of
modification can be performed using standard molecular biology
techniques, such as by PCR mutagenesis, targeting individual
contact or hypermutation amino acid residues in the light chain
and/or heavy chain CDRs-, followed by kinetic and functional
analysis of the modified antibodies as described herein (e.g.,
neutralization assays described in Example 3, and by BIAcore
analysis, as described in Example 5).
[0383] Accordingly, in another aspect the invention features an
isolated human antibody, or an antigen-binding portion thereof,
which
[0384] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-6 M or
less;
[0385] b) comprises a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 3 and a heavy chain CDR1 comprising the
amino acid sequence of SEQ ID NO: 5, or a mutant thereof having one
or more amino acid substitutions at a preferred selective
mutagenesis position or a hypermutation position, wherein said
mutant has a k.sub.off rate no more than 10-fold higher than the
antibody comprising a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 3, and a heavy chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 5; and
[0386] c) comprises a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 2, a light chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 4, and a light chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 6, or a mutant thereof having
one or more amino acid substitutions at a preferred selective
mutagenesis position or a hypermutation position, wherein said
mutant has a k.sub.off rate no more than 10-fold higher than the
antibody comprising a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 2, a light chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 4, and a light chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 6.
[0387] In another aspect the invention features an isolated human
antibody, or an antigen-binding portion thereof, which
[0388] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less;
[0389] b) comprises a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 11 and a heavy chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 13, or a mutant thereof
having one or more amino acid substitutions at a preferred
selective mutagenesis position, contact position or a hypermutation
position, wherein said mutant has a k.sub.off rate no more than
10-fold higher than the antibody comprising a heavy chain CDR3
comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a
heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:
13; and
[0390] c) comprises a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 10, a light chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 12, and a light chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 14, or a mutant thereof
having one or more amino acid substitutions at a preferred
selective mutagenesis position, contact position or a hypermutation
position, wherein said mutant has a k.sub.off rate no more than
10-fold higher than the antibody comprising a light chain CDR3
comprising the amino acid sequence of SEQ ID NO: 10, a light chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a
light chain CDR1 comprising the amino acid sequence of SEQ ID NO:
14.
[0391] An ordinarily skilled artisan will also appreciate that
additional mutations to the CDR regions of an antibody of the
invention, for example in Y61 or in J695, can be made to provide
additional anti-IL-12 antibodies of the invention. Such methods of
modification can be performed using standard molecular biology
techniques, as described above. The functional and kinetic analysis
of the modified antibodies can be performed as described in Example
3 and Example 5, respectively. Modifications of individual residues
of Y61 that led to the identification of J695 are shown in FIGS.
2A-2H and are described in Example 2.
[0392] Accordingly, in another aspect the invention features an
isolated human antibody, or an antigen-binding portion thereof,
which
[0393] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less;
[0394] b) comprises a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 19 and a heavy chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 21, or a mutant thereof
having one or more amino acid substitutions at a preferred
selective mutagenesis position or a hypermutation position, wherein
said mutant has a k.sub.off rate no more than 10-fold higher than
the antibody comprising a heavy chain CDR3 comprising the amino
acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the
amino acid sequence of SEQ ID NO: 19, and a heavy chain CDR1
comprising the amino acid sequence of SEQ ID NO: 21; and
[0395] c) comprises a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 18, a light chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 20, and a light chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 22, or a mutant thereof
having one or more amino acid substitutions at a preferred
selective mutagenesis position or a hypermutation position, wherein
said mutant has a k.sub.off rate no more than 10-fold higher than
the antibody comprising a light chain CDR3 comprising the amino
acid sequence of SEQ ID NO: 18, a light chain CDR2 comprising the
amino acid sequence of SEQ ID NO: 20, and a light chain CDR1
comprising the amino acid sequence of SEQ ID NO: 22.
[0396] In another aspect the invention features an isolated human
antibody, or an antigen-binding portion thereof, which
[0397] a) inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or
less;
[0398] b) comprises a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 27 and a heavy chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 29, or a mutant thereof
having one or more amino acid substitutions at a preferred
selective mutagenesis position or a hypermutation position, wherein
said mutant has a k.sub.off rate no more than 10-fold higher than
the antibody comprising a heavy chain CDR3 comprising the amino
acid sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the
amino acid sequence of SEQ ID NO: 27, and a heavy chain CDR1
comprising the amino acid sequence of SEQ ID NO: 29; and
[0399] c) comprises a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino
acid sequence of SEQ ID NO: 28, and a light chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 30, or a mutant thereof
having one or more amino acid substitutions at a preferred
selective mutagenesis position or a hypermutation position, wherein
said mutant has a k.sub.off rate no more than 10-fold higher than
the antibody comprising a light chain CDR3 comprising the amino
acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the
amino acid sequence of SEQ ID NO: 28, and a light chain CDR1
comprising the amino acid sequence of SEQ ID NO: 30.
[0400] In yet another embodiment, the invention provides isolated
human antibodies, or antigen-binding portions thereof, that
neutralize the activity of human IL-12, and at least one additional
primate IL-12 selected from the group consisting of baboon IL-12,
marmoset IL-12, chimpanzee IL-12, cynomolgus IL-12 and rhesus
IL-12, but which do not neutralize the activity of the mouse
IL-12.
II Selection of Recombinant Human Antibodies
[0401] Recombinant human antibodies of the invention can be
isolated by screening of a recombinant combinatorial antibody
library, preferably a scFv phage display library, prepared using
human VL and VH cDNAs prepared from mRNA derived from human
lymphocytes. Methodologies for preparing and screening such
libraries are known in the art. In addition to commercially
available kits for generating phage display libraries (e.g., the
Pharmacia Recombinant Phage Antibody System, catalog no.
27-9400-01; and the Stratagene SurfZAP.TM. phage display kit,
catalog no. 240612), examples of methods and reagents particularly
amenable for use in generating and screening antibody display
libraries can be found in, for example, Kang et al. PCT Publication
No. WO 92/18619; Winter et al. PCT Publication No. WO 92/20791;
Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al.
PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No.
WO 92/09690; Fuchs et al. (1991) Bio/Technology 2:1370-1372; Hay et
al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989)
Science 246:1275-1281; McCafferty et al., Nature (1990)
348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et
al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature
352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al.
(1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc
Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS
88:7978-7982.
[0402] The antibody libraries used in this method are preferably
scFv libraries prepared from human VL and VH cDNAs. The scFv
antibody libraries are preferably screened using recombinant human
IL-12 as the antigen to select human heavy and light chain
sequences having a binding activity toward IL-12. To select for
antibodies specific for the p35 subunit of IL-12 or the p70
heterodimer, screening assays were performed in the presence of
excess free p40 subunit. Subunit preferences can be determined, for
example by, micro-Friguet titration, as described in Example 1.
[0403] Once initial human VL and VH segments are selected, "mix and
match" experiments, in which different pairs of the selected VL and
VH segments are screened for IL-12 binding, are performed to select
preferred VL/VH pair combinations (see Example 1). Additionally, to
further improve the affinity and/or lower the off rate constant for
hIL-12 binding, the VL and VH segments of the preferred VL/VH
pair(s) can be randomly mutated, preferably within the CDR3 region
of VH and/or VL, in a process analogous to the in vivo somatic
mutation process responsible for affinity maturation of antibodies
during a natural immune response. This in vitro affinity maturation
can be accomplished by amplifying VH and VL regions using PCR
primers complimentary to the VH CDR3 or VL CDR3, respectively,
which primers have been "spiked" with a random mixture of the four
nucleotide bases at certain positions such that the resultant PCR
products encode VH and VL segments into which random mutations have
been introduced into the VH and/or VL CDR3 regions. These randomly
mutated VH and VL segments can be reselected and rescreened for
binding to hIL-12 and sequences that exhibit high affinity and a
low off rate for IL-12 binding can be selected. Table 2 (see
Appendix A) shows antibodies that displayed altered binding
specificity/affinity produced as a result of in vitro affinity
maturation.
[0404] Following selection, isolation and screening of an
anti-hIL-12 antibody of the invention from a recombinant
immunoglobulin display library, nucleic acid encoding the selected
antibody can be recovered from the phage particle(s) (e.g. from the
phage genome) and subcloned into other expression vectors by
standard recombinant DNA techniques. If desired, the nucleic acid
can be further manipulated to create other antibody forms of the
invention (e.g., linked to nucleic acid encoding additional
immunoglobulin domains, such as additional constant regions). To
express a recombinant human antibody isolated by screening of a
combinatorial library, the DNA encoding the antibody is cloned into
a recombinant expression vector and introduced into a mammalian
host cells, as described in further detail in Section IV below.
[0405] Methods for selecting human IL-12 binding antibodies by
phage display technology, and affinity maturation of selected
antibodies by random or site-directed mutagenesis of CDR regions
are described in further detail in Example 1.
[0406] As described in Example 1, screening of human VL and VH cDNA
libraries identified a series of anti-IL-12 antibodies, of which
the Joe 9 antibody was selected for further development. A
comparison of the heavy chain variable region of Joe 9 with the
heavy chain germline sequences selected from the VBASE database,
revealed that Joe 9 was similar to the COS-3 germline sequence.
COS-3 belongs to the V.sub.H3 family of germline sequences.
[0407] The V.sub.H3 family is part of the human VH germline
repertoire which is grouped into seven families, V.sub.H1-V.sub.H7,
based on nucleotide sequence homology (Tomlinson et al. (1992) J.
Mol. Biol., 227, 776-798 and Cook et al. (1995) Immunology Today,
16, 237-242). The V.sub.H3 family contains the highest number of
members and makes the largest contribution to the germline
repertoire. For any given human V.sub.H3-germline antibody
sequence, the amino acid sequence identity within the entire
V.sub.H3 family is high (See e.g., Tomlinson et al. (1992) J. Mol.
Biol., 227, 776-798 and Cook et al. (1995) Immunology Today, 16,
237-242). The range of amino acid sequence identity between any two
germline VH sequences of the V.sub.H3 family varies from 69-98
residues out of approximately 100 VH residues, (i.e., 69-98% amino
acid sequence homology between any two germline VH sequences). For
most pairs of germline sequences there is at least 80 or more
identical amino acid residues, (i.e., at least 80% amino acid
sequence homology). The high degree of amino acid sequence homology
between the V.sub.H3 family members results in certain amino acid
residues being present at key sites in the CDR and framework
regions of the VH chain. These amino acid residues confer
structural features upon the CDRs.
[0408] Studies of antibody structures have shown that CDR
conformations can be grouped into families of canonical CDR
structures based on the key amino acid residues that occupy certain
positions in the CDR and framework regions. Consequently, there are
similar local CDR conformations in different antibodies that have
canonical structures with identical key amino acid residues
(Chothia et al. (1987) J. Mol. Biol., 196, 901-917 and Chothia et
al. (1989) Nature, 342, 877-883). Within the V.sub.H3 family there
is a conservation of amino acid residue identity at the key sites
for the CDR1 and CDR2 canonical structures (Chothia et al. (1992)
J. Mol. Biol., 227, 799-817).
[0409] The COS-3 germline VH gene, is a member of the V.sub.H3
family and is a variant of the 3-30 (DP-49) germline VH allele.
COS-3, differs from Joe9 VH amino acid sequences at only 5
positions. The high degree of amino acid sequence homology between
Joe9 VH and COS-3, and between Joe9 VH and the other V.sub.H3
family members also confers a high degree of CDR structural
homology (Chothia et al. (1992) J. Mol. Biol., 227, 799-817;
Chothia et al. (1987) J. Mol. Biol., 196, 901-917 and Chothia et
al. (1989) Nature, 342, 877-883).
[0410] The skilled artisan will appreciate that based on the high
amino acid sequence and canonical structural similarity to Joe 9,
other V.sub.H3 family members could also be used to generate
antibodies that bind to human IL-12. This can be performed, for
example, by selecting an appropriate VL by chain-shuffling
techniques (Winter et al. (1994) Annual Rev. Immunol., 12, 433-55),
or by the grafting of CDRs from a rodent or other human antibody
including CDRs from antibodies of this invention onto a V.sub.H3
family framework.
[0411] The human V lambda germline repertoire is grouped into 10
families based on nucleotide sequence homology (Williams et al.
(1996) J. Mol. Biol., 264, 220-232). A comparison of the light
chain variable region of Joe 9 with the light chain germline
sequences selected from the VBASE database, revealed that Joe 9 was
similar to the DPL8 lambda germline. The Joe9 VL differs from DPL8
sequence at only four framework positions, and is highly homologous
to the framework sequences of the other V.sub..lamda.1 family
members. Based on the high amino acid sequence homology and
canonical structural similarity to Joe 9, other V.sub..lamda.1
family members may also be used to generate antibodies that bind to
human IL-12. This can be performed, for example, by selecting an
appropriate VH by chain-shuffling techniques (Winter et al. Supra,
or by the grafting of CDRs from a rodent or other human antibody
including CDRs from antibodies of this invention onto a
V.sub..lamda.1 family framework.
[0412] The methods of the invention are intended to include
recombinant antibodies that bind to hIL-12, comprising a heavy
chain variable region derived from a member of the V.sub.H3 family
of germline sequences, and a light chain variable region derived
from a member of the V.sub..lamda.1 family of germline sequences.
Moreover, the skilled artisan will appreciate that any member of
the V.sub.H3 family heavy chain sequence can be combined with any
member of the V.sub..lamda.1 family light chain sequence.
[0413] Those skilled in the art will also appreciate that DNA
sequence polymorphisms that lead to changes in the amino acid
sequences of the germline may exist within a population (e.g., the
human population). Such genetic polymorphism in the germline
sequences may exist among individuals within a population due to
natural allelic variation. Such natural allelic variations can
typically result in 1-5% variance in the nucleotide sequence of the
a gene. Any and all such nucleotide variations and resulting amino
acid polymorphisms in germline sequences that are the result of
natural allelic variation are intended to be within the scope of
the invention.
[0414] Accordingly, in one aspect, the invention features an
isolated human antibody, or an antigen-binding portion thereof,
which has the following characteristics: [0415] a) that binds to
human IL-12 and dissociates from human IL-12 with a k.sub.off rate
constant of 0.1 s.sup.-1 or less, as determined by surface plasmon
resonance, or which inhibits phytohemagglutinin blast proliferation
in an in vitro phytohemagglutinin blast proliferation assay (PHA
assay) with an IC.sub.50 of 1.times.10.sup.-6M or less. [0416] b)
has a heavy chain variable region comprising an amino acid sequence
selected from a member of the V.sub.H3 germline family, wherein the
heavy chain variable region has a mutation at a contact or
hypermutation position with an activity enhancing amino acid
residue. [0417] c) has a light chain variable region comprising an
amino acid sequence selected from a member of the V.sub..lamda.1
germline family, wherein the light chain variable region has a
mutation at a preferred selective mutagenesis position, contact or
hypermutation position with an activity enhancing amino acid
residue.
[0418] In a preferred embodiment, the isolated human antibody, or
antigen binding has mutation in the heavy chain CDR3.
[0419] In another preferred embodiment, the isolated human
antibody, or antigen binding has mutation in the light chain
CDR3.
[0420] In another preferred embodiment, the isolated human
antibody, or antigen binding has mutation in the heavy chain
CDR2.
[0421] In another preferred embodiment, the isolated human
antibody, or antigen binding has mutation in the light chain
CDR2.
[0422] In another preferred embodiment, the isolated human
antibody, or antigen binding has mutation in the heavy chain
CDR1.
[0423] In another preferred embodiment, the isolated human
antibody, or antigen binding has mutation in the light chain
CDR1.
[0424] An ordinarily skilled artisan will appreciate that based on
the high amino acid sequence similarity between members of the
V.sub.H3 germline family, or between members of the light chain
V.sub..lamda.1 germline family, that mutations to the germlines
sequences can provide additional antibodies that bind to human
IL-12. Table 1 (see Appendix A) shows the germline sequences of the
V.sub.H3 family members and demonstrates the significant sequence
homology within the family members. Also shown in Table 1 are the
germline sequences for V.sub..lamda.1 family members. The heavy and
light chain sequences of Joe 9 are provided as a comparison.
Mutations to the germline sequences of V.sub.H3 or V.sub..lamda.1
family members may be made, for example, at the same amino acid
positions as those made in the antibodies of the invention (e.g.
mutations in Joe 9). The modifications can be performed using
standard molecular biology techniques, such as by PCR mutagenesis,
targeting individual amino acid residues in the germline sequences,
followed by kinetic and functional analysis of the modified
antibodies as described herein (e.g., neutralization assays
described in Example 3, and by BIAcore analysis, as described in
Example 5).
[0425] Accordingly, in one aspect, the invention features isolated
human antibody, or an antigen-binding portion thereof, which has
the following characteristics: [0426] a) has a heavy chain variable
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 595-667, wherein the heavy chain variable
region has a mutation at a preferred selective mutagenesis
position, contact or hypermutation position with an activity
enhancing amino acid residue. [0427] b) has a light chain variable
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 669-675, wherein the light chain variable
region has a mutation at a preferred selective mutagenesis
position, contact or hypermutation position with an activity
enhancing amino acid residue.
[0428] An ordinarily skilled artisan will appreciate that based on
the high amino acid sequence similarity between Joe 9 and COS-3
heavy chain germline sequence, and between Joe 9 and DPL8 lambda
germline sequence, that other mutations to the CDR regions of these
germlines sequences can provide additional antibodies that bind to
human IL-12. Such methods of modification can be performed using
standard molecular biology techniques as described above.
[0429] Accordingly, in one aspect, the invention features isolated
human antibody, or an antigen-binding portion thereof, which has
the following characteristics: [0430] a) that binds to human IL-12
and dissociates from human IL-12 with a k.sub.off rate constant of
0.1 s.sup.-1 or less, as determined by surface plasmon resonance,
or which inhibits phytohemagglutinin blast proliferation in an in
vitro phytohemagglutinin blast proliferation assay (PHA assay) with
an IC.sub.50 of 1.times.10.sup.-6M or less. [0431] b) has a heavy
chain variable region comprising the COS-3 germline amino acid
sequence, wherein the heavy chain variable region has a mutation at
a preferred selective mutagenesis position, contact or
hypermutation position with an activity enhancing amino acid
residue. [0432] c) has a light chain variable region comprising the
DPL8 germline amino acid sequence, wherein the light chain variable
region has a mutation at a preferred selective mutagenesis
position, contact or hypermutation position with an activity
enhancing amino acid residue.
[0433] Due to certain amino acid residues occupying key sites in
the CDR and framework regions in the light and heavy chain variable
region, structural features are conferred at these regions. In
particular, the CDR2 and CDR1 regions are subject to canonical
structural classifications. Since there is a high degree of amino
acids sequence homology between family members, these canonical
features are present between family members. The skilled artisan
will appreciate that modifications at the amino acid residues that
confer these canonical structures would produce additional
antibodies that bind to IL-12. The modifications can be performed
using standard molecular biology techniques as described above.
[0434] Accordingly, in another aspect, the invention features an
isolated human antibody, or an antigen-binding portion thereof,
which has the following characteristics: [0435] a) that binds to
human IL-12 and dissociates from human IL-12 with a k.sub.off rate
constant of 0.1 s.sup.-1 or less, as determined by surface plasmon
resonance, or which inhibits phytohemagglutinin blast proliferation
in an in vitro phytohemagglutinin blast proliferation assay (PHA
assay) with an IC.sub.50 of 1.times.10.sup.-6M or less. [0436] b)
has a heavy chain variable region comprising an amino acid sequence
selected from a member of the V.sub.H3 germline family, wherein the
heavy chain variable region comprises a CDR2 that is structurally
similar to CDR2s from other V.sub.H3 germline family members, and a
CDR1 that is structurally similar to CDR1s from other V.sub.H3
germline family members, and wherein the heavy chain variable
region has a mutation at a preferred selective mutagenesis
position, contact or hypermutation position with an activity
enhancing amino acid residue; [0437] c) has a light chain variable
region comprising an amino acid sequence selected from a member of
the V.sub..lamda.1 germline family, wherein the light chain
variable region comprises a CDR2 that is structurally similar to
CDR2s from other V.sub..lamda.1 germline family members, and a CDR1
that is structurally similar to CDR1s from other V.sub..lamda.1
germline family members, and wherein the light chain variable
region has a mutation at a preferred selective mutagenesis
position, contact or hypermutation position with an activity
enhancing amino acid residue.
[0438] Recombinant human antibodies of the invention have variable
and constant regions which are homologous to human germline
immunoglobulin sequences selected from the VBASE database.
Mutations to the recombinant human antibodies (e.g., by random
mutagenesis or PCR mutagenesis) result in amino acids that are not
encoded by human germline immunoglobulin sequences. Also, libraries
of recombinant antibodies which were derived from human donors will
contain antibody sequences that differ from their corresponding
germline sequences due to the normal process of somatic mutation
that occurs during B-cell development. It should be noted that if
the "germline" sequences obtained by PCR amplification encode amino
acid differences in the framework regions from the true germline
configuration (i.e., differences in the amplified sequence as
compared to the true germline sequence), it may be desirable to
change these amino acid differences back to the true germline
sequences (i.e., "backmutation" of framework residues to the
germline configuration). Thus, the present invention can optionally
include a backmutation step. To do this, the amino acid sequences
of heavy and light chain encoded by the germline (as found as
example in VBASE database) are first compared to the mutated
immunoglobulin heavy and light chain framework amino acid sequences
to identify amino acid residues in the mutated immunoglobulin
framework sequence that differ from the closest germline sequences.
Then, the appropriate nucleotides of the mutated immunoglobulin
sequence are mutated back to correspond to the germline sequence,
using the genetic code to determine which nucleotide changes should
be made. Mutagenesis of the mutated immunoglobulin framework
sequence is carried out by standard methods, such as PCR-mediated
mutagenesis (in which the mutated nucleotides are incorporated into
the PCR primers such that the PCR product contains the mutations)
or site-directed mutagenesis. The role of each amino acid
identified as candidate for backmutation should be investigated for
a direct or indirect role in antigen binding and any amino acid
found after mutation to affect any desirable characteristic of the
human antibody should not be included in the final human antibody;
as an example, activity enhancing amino acids identified by the
selective mutagenesis approach will not be subject to backmutation.
Assays to determine the characteristics of the antibody resulting
from mutagenesis can include ELISA, competitive ELISA, in vitro and
in vivo neutralization assays and/or (see e.g. Example 3)
immunohistochemistry with tissue sections from various sources
(including human, primate and/or other species).
[0439] To minimize the number of amino acids subject to
backmutation those amino acid positions found to be different from
the closest germline sequence but identical to the corresponding
amino acid in a second germline sequence can remain, provided that
the second germline sequence is identical and colinear to the
sequence of the human antibody of the invention for at least 10,
preferably 12 amino acids, on both sides of the amino acid in
question. This would assure that any peptide epitope presented to
the immune system by professional antigen presenting cells in a
subject treated with the human antibody of the invention would not
be foreign but identical to a self-antigen, i.e. the immunoglobulin
encoded by that second germline sequence. Backmutation may occur at
any stage of antibody optimization; preferably, backmutation occurs
directly before or after the selective mutagenesis approach. More
preferably, backmutation occurs directly before the selective
mutagenesis approach.
III. Modifications to Preferred Selective Mutagenesis Positions,
Contact and/or Hypermutation Positions
[0440] Typically, selection of antibodies with improved affinities
can be carried out using phage display methods, as described in
section II above. This can be accomplished by randomly mutating
combinations of CDR residues and generating large libraries
containing antibodies of different sequences. However, for these
selection methods to work, the antibody-antigen reaction must tend
to equilibrium to allow, over time, preferential binding of higher
affinity antibodies to the antigen. Selection conditions that would
allow equilibrium to be established could not be determined
(presumably due to additional non-specific interactions between the
antigen and phage particle) when phage display methods were used to
improve the affinity of selected anti-IL-12 antibodies, upon
attaining a certain level of affinity achieved (i.e., that of
antibody Y61). Accordingly, antibodies with even higher affinities
could not be selected by phage display methods. Thus, for at least
certain antibodies or antigens, phage display methods are limiting
in their ability to select antibodies with a highly improved
binding specificity/affinity. Accordingly, a method termed
Selective Mutagenesis Approach which does not require phage display
affinity maturation of antibodies, was established to overcome this
limitation and is provided by the invention. Although this
Selective Mutagenesis Approach was developed to overcome
limitations using the phage display system, it should be noted that
this method can also be used with the phage display system.
Moreover, the selective mutagenesis approach can be used to improve
the activity of any antibody.
[0441] To improve the activity (e.g., affinity or neutralizing
activity) of an antibody, ideally one would like to mutate every
CDR position in both the heavy and light chains to every other
possible amino acid residue. However, since there are, on average,
70 CDR positions within an antibody, such an approach would be very
time consuming and labor intensive. Accordingly, the method of the
invention allows one to improve the activity of the antibody by
mutating only certain selected residues within the heavy and/or
light chain CDRs. Furthermore, the method of the invention allows
improvement in activity of the antibody without affecting other
desirable properties of the antibody.
[0442] Determining which amino acid residues of an antibody
variable region are in contact with an antigen cannot be accurately
predicted based on primary sequence or their positions within the
variable region. Nevertheless, alignments of sequences from
antibodies with different specificities conducted by Kabat et al.
have identified the CDRs as local regions within the variable
regions which differ significantly among antibodies (Kabat et al.
(1971) Ann. NY Acad, Sci. 190:382-393, 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). Structural studies have shown that the antigen binding
surface is formed by amino acid residues present in the CDRs. Other
amino acid residues outside the CDR are also known to play
structural roles or be directly involved in antigen binding.
Therefore, for each antigen-antibody pair, amino acid residues
within and outside of the CDRs may be important.
[0443] The sequence alignment studies by Tomlison et al identified
a number of positions in the heavy and light chain CDR1 and CDR2,
and in a portion of the kappa chain CDR3 which are frequent sites
of somatic mutation. (Tomlison et al (1996) J. Mol. Biol. 256:
813-817). In particular, positions H31, H31B, H33, H33B, H.sub.52B,
H56, H58, L30, L31, L31A, L50, L53, L91, L92, L93 and L94 were
identified as frequent sites for somatic mutation. However, this
analysis excludes the important heavy chain CDR3 regions, and
sections of the light chain CDR3 which are known to lie in the
center of an antibody binding site, and potentially provide
important interactions with an antigen. Furthermore, Tomlison et
al. propose that somatic diversity alone does not necessarily
predict a role of a specific amino acid in antigen binding, and
suggest conserved amino acid residues that contact the antigen, and
diverse amino acid residues which do not contact the antigen. This
conclusion is further supported by mutational studies on the role
of somatic mutations to antibody affinity (Sharon, (1990), PNAS,
87:4814-7). Nineteen somatic mutations in a high-affinity
anti-p-azophenylarsonate (Ars) antibody were simultaneously
replaced with their corresponding germline residues, generating a
germline version of the anti-Ars antibody which had a two-hundred
fold loss in activity. The full affinity of the anti-Ars antibody
could be recovered by restoring only three of the nineteen somatic
mutations, demonstrating that many somatic mutations may be
permitted that do not contribute to antigen binding activity.
[0444] The result can be explained in part by the nature of
antibody diversity itself. Immature B-cells may produce initially
low affinity antibodies that recognize a number of self or non-self
antigens. Moreover, antibodies may undergo in the course of
affinity maturation sequence variations that may cause
self-reactivity. Hypermutation of such low affinity antibodies may
serve to abolish self-reactivity ("negative selection") and
increase affinity for the foreign antigen. Therefore, the analysis
of primary and structural data of a large number of antibodies does
not provide a method of predicting either (1) the role of somatic
hyper-mutation sites in the affinity maturation process versus the
process of decreasing affinity towards unwanted antigens, or (2)
how a given amino acid contributes to the properties of a specific
antigen-antibody pair.
[0445] Other attempts to address the role of specific amino acid
residues in antigen recognition were made by analyzing a number of
crystal structures of antigen-antibody complexes (MacCallum et al.
(1996) J. Mol. Biol. 262: 732-745). The potential role of positions
located within and outside the CDRs was indicated. Positions in
CDRs involved in antigen binding in more than 10 of 26 analyzed
structures included H31, H33, H50, H52, H53, H54, H56, H58, H95,
H96, H97, H98 and H100 in the heavy chain and L30A, L32, L91, L92,
L93, L94, L96 in the light chain. However, the authors noted that
prediction of antigen contacts using these and other structural
data may over and under predict contact positions, leading to the
speculation that a different strategy may have to be applied to
different antigens.
[0446] Pini et al. describe randomizing multiple residues in
antibody CDR sequences in a large phage display library to rapidly
increase antibody affinity (Pini et al. (1998) J. Biol. Chem. 273:
21769-21776). However, the high affinity antibodies discussed by
Pini et al. had mutations in a total of eight positions, and a
reductionary analysis of which changes are absolutely required to
improve affinity of the antibody becomes impractical because of the
large number of possible combinations to be tested for the smallest
number of amino acids required.
[0447] Furthermore, randomizing multiple residues may not
necessarily preserve other desired properties of the antibody.
Desirable properties or characteristics of an antibody are
art-recognized and include for example, preservation of non-cross
reactivity, e.g., with other proteins or human tissues and
preservation of antibody sequences that are close to human germline
immunoglobulin sequences improvement of neutralization potency.
Other desirable properties or characteristics include ability to
preserve species cross reactivity, ability to preserve epitope
specificity and ability to preserve high expression levels of
protein in mammalian cells. The desirable properties or
characteristics can be observed or measured using art-recognized
techniques including but not limited to ELISA, competitive ELISA,
in vitro and in vivo neutralization assays (see e.g. Example 3),
immunohistochemistry with tissue sections from different sources
including human, primate or other sources as the need may be, and
studies to expression in mammalian cells using transient expression
or stable expression.
[0448] In addition, the method of Pini et al may introduce more
changes than the minimal number actually required to improve
affinity and may lead to the antibodies triggering
anti-human-antibody (HAMA) formation in human subjects. Further, as
discussed elsewhere, the phage display as demonstrated here, or
other related method including ribosome display may not work
appropriately upon reaching certain affinities between antibody and
antigen and the conditions required to reach equilibrium may not be
established in a reasonable time frame because of additional
interactions including interactions with other phage or ribosome
components and the antigen.
[0449] The ordinarily skilled artisan may glean interesting
scientific information on the origin of antibody diversity from the
teachings of the references discussed above. The present invention,
however, provides a method for increasing antibody affinity of a
specific antigen-antibody pair while preserving other relevant
features or desirable characteristics of the antibody. This is
especially important when considering the desirability of imparting
a multitude of different characteristics on a specific antibody
including antigen binding.
[0450] If the starting antibody has desirable properties or
characteristics which need to be retained, a selective mutagenesis
approach can be the best strategy for preserving these desirable
properties while improving the activity of the antibody. For
example, in the mutagenesis of Y61, the aim was to increase
affinity for hIL-12, and to improve the neutralization potency of
the antibody while preserving desired properties. Desired
properties of Y61 included (1) preservation of non-cross reactivity
with other proteins or human tissues, (2) preservation of fine
epitope specificity, i.e. recognizing a p40 epitope preferably in
the context of the p70 (p40/p35) heterodimer, thereby preventing
binding interference from free soluble p40; and (3) generation of
an antibody with heavy and light chain amino acid sequences that
were as close as possible to their respective germline
immunoglobulin sequences.
[0451] In one embodiment, the method of the invention provides a
selective mutagenesis approach as a strategy for preserving the
desirable properties or characteristics of the antibody while
improving the affinity and/or neutralization potency. The term
"selective mutagenesis approach" is as defined above and includes a
method of individually mutating selected amino acid residues. The
amino acid residues to be mutated may first be selected from
preferred selective mutagenesis positions, then from contact
positions, and then from hypermutation positions. The individual
selected position can be mutated to at least two other amino acid
residue and the effect of the mutation both on the desired
properties of the antibody, and improvement in antibody activity is
determined.
[0452] The Selective Mutagenesis approach comprises the steps
of:
[0453] selecting candidate positions in the order 1) preferred
selective mutagenesis positions; 2) contact positions; 3)
hypermutation positions and ranking the positions based on the
location of the position within the heavy and light chain variable
regions of an antibody (CDR3 preferred over CDR2 preferred over
CDR1);
[0454] individually mutating candidate preferred selective
mutagenesis positions, hypermutation and/or contact positions in
the order of ranking, to all possible other amino acid residues and
analyzing the effect of the individual mutations on the activity of
the antibody in order to determine activity enhancing amino acid
residues;
[0455] if necessary, making stepwise combinations of the individual
activity enhancing amino acid residues and analyzing the effect of
the various combinations on the activity of the antibodies;
selecting mutant antibodies with activity enhancing amino acid
residues and ranking the mutant antibodies based on the location
and identity of the amino acid substitutions with regard to their
immunogenic potential. Highest ranking is given to mutant
antibodies that comprise an amino acid sequence which nearly
identical to a variable region sequence that is described in a
germline database, or has an amino acid sequence that is comparable
to other human antibodies. Lower ranking is given to mutant
antibodies containing an amino acid substitution that is rarely
encountered in either germline sequences or the sequences of other
human antibodies. The lowest ranking is given to mutant antibodies
with an amino acid substitution that has not been encountered in a
germline sequence or the sequence of another human antibody. As set
forth above, mutant antibodies comprising at least one activity
enhancing amino acid residue located in CDR3 is preferred over CDR2
which is preferred over CDR1. The CDRs of the heavy chain variable
regions are preferred over those of the light chain variable
region.
[0456] The mutant antibodies can also be studied for improvement in
activity, e.g. when compared to their corresponding parental
antibody. The improvement in activity of the mutant antibody can be
determined for example, by neutralization assays, or binding
specificity/affinity by surface plasmon resonance analysis (see
Example 3). Preferably, the improvement in activity can be at least
2-20 fold higher than the parental antibody. The improvement in
activity can be at least "x.sub.1" to "x.sub.2" fold higher than
the parental antibody wherein "x.sub.1" and "x.sub.2" are integers
between and including 2 to 20, including ranges within the state
range, e.g. 2-15, e.g. 5-10.
[0457] The mutant antibodies with the activity enhancing amino acid
residue also can be studied to determine whether at least one other
desirable property has been retained after mutation. For example,
with anti-hIL-12 antibodies testing for, (1) preservation of
non-cross reactivity with other proteins or human tissues, (2)
preservation of epitope recognition, i.e. recognizing a p40 epitope
preferably in the context of the p70 (p40/p35) heterodimer, thereby
preventing binding interference from free soluble p40; and (3)
generation of antibodies with heavy and light chain amino acid
sequences that were as close as possible to their respective
germline immunoglobulin sequences, and determining which would be
least likely to elicit a human immune response based on the number
of differences from the germline sequence. The same observations
can be made on an antibody having more than one activity enhancing
amino acid residues, e.g. at least two or at least three activity
enhancing amino acid residues, to determine whether retention of
the desirable property or characteristic has occurred.
[0458] An example of the use of a "selective mutagenesis approach",
in the mutagenesis of Y61 is described below. The individual
mutations H31S.fwdarw.E, L50.fwdarw.Y, or L94G.fwdarw.Y each
improved neutralization activity of the antibody. However, when
combination clones were tested, the activity of the combined clone
H31S.fwdarw.E+L50.fwdarw.Y+L94G.fwdarw.Y was no better than
L50.fwdarw.Y+L94G.fwdarw.Y (J695). Therefore, changing the germline
amino acid residue Ser to Glu at position 31 of CDR1 was
unnecessary for the improved activity of J695 over Y61. The
selective mutagenesis approach therefore, identified the minimal
number of changes that contributed to the final activity, thereby
reducing the immunogenic potential of the final antibody and
preserving other desired properties of the antibody.
[0459] Isolated DNA encoding the VH and VL produced by the selected
mutagenesis approach can be converted into full length antibody
chain genes, to Fab fragment genes as to a scFV gene, as described
in section IV. For expression of VH and VL regions produced by the
selected mutagenesis approach, expression vectors encoding the
heavy and light chain can be transfected into variety host cells as
described in detail in section IV. Preferred host cells include
either prokaryotic host cells, for example, E. coli, or eukaryotic
host cells, for example, yeast cells, e.g., S. cerevisae. Most
preferred eukaryotic host cells are mammalian host cells, described
in detail in section IV.
[0460] The selective mutagenesis approach provides a method of
producing antibodies with improved activities without prior
affinity maturation of the antibody by other means. The selective
mutagenesis approach provides a method of producing antibodies with
improved affinities which have been subject to back mutations. The
selective mutagenesis approach also provides a method of improving
the activity of affinity matured antibodies.
[0461] The skilled artisan will recognize that the selective
mutagenesis approach can be used in standard antibody manipulation
techniques known in the art. Examples include, but are not limited
to, CDR grafted antibodies, chimeric antibodies, scFV fragments,
Fab fragments of a full length antibodies and human antibodies from
other sources, e.g., transgenic mice.
[0462] Rapid large scale mutational analysis of antibodies include
in vitro transcription and translation using ribosome display
technology (see e.g., Hanes et al., (1997) Proc. Natl. Acad. Sci.
94: 4937-4942; Dall Acqua et al., (1998) Curr. Opin. Struc. Biol.
8: 443-450; He et al., (1997) Nucleic Acid Res. 25: 5132-5134), and
U.S. Pat. Nos. 5,643,768 and 5,658,754 issued to Kawasaki. The
selective mutagenesis approach also provides a method of producing
antibodies with improved activities that can be selected using
ribosomal display techniques.
[0463] In the methods of the invention, antibodies or antigen
binding portions thereof are further modified by altering
individual positions in the CDRs of the HCVR and/or LCVR. Although
these modifications can be made in phage-displayed antibodies, the
method is advantageous in that it can be performed with antibodies
that are expressed in other types of host systems, such as
bacterial, yeast or mammalian cell expression systems. The
individual positions within the CDRs selected for modification are
based on the positions being a contact and/or hypermutation
position.
[0464] Preferred contact positions and hypermutation positions as
defined herein are shown in Table 3 (see Appendix A) and their
modification in accordance with the method of the invention is
described in detail in Example 2. Preferred contact positions are
selected from the group consisting of H30, H31, H31B, H32, H33,
H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101,
L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96.
Preferred hypermutation positions are selected from the group
consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32,
L53 and L93. More preferred amino acid residues (referred to as
"preferred selective mutagenesis positions") are both contact and
hypermutation positions and are selected from the group consisting
of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50,
L91, L92, L93, L94. Particularly preferred contact positions are
selected from the group consisting of L50 and L94.
[0465] Preferred activity enhancing amino acid residues replace
amino acid residues located at positions selected from the group
consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,
H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50,
L52, L53, L55, L91, L92, L93, L94, and L96. More preferred activity
enhancing amino acid residues replace amino acid residues located
at positions H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,
L32, L50, L91, L92, L93, L94. Particularly, preferred activity
enhancing amino acid residues replace amino acid residues located
at positions selected from the group consisting of L50 and L94.
[0466] In general, the method of the invention involves selecting a
particular preferred selective mutagenesis position, contact and/or
hypermutation position within a CDR of the heavy or light chain of
a parent antibody of interest, or antigen binding portion thereof,
randomly mutagenizing that individual position (e.g., by genetic
means using a mutagenic oligonucleotide to generate a
"mini-library" of modified antibodies), or mutating a position to
specific desired amino acids, to identify activity enhancing amino
acid residues expressing, and purifying the modified antibodies
(e.g., in a non-phage display host system), measuring the activity
of the modified antibodies for antigen (e.g., by measuring
k.sub.off rates by BIAcore analysis), repeating these steps for
other CDR positions, as necessary, and combining individual
mutations shown to have improved activity and testing whether the
combination(s) generate an antibody with even greater activity
(e.g., affinity or neutralizing potency) than the parent antibody,
or antigen-binding portion thereof.
[0467] Accordingly, in one embodiment, the invention provides a
method for improving the activity of an antibody, or
antigen-binding portion thereof, comprising:
[0468] a) providing a parent antibody or antigen-binding portion
thereof;
[0469] b) selecting in order a 1) preferred selective mutagenesis
position, 2) contact position, or 3) hypermutation position within
a complementarity determining region (CDR) for mutation, thereby
identifying a selected preferred selective mutagenesis position,
contact or hypermutation position;
[0470] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;
[0471] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof;
[0472] e) optionally, repeating steps a) through d) for at least
one other preferred selective mutagenesis position, contact or
hypermutation position;
[0473] f) combining, in the parent antibody, or antigen-binding
portion thereof, individual mutations shown to have improved
activity, to form combination antibodies, or antigen-binding
portions thereof; and
[0474] g) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof; until an antibody, or
antigen-binding portion thereof, with an improved activity,
relative to the parent antibody, or antigen-binding portion
thereof, is obtained. Preferably, the selected antibody or
antibodies have an improved activity without loss or with retention
of at least one desirable characteristic or property of the
parental antibody as described above. The desirable characteristic
or property can be measured or observed by the ordinarily skilled
artisan using art-recognized techniques.
[0475] Preferred contact positions are selected from the group
consisting of H30, H31, H.sub.31B, H32, H33, H35, H50, H52, H52A,
H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34,
L50, L52, L53, L55, L91, L92, L93, L94 and L96. Preferred
hypermutation positions are selected from the group consisting of
H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93.
More preferred selective mutagenesis positions are selected from
the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58,
L30, L31, L32, L50, L91, L92, L93 and L94. Particularly preferred
contact positions are selected from the group consisting of L50 and
L94.
[0476] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0477] a) providing a parent antibody or antigen-binding portion
thereof;
[0478] b) selecting a preferred selective mutagenesis position,
contact or hypermutation position within a complementarity
determining region (CDR) for mutation;
[0479] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;
[0480] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof, thereby
identifying an activity enhancing amino acid residue;
[0481] e) optionally, repeating steps a) through d) for at least
one other preferred selective mutagenesis position, contact or
hypermutation position;
[0482] f) combining, in the parent antibody, or antigen-binding
portion thereof, two individual activity enhancing amino acid
residues shown to have improved activity, to form combination
antibodies, or antigen-binding portions thereof; and
[0483] g) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino
acid residues, relative to the parent antibody or antigen-binding
portion thereof;
until an antibody, or antigen-binding portion thereof, with an
improved activity, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.
[0484] Preferred contact positions are selected from the group
consisting of H30, H31, H.sub.31B, H32, H33, H35, H50, H52, H52A,
H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34,
L50, L52, L53, L55, L91, L92, L93, L94 and L96. Preferred
hypermutation positions are selected from the group consisting of
H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93.
More preferred selective mutagenesis positions are selected from
the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58,
L30, L31, L32, L50, L91, L92, L93 and L94. Particularly preferred
contact positions are selected from the group consisting of L50 and
L94.
[0485] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0486] a) providing a parent antibody or antigen-binding portion
thereof;
[0487] b) selecting a preferred selective mutagenesis position,
contact or hypermutation position within a complementarity
determining region (CDR) for mutation;
[0488] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;
[0489] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof, thereby
identifying an activity enhancing amino acid residue;
[0490] e) optionally, repeating steps a) through d) for at least
one other preferred selective mutagenesis position, contact or
hypermutation position;
[0491] f) combining, in the parent antibody, or antigen-binding
portion thereof, three individual activity enhancing amino acid
residues shown to have improved activity, to form combination
antibodies, or antigen-binding portions thereof; and
[0492] g) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino
acid residues, relative to the parent antibody or antigen-binding
portion thereof;
until an antibody, or antigen-binding portion thereof, with an
improved activity, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.
[0493] Preferably, the activity enhancing amino acid residue
replaces amino acid residues located at positions selected from the
group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A,
H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34,
L50, L52, L53, L55, L91, L92, L93, L94 and L96.
[0494] Following mutagenesis of individual selected positions,
mutated clones can be sequenced to identify which amino acid
residues have been introduced into the selected position in each
clone. A small number of clones (e.g., about 24) can be selected
for sequencing, which statistically should yield 10-15 unique
antibodies, whereas larger numbers of clones (e.g., greater than
60) can be sequenced to ensure that antibodies with every possible
substitution at the selected position are identified.
[0495] In one embodiment, contact and/or hypermutation positions
within the CDR3 regions of the heavy and/or light chains are first
selected for mutagenesis. However, for antibodies that have already
been affinity matured in vitro by random mutagenesis of the CDR3
regions via phage display selection, it may be preferably to first
select contact and/or hypermutation positions within CDR1 or CDR2
of the heavy and/or light chain.
[0496] In a more preferred embodiment, preferred selective
mutagenesis positions within the CDR3 regions of the heavy and/or
light chains are first selected for mutagenesis. However, for
antibodies that have already been affinity matured in vitro by
random mutagenesis of the CDR3 regions via phage display selection,
it may be preferably to first select preferred selective
mutagenesis positions within CDR1 or CDR2 of the heavy and/or light
chain.
[0497] In another preferred embodiment, the optimization of a
selected antibody by the selective mutagenesis approach is done
sequentially as follows: preferred selective mutagenesis positions
selected from the group consisting of H30, H31, H31B, H32, H33,
H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 are mutated
first to at least 2 other amino acids each (preferably 5-14 other
amino acids) and the resulting antibodies are characterized for
increased affinity, neutralization potency (and possibly also for
at least one other retained characteristic or property discussed
elsewhere). If a mutation of a single preferred selective
mutagenesis position does not increase the affinity or
neutralization potency at all or sufficiently and if even the
combination of multiple activity enhancing amino acids replacing
amino acids in preferred selective mutagenesis positions does not
result in an combination antibody which meets the target activity
(including affinity and/or neutralization potency), additional
amino acid residues will be selected for selective mutagenesis from
the group consisting of H35, H50, H53, H54, H95, H96, H97, H98,
L30A and L96 are mutated to at least 2 other amino acids each
(preferably 5-14 other amino acids) and the resulting antibodies
are characterized for increased affinity, neutralization potency
(and possibly also for at least one other retained characteristic
or property discussed elsewhere).
[0498] If a mutation of a single amino acid residue selected from
the group consisting of H35, H50, H53, H54, H95, H96, H97, H98,
L30A and L96 does not increase the activity (including affinity
and/or neutralization potency) at all or not sufficiently and if
even the combination of multiple activity enhancing amino acids
replacing amino acids in those positions does not result in an
combination antibody which meets the targeted activity (including
affinity and/or target neutralization potency), additional amino
acid residues will be selected for selective mutagenesis from the
group consisting of H33B, H52B, L31A and are mutated to at least 2
other amino acids each (preferably 5-14 other amino acids) and the
resulting antibodies are characterized for increased affinity,
neutralization potency (and possibly also for at least one other
retained characteristic or property discussed elsewhere).
[0499] It should be understood that the sequential selective
mutagenesis approach may end at any of the steps outline above as
soon as an antibody with the desired activity (including affinity
and neutralization potency) has been identified. If mutagenesis of
the preselected positions has identified activity enhancing amino
acids residues but the combination antibody still do not meet the
targets set for activity (including affinity and neutralization
potency) and/or if the identified activity enhancing amino acids
also affect other desired characteristics and are therefore not
acceptable, the remaining CDR residues may be subjected to
mutagenesis (see section IV).
[0500] The method of the invention can be used to improve activity
of an antibody, or antigen binding portion thereof, to reach a
predetermined target activity (e.g. a predetermined affinity and/or
neutralization potency, and/or a desired property or
characteristic).
[0501] Accordingly, the invention provides a method of improving
the activity of an antibody, or antigen-binding portion thereof, to
attain a predetermined target activity, comprising:
[0502] a) providing a parent antibody a antigen-binding portion
thereof;
[0503] b) selecting a preferred selective mutagenesis position
selected from group consisting of H30, H31, H31B, H32, H33, H52,
H56, H58, L30, L31, L32, L50, L91, L92, L93, L94.
[0504] c) individually mutating the selected preferred selective
mutagenesis position to at least two other amino acid residues to
hereby create a first panel of mutated antibodies, or antigen
binding portions thereof;
[0505] d) evaluating the activity of the first panel of mutated
antibodies, or antigen binding portions thereof to determined if
mutation of a single selective mutagenesis position produces an
antibody or antigen binding portion thereof with the predetermined
target activity or a partial target activity;
[0506] e) combining in a stepwise fashion, in the parent antibody,
or antigen binding portion thereof, individual mutations shown to
have an improved activity, to form combination antibodies, or
antigen binding portions thereof.
[0507] f) evaluating the activity of the combination antibodies, or
antigen binding portions thereof to determined if the combination
antibodies, or antigen binding portions thereof have the
predetermined target activity or a partial target activity.
[0508] g) if steps d) or f) do not result in an antibody or antigen
binding portion thereof having the predetermined target activity,
or result an antibody with only a partial activity, additional
amino acid residues selected from the group consisting of H35, H50,
H53, H54, H95, H96, H97, H98, L30A and L96 are mutated to at least
two other amino acid residues to thereby create a second panel of
mutated antibodies or antigen-binding portions thereof;
[0509] h) evaluating the activity of the second panel of mutated
antibodies or antigen binding portions thereof, to determined if
mutation of a single amino acid residue selected from the group
consisting of H35, H50, H53, H54, H95, H96, H97, H98, L30A and L96
results an antibody or antigen binding portion thereof, having the
predetermined target activity or a partial activity;
[0510] i) combining in stepwise fashion in the parent antibody, or
antigen-binding portion thereof, individual mutations of step g)
shown to have an improved activity, to form combination antibodies,
or antigen binding portions thereof;
[0511] j) evaluating the activity of the combination antibodies or
antigen binding portions thereof, to determined if the combination
antibodies, or antigen binding portions thereof have the
predetermined target activity or a partial target activity;
[0512] k) if steps h) or j) do not result in an antibody or antigen
binding portion thereof having the predetermined target activity,
or result in an antibody with only a partial activity, additional
amino acid residues selected from the group consisting of H33B,
H52B and L31A are mutated to at least two other amino acid residues
to thereby create a third panel of mutated antibodies or antigen
binding portions thereof;
[0513] l) evaluating the activity of the third panel of mutated
antibodies or antigen binding portions thereof, to determine if a
mutation of a single amino acid residue selected from the group
consisting of H33B, H52B and L31A resulted in an antibody or
antigen binding portion thereof, having the predetermined target
activity or a partial activity;
[0514] m) combining in a stepwise fashion in the parent antibody,
or antigen binding portion thereof, individual mutation of step k)
shown to have an improved activity, to form combination antibodies,
or antigen binding portions, thereof,
[0515] n) evaluating the activity of the combination antibodies or
antigen-binding portions thereof, to determine if the combination
antibodies, or antigen binding portions thereof have the
predetermined target activity to thereby produce an antibody or
antigen binding portion thereof with a predetermined target
activity.
[0516] A number of mutagenesis methods can be used, including PCR
assembly, Kunkel (dut-ung-) and thiophosphate (Amersham Sculptor
kit) oligonucleotide-directed mutagenesis.
[0517] A wide variety of host expression systems can be used to
express the mutated antibodies, including bacterial, yeast,
baculoviral and mammalian expression systems (as well as phage
display expression systems). An example of a suitable bacterial
expression vector is pUC119(Sfi). Other antibody expression systems
are known in the art and/or are described below in section IV.
[0518] The modified antibodies, or antigen binding portions
thereof, produced by the method of the invention can be identified
without the reliance on phage display methods for selection.
Accordingly, the method of the invention is particularly
advantageous for improving the activity of a recombinant parent
antibody or antigen-binding portion thereof, that was obtained by
selection in a phage-display system but whose activity cannot be
further improved by mutagenesis in the phage-display system.
[0519] Accordingly, in another embodiment, the invention provides a
method for improving the affinity of an antibody, or
antigen-binding portion thereof, comprising:
[0520] a) providing a recombinant parent antibody or
antigen-binding portion thereof; that was obtained by selection in
a phage-display system but whose activity cannot be further
improved by mutagenesis in said phage-display system;
[0521] b) selecting a preferred selective mutagenesis position,
contact or hypermutation position within a complementarity
determining region (CDR) for mutation, thereby identifying a
selected contact or hypermutation position;
[0522] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof, and expressing
said panel in a non-phage display system;
[0523] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof;
[0524] e) optionally repeating steps b) through d) for at least one
other preferred selective mutagenesis position, contact or
hypermutation position;
[0525] f) combining, in the parent antibody, or antigen-binding
portion thereof, individual mutations shown to have improved
activity, to form combination antibodies, or antigen-binding
portions thereof; and
[0526] g) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof; until an antibody, or
antigen-binding portion thereof, with an improved activity,
relative to the parent antibody, or antigen-binding portion
thereof, is obtained.
[0527] Preferred contact positions are selected from the group
consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,
H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50,
L52, L53, L55, L91, L92, L93, L94 and L96. Preferred hypermutation
positions are selected from the group consisting of H30, H31, H31B,
H32, H52, H56, H58, L30, L31, L32, L53 and L93. More preferred
selective mutagenesis positions are selected from the group
consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,
L32, L50, L91, L92, L93 and L94. Particularly preferred contact
positions are selected from the group consisting of L50 and
L94.
[0528] With available methods it is not possible or it is extremely
laborious to derive an antibody with increased binding affinity and
neutralization potency while retaining other properties or
characteristics of the antibodies as discussed above. The method of
this invention, however, can readily identify such antibodies. The
antibodies subjected to the method of this invention can come from
any source.
[0529] Therefore, in another embodiment, the invention provides a
method for improving the activity of an antibody, or
antigen-binding portion thereof, comprising:
[0530] a) providing a recombinant parent antibody or
antigen-binding portion thereof
[0531] b) selecting a preferred selective mutagenesis position,
contact or hypermutation position within a complementarity
determining region (CDR) for mutation, thereby identifying a
selected preferred selective mutagenesis position, contact or
hypermutation position;
[0532] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expressing said
panel in an appropriate expression system;
[0533] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof, thereby
identifying an activity enhancing amino acid residue;
[0534] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof for at least one other property
or characteristics, wherein the property or characteristic is one
that needs to be retained in the antibody; until an antibody, or
antigen-binding portion thereof, with an improved activity and at
least one retained property or characteristic, relative to the
parent antibody, or antigen-binding portion thereof, is
obtained.
[0535] In a preferred embodiment, the contact positions are
selected from the group consisting of H30, H31, H31B, H32, H33,
H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101,
L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96
and the other characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0536] In another preferred embodiment, the hypermutation positions
are selected from the group consisting of H30, H31, H31B, H32, H52,
H56, H58, L30, L31, L32, L53 and L93 and the other characteristic
is selected from 1) preservation of non-crossreactivity with other
proteins or human tissues, 2) preservation of epitope recognition,
i.e. recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence.
[0537] In a more preferred embodiment the residues for selective
mutagenesis are selected from the preferred selective mutagenesis
positions from the group consisting of H30, H31, H31B, H32, H33,
H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other
characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0538] In a more preferred embodiment, the contact positions are
selected from the group consisting of L50 and L94 and the other
characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0539] If therefore, the affinity of an antibody for a specific
antigen should be improved, but where the phage display (or related
system including ribosome display) method is no longer applicable,
and other desirable properties or characteristics should be
retained, the method of the invention can be used. Accordingly, in
another embodiment, the invention provides a method for improving
the activity of an antibody, or antigen-binding portion thereof,
comprising:
[0540] a) providing a recombinant parent antibody or
antigen-binding portion thereof; that was obtained by selection in
a phage-display system but whose activity cannot be further
improved by mutagenesis in said phage-display system;
[0541] b) selecting a preferred selective mutagenesis position,
contact or hypermutation position within a complementarity
determining region (CDR) for mutation, thereby identifying a
selected preferred selective mutagenesis position, contact or
hypermutation position;
[0542] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof, and expressing
said panel in a non-phage display system;
[0543] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0544] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof for at least one other property
or characteristic, wherein the property or characteristic is one
that needs to be retained, until an antibody, or antigen-binding
portion thereof, with an improved activity and at least one
retained property or characteristic, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.
[0545] f) optionally, repeating steps a) through e) for at least
one other preferred selective mutagenesis position, contact or
hypermutation position;
[0546] g) combining, in the parent antibody, or antigen-binding
portion thereof, at least two individual activity enhancing amino
acid residues shown to have improved activity and at least one
retained property or characteristic, to form combination
antibodies, or antigen-binding portions thereof; and
[0547] h) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof; until an antibody, or
antigen-binding portion thereof, with an improved activity and at
least one retained other property or characteristic, relative to
the parent antibody, or antigen-binding portion thereof, is
obtained.
[0548] In a preferred embodiment, the contact positions are
selected from the group consisting of H30, H31, H31B, H32, H33,
H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101,
L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96
and the other characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0549] In another preferred embodiment, the hypermutation positions
are selected from the group consisting of H30, H31, H31B, H32, H52,
H56, H58, L30, L31, L32, L53 and L93 and the other characteristic
is selected from 1) preservation of non-crossreactivity with other
proteins or human tissues, 2) preservation of epitope recognition,
i.e. recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence.
[0550] In a more preferred embodiment the residues for selective
mutagenesis are selected from the preferred selective mutagenesis
positions from the group consisting of H30, H31, H31B, H32, H33,
H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other
characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0551] In a more preferred embodiment, the contact positions are
selected from the group consisting of L50 and L94 and the other
characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0552] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0553] a) providing a recombinant parent antibody or
antigen-binding portion thereof; that was obtained by selection in
a phage-display system but whose activity cannot be further
improved by mutagenesis in said phage-display system;
[0554] b) selecting a preferred selective mutagenesis position,
contact or hypermutation position within a complementarity
determining region (CDR) for mutation, thereby identifying a
selected contact or hypermutation position;
[0555] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof, and expressing
said panel in a non-phage display system;
[0556] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0557] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof for at least one other property
or characteristic, wherein the property or characteristic is one
that needs to be retained, until an antibody, or antigen-binding
portion thereof, with an improved activity and at least one
retained property or characteristic, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.
[0558] In a preferred embodiment, the contact positions are
selected from the group consisting of H30, H31, H31B, H32, H33,
H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101,
L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96
and the other characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0559] In another preferred embodiment, the hypermutation positions
are selected from the group consisting of H30, H31, H31B, H32, H52,
H56, H58, L30, L31, L32, L53 and L93 and the other characteristic
is selected from 1) preservation of non-crossreactivity with other
proteins or human tissues, 2) preservation of epitope recognition,
i.e. recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence.
[0560] In a more preferred embodiment the residues for selective
mutagenesis are selected from the preferred selective mutagenesis
positions from the group consisting of H30, H31, H31B, H32, H33,
H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other
characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0561] In a more preferred embodiment, the contact positions are
selected from the group consisting of L50 and L94 and the other
characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0562] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:
[0563] a) providing a recombinant parent antibody or
antigen-binding portion thereof; that was obtained by selection in
a phage-display system but whose activity cannot be further
improved by mutagenesis in said phage-display system;
[0564] b) selecting a preferred selective mutagenesis position,
contact or hypermutation position within a complementarity
determining region (CDR) for mutation, thereby identifying a
selected contact or hypermutation position;
[0565] c) individually mutating said selected preferred selective
mutagenesis positions, contact or hypermutation position to at
least two other amino acid residues to thereby create a panel of
mutated antibodies, or antigen-binding portions thereof, and
expressing said panel in a non-phage display system;
[0566] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0567] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof for at least one other property
or characteristic, wherein the property or characteristic is one
that needs to be retained, until an antibody, or antigen-binding
portion thereof, with an improved activity and at least one
retained characteristic, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.
[0568] f) optionally, repeating steps a) through e) for at least
one other preferred selective mutagenesis position, contact or
hypermutation position;
[0569] g) combining, in the parent antibody, or antigen-binding
portion thereof, at least two individual activity enhancing amino
acid residues shown to have improved activity and at least on
retained other characteristic, to form combination antibodies, or
antigen-binding portions thereof; and
[0570] h) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof; until an antibody, or
antigen-binding portion thereof, with an improved activity and at
least one retained property or characteristic, relative to the
parent antibody, or antigen-binding portion thereof, is
obtained.
[0571] In a preferred embodiment, the contact positions are
selected from the group consisting of H30, H31, H31B, H32, H33,
H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101,
L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96
and the other characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0572] In another preferred embodiment, the hypermutation positions
are selected from the group consisting of H30, H31, H31B, H32, H52,
H56, H58, L30, L31, L32, L53 and L93 and the other characteristic
is selected from 1) preservation of non-crossreactivity with other
proteins or human tissues, 2) preservation of epitope recognition,
i.e. recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence.
[0573] In a more preferred embodiment the residues for selective
mutagenesis are selected from the preferred selective mutagenesis
positions from the group consisting of H30, H31, H31B, H32, H33,
H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other
characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
[0574] In a more preferred embodiment, the contact positions are
selected from the group consisting of L50 and L94 and the other
characteristic is selected from 1) preservation of
non-crossreactivity with other proteins or human tissues, 2)
preservation of epitope recognition, i.e. recognizing p40 epitope
preferably in the context of the p70 p40/p35 heterodimer preventing
binding interference from free, soluble p40 and/or 3) to produce an
antibody with a close to germline immunoglobulin sequence.
IV. Modifications of Other CDR Residues
[0575] Ultimately, all CDR residues in a given antibody-antigen
pair identified by any means to be required as activity enhancing
amino acid residues and/or required directly or indirectly for
binding to the antigen and/or for retaining other desirable
properties or characteristics of the antibody. Such CDR residues
are referred to as "preferred selective mutagenesis positions". It
should be noted that in specific circumstances that preferred
selective mutagenesis residues can be identified also by other
means including co-crystallization of antibody and antigen and
molecular modeling.
[0576] If the preferred attempts to identify activity enhancing
amino acids focussing on the preferred selective mutagenesis
positions, contact or hypermutation positions described above are
exhausted, or if additional improvements are required, the
remaining CDR residues may be modified as described below. It
should be understood that the antibody could already be modified in
any one or more contact or hypermutation positions according to the
embodiments discussed above but may require further improvements.
Therefore, in another embodiment, the invention provides a method
for improving the activity of an antibody, or antigen-binding
portion thereof, comprising:
[0577] a) providing a parent antibody or antigen-binding portion
thereof;
[0578] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and L96;
[0579] c) individually mutating said selected position e.g., to at
least two other amino acid residues to thereby create a mutated
antibody or a panel of mutated antibodies, or antigen-binding
portions thereof;
[0580] d) evaluating the activity of the mutated antibody or the
panel of mutated antibodies, or antigen-binding portions thereof,
relative to the parent antibody or antigen-binding portion thereof
thereby identifying an activity enhancing amino acid residue;
[0581] e) evaluating the mutated antibody or the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof, for changes in
at least one other property or characteristic until an antibody, or
antigen-binding portion thereof, with an improved activity,
relative to the parent antibody, or antigen-binding portion
thereof, is obtained.
[0582] Preferably, the other characteristic or property is selected
from 1) preservation of non-crossreactivity with other proteins or
human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence
[0583] If mutagenesis of a single residue is not sufficient other
residues can be included; therefore, in another embodiment, the
invention provides a method for improving the activity of an
antibody, or antigen-binding portion thereof, comprising:
[0584] a) providing a parent antibody or antigen-binding portion
thereof;
[0585] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and L96;
[0586] c) individually mutating said selected position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;
[0587] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof, thereby
identifying an activity enhancing amino acid residue;
[0588] e) repeating steps b) through d) for at least one other CDR
position which is neither the position selected under b) nor a
position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,
H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50,
L52, L53, L55, L91, L92, L93, L94 and L96;
[0589] f) combining, in the parent antibody, or antigen-binding
portion thereof, at least two individual activity enhancing amino
acid residues shown to have improved activity, to form combination
antibodies, or antigen-binding portions thereof; and
[0590] g) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino
acid residues, relative to the parent antibody or antigen-binding
portion thereof until an antibody, or antigen-binding portion
thereof, with an improved activity, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.
[0591] If the preferred attempts to identify activity enhancing
amino acids focussing on the contact or hypermutation positions
described above are exhausted, or if additional improvements are
required, and the antibody in question can not further be optimized
by mutagenesis and phage display (or related ribosome display)
methods the remaining CDR residues may be modified as described
below. It should be understood that the antibody could already be
modified in any one or more preferred selective mutagenesis
position, contact or hypermutation positions according to the
embodiments discussed above but may require further
improvements.
[0592] Therefore, in another embodiment, the invention provides a
method for improving the activity of an antibody, or
antigen-binding portion thereof, comprising:
[0593] a) providing a recombinant parent antibody or
antigen-binding portion thereof; that was obtained by selection in
a phage-display system but whose activity cannot be further
improved by mutagenesis in said phage-display system;
[0594] b) selecting a selecting an amino acid residue within a
complementarity determining region (CDR) for mutation other than
H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58,
H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55,
L91, L92, L93, L94 and;
[0595] c) individually mutating said selected contact or
hypermutation position to at least two other amino acid residues to
thereby create a panel of mutated antibodies, or antigen-binding
portions thereof, and expressing said panel in a non-phage display
system;
[0596] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0597] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof, for changes in at least one
other property or characteristic, until an antibody, or
antigen-binding portion thereof, with an improved activity,
relative to the parent antibody, or antigen-binding portion
thereof, is obtained.
[0598] Preferably, the other characteristic or property is selected
from 1) preservation of non-crossreactivity with other proteins or
human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence.
[0599] If a single mutagenesis is not sufficient to increase the
affinity of the antibody other residues may be included in the
mutagenesis. Therefore, in another embodiment, the invention
provides a method for improving the activity of an antibody, or
antigen-binding portion thereof, comprising:
[0600] a) providing a parent antibody or antigen-binding portion
thereof that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in
said phage-display system;
[0601] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and L96;
[0602] c) individually mutating said selected position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expression in a
non-phage display system;
[0603] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0604] e) repeating steps b) through d) for at least one other
position which is neither the position selected under b) nor a
position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,
H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50,
L52, L53, L55, L91, L92, L93, L94;
[0605] g) combining, in the parent antibody, or antigen-binding
portion thereof, at least two individual activity enhancing amino
acid residues shown to have improved activity, to form combination
antibodies, or antigen-binding portions thereof; and
[0606] h) evaluating the activity and other property or
characteristic of the combination antibodies, or antigen-binding
portions thereof with two activity enhancing amino acid residues,
relative to the parent antibody or antigen-binding portion thereof;
until an antibody, or antigen-binding portion thereof, with an
improved activity, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.
[0607] Preferably, the other characteristic or property is selected
from 1) preservation of non-crossreactivity with other proteins or
human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence
[0608] The preferred attempts to identify activity enhancing amino
acids focussing on the preferred selective mutagenesis positions,
contact or hypermutation positions described may be exhausted, or
additional improvements may be required, and it is important to
retain other properties or characteristics of the antibody.
[0609] Therefore, in another embodiment, the invention provides a
method for improving the activity of an antibody, or
antigen-binding portion thereof, without affecting other
characteristics, comprising:
[0610] a) providing a parent antibody or antigen-binding portion
thereof;
[0611] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and L96;
[0612] c) individually mutating said selected position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;
[0613] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0614] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof, for changes in at least one
other property or characteristic until an antibody, or
antigen-binding portion thereof, with an improved activity and
retained other property or characteristic, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.
[0615] Preferably, the other characteristic or property is selected
from 1) preservation of non-crossreactivity with other proteins or
human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence
[0616] If mutagenesis of a single residue is not sufficient other
residues can be included; therefore, in another embodiment, the
invention provides a method for improving the activity of an
antibody, or antigen-binding portion thereof, comprising:
[0617] a) providing a parent antibody or antigen-binding portion
thereof;
[0618] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and L96;
[0619] c) individually mutating said selected position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;
[0620] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof, thereby
identifying an activity enhancing amino acid residue;
[0621] e) evaluating the panel of mutated antibodies or
antigen-binding portions thereof, relative to the parent antibody
or antigen-portion thereof, for changes in at least one other
characteristic or property;
[0622] e) repeating steps b) through e) for at least one other CDR
position which is neither the position selected under b) nor a
position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,
H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50,
L52, L53, L55, L91, L92, L93, L94 and L96;
[0623] f) combining, in the parent antibody, or antigen-binding
portion thereof, at least two individual activity enhancing amino
acid residues shown to have improved activity and not affecting at
least one other property or characteristic, to form combination
antibodies, or antigen-binding portions thereof, and
[0624] g) evaluating the activity and the retention of at least one
other property or characteristic of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino
acid residues, relative to the parent antibody or antigen-binding
portion thereof until an antibody, or antigen-binding portion
thereof, with an improved activity and at least one retained other
property or characteristic, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.
[0625] Mutagenesis of the preferred selective mutagenesis position,
contact and hypermutation residues may not have increased the
affinity of the antibody sufficiently, and mutagenesis and the
phage display method (or related ribosome display method) may no
longer be useful and at least one other characteristic or property
of the antibody should be retained.
[0626] Therefore, in another embodiment the invention provides a
method to improve the affinity of an antibody or antigen-binding
portion thereof, comprising:
[0627] a) providing a parent antibody or antigen-binding portion
thereof that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in
said phage-display system;
[0628] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and L96;
[0629] c) individually mutating said selected position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expression in a
non-phage display system;
[0630] d) evaluating the activity of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby
identifying an activity enhancing amino acid residue;
[0631] e) evaluating the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody
or antigen-binding portion thereof, for changes in at least one
other property or characteristic until an antibody, or
antigen-binding portion thereof, with an improved activity,
relative to the parent antibody, or antigen-binding portion
thereof, is obtained.
[0632] Preferably, the other characteristic or property is selected
from 1) preservation of non-crossreactivity with other proteins or
human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70
p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to
germline immunoglobulin sequence
[0633] If mutagenesis of a single residue is not sufficient other
residues can be included; therefore, in another embodiment, the
invention provides a method for improving the activity of an
antibody, or antigen-binding portion thereof, comprising:
[0634] a) providing a parent antibody or antigen-binding portion
thereof that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in
said phage-display system;
[0635] b) selecting an amino acid residue within a complementarity
determining region (CDR) for mutation other than H30, H31, H31B,
H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97,
H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93,
L94 and L96;
[0636] c) individually mutating said selected position to at least
two other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expression in a
non-phage display system;
[0637] d) evaluating the activity and retention of at least one
other property or characteristic of the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof, thereby
identifying an activity enhancing amino acid residue;
[0638] e) repeating steps b) through d) for at least one other CDR
position which is neither the position selected under b) nor a
position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53,
H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50,
L52, L53, L55, L91, L92, L93, L94 and L96;
[0639] f) combining, in the parent antibody, or antigen-binding
portion thereof, at least two individual activity enhancing amino
acid residues shown to have improved activity and not to affect at
least one other property or characteristic, to form combination
antibodies, or antigen-binding portions thereof, and
[0640] g) evaluating the activity and retention of at least one
property or characteristic of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino
acid residues, relative to the parent antibody or antigen-binding
portion thereof until an antibody, or antigen-binding portion
thereof, with an improved activity and at least one other retained
characteristic or property, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.
V. Expression of Antibodies
[0641] An antibody, or antibody portion, of the invention can be
prepared by recombinant expression of immunoglobulin light and
heavy chain genes in a host cell. To express an antibody
recombinantly, a host cell is transfected with one or more
recombinant expression vectors carrying DNA fragments encoding the
immunoglobulin light and heavy chains of the antibody such that the
light and heavy chains are expressed in the host cell and,
preferably, secreted into the medium in which the host cells are
cultured, from which medium the antibodies can be recovered.
Standard recombinant DNA methodologies are used to obtain antibody
heavy and light chain genes, incorporate these genes into
recombinant expression vectors and introduce the vectors into host
cells, such as those described in Sambrook, Fritsch and Maniatis
(eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold
Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current
Protocols in Molecular Biology, Greene Publishing Associates,
(1989) and in U.S. Pat. No. 4,816,397 by Boss et al.
[0642] To obtain a DNA fragment encoding the heavy chain variable
region of Joe 9 wt or a Joe 9 wt-related antibody, antibodies
specific for human IL-12 were screened from human libraries and
mutated, as described in section II. Once DNA fragments encoding
Joe 9 wt or Joe 9 wt-related VH and VL segments are obtained,
mutagenesis of these sequences is carried out by standard methods,
such as PCR site directed mutagenesis (PCR-mediated mutagenesis in
which the mutated nucleotides are incorporated into the PCR primers
such that the PCR product contains the mutations) or other
site-directed mutagenesis methods. Human IL-12 antibodies that
displayed a level of activity and binding specificity/affinity that
was desirable, for example J695, were further manipulated by
standard recombinant DNA techniques, for example to convert the
variable region genes to full-length antibody chain genes, to Fab
fragment genes or to a scFv gene. In these manipulations, a VL- or
VH-encoding DNA fragment is operatively linked to another DNA
fragment encoding another protein, such as an antibody constant
region or a flexible linker. The term "operatively linked", as used
in this context, is intended to mean that the two DNA fragments are
joined such that the amino acid sequences encoded by the two DNA
fragments remain in-frame.
[0643] The isolated DNA encoding the VH region can be converted to
a full-length heavy chain gene by operatively linking the
VH-encoding DNA to another DNA molecule encoding heavy chain
constant regions (CH1, CH2 and CH3). The sequences of human heavy
chain constant region genes are known in the art (see e.g., 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) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region and any
allotypic variant therein as described in Kabat (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), but most preferably is an IgG1 or IgG4
constant region. For a Fab fragment heavy chain gene, the
VH-encoding DNA can be operatively linked to another DNA molecule
encoding only the heavy chain CH1 constant region.
[0644] The isolated DNA encoding the VL region can be converted to
a full-length light chain gene (as well as a Fab light chain gene)
by operatively linking the VL-encoding DNA to another DNA molecule
encoding the light chain constant region, CL. The sequences of
human light chain constant region genes are known in the art (see
e.g., 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) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The light chain constant region can be a kappa or
lambda constant region, but most preferably is a lambda constant
region.
[0645] To create a scFv gene, the VH- and VL-encoding DNA fragments
are operatively linked to another fragment encoding a flexible
linker, e.g., encoding the amino acid sequence
(Gly.sub.4-Ser).sub.3, such that the VH and VL sequences can be
expressed as a contiguous single-chain protein, with the VL and VH
regions joined by the flexible linker (see e.g., Bird et al. (1988)
Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci.
USA 85:5879-5883; McCafferty et al., Nature (1990)
348:552-554).
[0646] To express the antibodies, or antibody portions of the
invention, DNAs encoding partial or full-length light and heavy
chains, obtained as described above, are inserted into expression
vectors such that the genes are operatively linked to
transcriptional and translational control sequences. In this
context, the term "operatively linked" is intended to mean that an
antibody gene is ligated into a vector such that transcriptional
and translational control sequences within the vector serve their
intended function of regulating the transcription and translation
of the antibody gene. The expression vector and expression control
sequences are chosen to be compatible with the expression host cell
used. The antibody light chain gene and the antibody heavy chain
gene can be inserted into separate vector or, more typically, both
genes are inserted into the same expression vector. The antibody
genes are inserted into the expression vector by standard methods
(e.g., ligation of complementary restriction sites on the antibody
gene fragment and vector, or blunt end ligation if no restriction
sites are present). Prior to insertion of the J695 or J695-related
light or heavy chain sequences, the expression vector may already
carry antibody constant region sequences. For example, one approach
to converting the J695 or J695-related VH and VL sequences to
full-length antibody genes is to insert them into expression
vectors already encoding heavy chain constant and light chain
constant regions, respectively, such that the VH segment is
operatively linked to the CH segment(s) within the vector and the
VL segment is operatively linked to the CL segment within the
vector. Additionally or alternatively, the recombinant expression
vector can encode a signal peptide that facilitates secretion of
the antibody chain from a host cell. The antibody chain gene can be
cloned into the vector such that the signal peptide is linked
in-frame to the amino terminus of the antibody chain gene. The
signal peptide can be an immunoglobulin signal peptide or a
heterologous signal peptide (i.e., a signal peptide from a
non-immunoglobulin protein).
[0647] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
The term "regulatory sequence" is intended to include promoters,
enhancers and other expression control elements (e.g.
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel; Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). It will be appreciated by those skilled in the art that the
design of the expression vector, including the selection of
regulatory sequences may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV) (such as the CMV
promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40
promoter/enhancer), adenovirus, (e.g., the adenovirus major late
promoter (AdMLP)) and polyoma. For further description of viral
regulatory elements, and sequences thereof, see e.g., U.S. Pat. No.
5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and
U.S. Pat. No. 4,968,615 by Schaffner et al., U.S. Pat. No.
5,464,758 by Bujard et al. and U.S. Pat. No. 5,654,168 by Bujard et
al.
[0648] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr.sup.- host
cells with methotrexate selection/amplification) and the neo gene
(for G418 selection).
[0649] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. Although it is theoretically possible to express the
antibodies of the invention in either prokaryotic or eukaryotic
host cells, expression of antibodies in eukaryotic cells, and most
preferably mammalian host cells, is the most preferred because such
eukaryotic cells, and in particular mammalian cells, are more
likely than prokaryotic cells to assemble and secrete a properly
folded and immunologically active antibody. Preferred mammalian
host cells for expressing the recombinant antibodies of the
invention include Chinese Hamster Ovary (CHO cells) (including
dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl.
Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker,
e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol.
Biol. 159:601-621), NS0 myeloma cells, COS cells and SP2 cells.
When recombinant expression vectors encoding antibody genes are
introduced into mammalian host cells, the antibodies are produced
by culturing the host cells for a period of time sufficient to
allow for expression of the antibody in the host cells or, more
preferably, secretion of the antibody into the culture medium in
which the host cells are grown. Antibodies can be recovered from
the culture medium using standard protein purification methods.
[0650] Host cells can also be used to produce portions of intact
antibodies, such as Fab fragments or scFv molecules. It will be
understood that variations on the above procedure are within the
scope of the present invention. For example, it may be desirable to
transfect a host cell with DNA encoding either the light chain or
the heavy chain (but not both) of an antibody of this invention.
Recombinant DNA technology may also be used to remove some or all
of the DNA encoding either or both of the light and heavy chains
that is not necessary for binding to hIL-12 The molecules expressed
from such truncated DNA molecules are also encompassed by the
antibodies of the invention. In addition, bifunctional antibodies
may be produced in which one heavy and one light chain are an
antibody of the invention and the other heavy and light chain are
specific for an antigen other than hIL-12 by crosslinking an
antibody of the invention to a second antibody by standard chemical
crosslinking methods.
[0651] In a preferred system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr-CHO
cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to enhancer/promoter regulatory
elements (e.g., derived from SV40, CMV, adenovirus and the like,
such as a CMV enhancer/AdMLP promoter regulatory element or an SV40
enhancer/AdMLP promoter regulatory element) to drive high levels of
transcription of the genes. The recombinant expression vector also
carries a DHFR gene, which allows for selection of CHO cells that
have been transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are
culture to allow for expression of the antibody heavy and light
chains and intact antibody is recovered from the culture medium.
Standard molecular biology techniques are used to prepare the
recombinant expression vector, transfect the host cells, select for
transformants, culture the host cells and recover the antibody from
the culture medium. Antibodies or antigen-binding portions thereof
of the invention can be expressed in an animal (e.g., a mouse) that
is transgenic for human immunoglobulin genes (see e.g., Taylor, L.
D. et al. (1992) Nucl. Acids Res. 20: 6287-6295). Plant cells can
also be modified to create transgenic plants that express the
antibody or antigen binding portion thereof, of the invention.
[0652] In view of the foregoing, another aspect of the invention
pertains to nucleic acid, vector and host cell compositions that
can be used for recombinant expression of the antibodies and
antibody portions of the invention. Preferably, the invention
features isolated nucleic acids that encode CDRs of J695, or the
full heavy and/or light chain variable region of J695. Accordingly,
in one embodiment, the invention features an isolated nucleic acid
encoding an antibody heavy chain variable region that encodes the
J695 heavy chain CDR3 comprising the amino acid sequence of SEQ ID
NO: 25. Preferably, the nucleic acid encoding the antibody heavy
chain variable region further encodes a J695 heavy chain CDR2 which
comprises the amino acid sequence of SEQ ID NO: 27. More
preferably, the nucleic acid encoding the antibody heavy chain
variable region further encodes a J695 heavy chain CDR1 which
comprises the amino acid sequence of SEQ ID NO: 29. Even more
preferably, the isolated nucleic acid encodes an antibody heavy
chain variable region comprising the amino acid sequence of SEQ ID
NO: 31 (the full VH region of J695).
[0653] In other embodiments, the invention features an isolated
nucleic acid encoding an antibody light chain variable region that
encodes the J695 light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 26. Preferably, the nucleic acid encoding
the antibody light chain variable region further encodes a J695
light chain CDR2 which comprises the amino acid sequence of SEQ ID
NO: 28. More preferably, the nucleic acid encoding the antibody
light chain variable region further encodes a J695 light chain CDR1
which comprises the amino acid sequence of SEQ ID NO: 30. Even more
preferably, the isolated nucleic acid encodes an antibody light
chain variable region comprising the amino acid sequence of SEQ ID
NO: 32 (the full VL region of J695).
[0654] The invention also provides recombinant expression vectors
encoding both an antibody heavy chain and an antibody light chain.
For example, in one embodiment, the invention provides a
recombinant expression vector encoding: [0655] a) an antibody heavy
chain having a variable region comprising the amino acid sequence
of SEQ ID NO: 31; and [0656] b) an antibody light chain having a
variable region comprising the amino acid sequence of SEQ ID NO:
32.
[0657] The invention also provides host cells into which one or
more of the recombinant expression vectors of the invention have
been introduced. Preferably, the host cell is a mammalian host
cell, more preferably the host cell is a CHO cell, an NS0 cell or a
COS cell. Still further the invention provides a method of
synthesizing a recombinant human antibody of the invention by
culturing a host cell of the invention in a suitable culture medium
until a recombinant human antibody of the invention is synthesized.
The method can further comprise isolating the recombinant human
antibody from the culture medium.
VI. Pharmaceutical Compositions and Pharmaceutical
Administration
[0658] The antibodies and antibody-portions of the invention can be
incorporated into pharmaceutical compositions suitable for
administration to a subject. Typically, the pharmaceutical
composition comprises an antibody or antibody portion of the
invention and a pharmaceutically acceptable carrier. As used
herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. Examples of pharmaceutically
acceptable carriers include one or more of water, saline, phosphate
buffered saline, dextrose, glycerol, ethanol and the like, as well
as combinations thereof. In many cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Pharmaceutically acceptable carriers may further comprise minor
amounts of auxiliary substances such as wetting or emulsifying
agents, preservatives or buffers, which enhance the shelf life or
effectiveness of the antibody or antibody portion.
[0659] The antibodies and antibody-portions of the invention can be
incorporated into a pharmaceutical composition suitable for
parenteral administration. Preferably, the antibody or
antibody-portions will be prepared as an injectable solution
containing 0.1-250 mg/ml antibody. The injectable solution can be
composed of either a liquid or lyophilized dosage form in a flint
or amber vial, ampule or pre-filled syringe. The buffer can be
L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0
(optimally pH 6.0). Other suitable buffers include but are not
limited to, sodium succinate, sodium citrate, sodium phosphate or
potassium phosphate. Sodium chloride can be used to modify the
toxicity of the solution at a concentration of 0-300 mM (optimally
150 mM for a liquid dosage form). Cryoprotectants can be included
for a lyophilized dosage form, principally 0-10% sucrose (optimally
0.5-1.0%). Other suitable cryoprotectants include trenhalose and
lactose. Bulking agents can be included for a lyophilized dosage
form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can
be used in both liquid and lyophilized dosage forms, principally
1-50 mM L-Methionine (optimally 5-10 mM). Other suitable bulking
agents include glycine, arginine, can be included as 0-0.05%
polysorbate-80 (optimally 0.005-0.01%). Additional surfactants
include but are not limited to polysorbate 20 and BRIJ
surfactants.
[0660] In a preferred embodiment, the pharmaceutical composition
includes the antibody at a dosage of about 0.01 mg/kg-10 mg/kg.
More preferred dosages of the antibody include 1 mg/kg administered
every other week, or 0.3 mg/kg administered weekly.
[0661] The compositions of this invention may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends on
the intended mode of administration and therapeutic application.
Typical preferred compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
passive immunization of humans with other antibodies. The preferred
mode of administration is parenteral (e.g., intravenous,
subcutaneous, intraperitoneal, intramuscular). In a preferred
embodiment, the antibody is administered by intravenous infusion or
injection. In another preferred embodiment, the antibody is
administered by intramuscular or subcutaneous injection.
[0662] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., antibody or antibody
portion) in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of
sterile, lyophilized powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are
vacuum drying and spray-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin.
[0663] The antibodies and antibody-portions of the present
invention can be administered by a variety of methods known in the
art, although for many therapeutic applications, the preferred
route/mode of administration is subcutaneous injection, intravenous
injection or infusion. As will be appreciated by the skilled
artisan, the route and/or mode of administration will vary
depending upon the desired results. In certain embodiments, the
active compound may be prepared with a carrier that will protect
the compound against rapid release, such as a controlled release
formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0664] In certain embodiments, an antibody or antibody portion of
the invention may be orally administered, for example, with an
inert diluent or an assimilable edible carrier. The compound (and
other ingredients, if desired) may also be enclosed in a hard or
soft shell gelatin capsule, compressed into tablets, or
incorporated directly into the subject's diet. For oral therapeutic
administration, the compounds may be incorporated with excipients
and used in the form of ingestible tablets, buccal tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, and the
like. To administer a compound of the invention by other than
parenteral administration, it may be necessary to coat the compound
with, or co-administer the compound with, a material to prevent its
inactivation.
[0665] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, an antibody or antibody
portion of the invention is coformulated with and/or coadministered
with one or more additional therapeutic agents that are useful for
treating disorders in which IL-12 activity is detrimental. For
example, an anti-hIL-12 antibody or antibody portion of the
invention may be coformulated and/or coadministered with one or
more additional antibodies that bind other targets (e.g.,
antibodies that bind other cytokines or that bind cell surface
molecules). Furthermore, one or more antibodies of the invention
may be used in combination with two or more of the foregoing
therapeutic agents. Such combination therapies may advantageously
utilize lower dosages of the administered therapeutic agents, thus
avoiding possible toxicities or complications associated with the
various monotherapies. It will be appreciated by the skilled
practitioner that when the antibodies of the invention are used as
part of a combination therapy, a lower dosage of antibody may be
desirable than when the antibody alone is administered to a subject
(e.g., a synergistic therapeutic effect may be achieved through the
use of combination therapy which, in turn, permits use of a lower
dose of the antibody to achieve the desired therapeutic
effect).
[0666] Interleukin 12 plays a critical role in the pathology
associated with a variety of diseases involving immune and
inflammatory elements. These diseases include, but are not limited
to, rheumatoid arthritis, osteoarthritis, juvenile chronic
arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis,
spondyloarthropathy, systemic lupus erythematosus, Crohn's disease,
ulcerative colitis, inflammatory bowel disease, insulin dependent
diabetes mellitus, thyroiditis, asthma, allergic diseases,
psoriasis, dermatitis scleroderma, atopic dermatitis, graft versus
host disease, organ transplant rejection, acute or chronic immune
disease associated with organ transplantation, sarcoidosis,
atherosclerosis, disseminated intravascular coagulation, Kawasaki's
disease, Grave's disease, nephrotic syndrome, chronic fatigue
syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea,
microscopic vasculitis of the kidneys, chronic active hepatitis,
uveitis, septic shock, toxic shock syndrome, sepsis syndrome,
cachexia, infectious diseases, parasitic diseases, acquired
immunodeficiency syndrome, acute transverse myelitis, Huntington's
chorea, Parkinson's disease, Alzheimer's disease, stroke, primary
biliary cirrhosis, hemolytic anemia, malignancies, heart failure,
myocardial infarction, Addison's disease, sporadic, polyglandular
deficiency type I and polyglandular deficiency type II, Schmidt's
syndrome, adult (acute) respiratory distress syndrome, alopecia,
alopecia greata, seronegative arthopathy, arthropathy, Reiter's
disease, psoriatic arthropathy, ulcerative colitic arthropathy,
enteropathic synovitis, chlamydia, yersinia and salmonella
associated arthropathy, spondyloarthopathy, atheromatous
disease/arteriosclerosis, atopic allergy, autoimmune bullous
disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,
linear IgA disease, autoimmune haemolytic anaemia, Coombs positive
haemolytic anaemia, acquired pernicious anaemia, juvenile
pernicious anaemia, myalgic encephalitis/Royal Free Disease,
chronic mucocutaneous candidiasis, giant cell arteritis, primary
sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired
Immunodeficiency Disease Syndrome, Acquired Immunodeficiency
Related Diseases, Hepatitis C, common varied immunodeficiency
(common variable hypogammaglobulinaemia), dilated cardiomyopathy,
female infertility, ovarian failure, premature ovarian failure,
fibrotic lung disease, cryptogenic fibrosing alveolitis,
post-inflammatory interstitial lung disease, interstitial
pneumonitis, connective tissue disease associated interstitial lung
disease, mixed connective tissue disease associated lung disease,
systemic sclerosis associated interstitial lung disease, rheumatoid
arthritis associated interstitial lung disease, systemic lupus
erythematosus associated lung disease, dermatomyositis/polymyositis
associated lung disease, Sjogren's disease associated lung disease,
ankylosing spondylitis associated lung disease, vasculitic diffuse
lung disease, haemosiderosis associated lung disease, drug-induced
interstitial lung disease, radiation fibrosis, bronchiolitis
obliterans, chronic eosinophilic pneumonia, lymphocytic
infiltrative lung disease, postinfectious interstitial lung
disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune
hepatitis (classical autoimmune or lupoid hepatitis), type-2
autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune
mediated hypoglycemia, type B insulin resistance with acanthosis
nigricans, hypoparathyroidism, acute immune disease associated with
organ transplantation, chronic immune disease associated with organ
transplantation, osteoarthrosis, primary sclerosing cholangitis,
idiopathic leucopenia, autoimmune neutropenia, renal disease NOS,
glomerulonephritides, microscopic vasulitis of the kidneys, lyme
disease, discoid lupus erythematosus, male infertility idiopathic
or NOS, sperm autoimmunity, multiple sclerosis (all subtypes),
insulin-dependent diabetes mellitus, sympathetic ophthalmia,
pulmonary hypertension secondary to connective tissue disease,
Goodpasture's syndrome, pulmonary manifestation of polyarteritis
nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's
disease, systemic sclerosis, Takayasu's disease/arteritis,
autoimmune thrombocytopenia, idiopathic thrombocytopenia,
autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune
hypothyroidism (Hashimoto's disease), atrophic autoimmune
hypothyroidism, primary myxoedema, phacogenic uveitis, primary
vasculitis and vitiligo. The human antibodies, and antibody
portions of the invention can be used to treat autoimmune diseases,
in particular those associated with inflammation, including,
rheumatoid spondylitis, allergy, autoimmune diabetes, autoimmune
uveitis.
[0667] Preferably, the antibodies of the invention or
antigen-binding portions thereof, are used to treat rheumatoid
arthritis, Crohn's disease, multiple sclerosis, insulin dependent
diabetes mellitus and psoriasis, as described in more detail in
section VII.
[0668] A human antibody, or antibody portion, of the invention also
can be administered with one or more additional therapeutic agents
useful in the treatment of autoimmune and inflammatory
diseases.
[0669] Antibodies of the invention, or antigen binding portions
thereof can be used alone or in combination to treat such diseases.
It should be understood that the antibodies of the invention or
antigen binding portion thereof can be used alone or in combination
with an additional agent, e.g., a therapeutic agent, said
additional agent being selected by the skilled artisan for its
intended purpose. For example, the additional agent can be a
therapeutic agent art-recognized as being useful to treat the
disease or condition being treated by the antibody of the present
invention. The additional agent also can be an agent which imparts
a beneficial attribute to the therapeutic composition e.g., an
agent which effects the viscosity of the composition.
[0670] It should further be understood that the combinations which
are to be included within this invention are those combinations
useful for their intended purpose. The agents set forth below are
illustrative for purposes and not intended to be limited. The
combinations which are part of this invention can be the antibodies
of the present invention and at least one additional agent selected
from the lists below. The combination can also include more than
one additional agent, e.g., two or three additional agents if the
combination is such that the formed composition can perform its
intended function.
[0671] Preferred combinations are non-steroidal anti-inflammatory
drug(s) also referred to as NSAIDS which include drugs like
ibuprofen. Other preferred combinations are corticosteroids
including prednisolone; the well known side-effects of steroid use
can be reduced or even eliminated by tapering the steroid dose
required when treating patients in combination with the anti-IL-12
antibodies of this invention. Non-limiting examples of therapeutic
agents for rheumatoid arthritis with which an antibody, or antibody
portion, of the invention can be combined include the following:
cytokine suppressive anti-inflammatory drug(s) (CSAIDs); antibodies
to or antagonists of other human cytokines or growth factors, for
example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16,
IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the invention,
or antigen binding portions thereof, can be combined with
antibodies to cell surface molecules such as CD2, CD3, CD4, CD8,
CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90,
or their ligands including CD154 (gp39 or CD40L).
[0672] Preferred combinations of therapeutic agents may interfere
at different points in the autoimmune and subsequent inflammatory
cascade; preferred examples include TNF antagonists like chimeric,
humanized or human TNF antibodies, D2E7, (U.S. application Ser. No.
08/599,226 filed Feb. 9, 1996), cA2 (Remicade.TM.), CDP 571,
anti-TNF antibody fragments (e.g., CDP870), and soluble p55 or p75
TNF receptors, derivatives thereof, (p75TNFR1gG (Enbrel.TM.) or
p55TNFR1gG (Lenercept), soluble IL-13 receptor (sIL-13), and also
TNF.alpha. converting enzyme (TACE) inhibitors; similarly IL-1
inhibitors (e.g., Interleukin-1-converting enzyme inhibitors, such
as Vx740, or IL-1RA etc.) may be effective for the same reason.
Other preferred combinations include Interleukin 11, anti-P7s and
p-selectin glycoprotein ligand (PSGL). Yet another preferred
combination are other key players of the autoimmune response which
may act parallel to, dependent on or in concert with IL-12
function; especially preferred are IL-18 antagonists including
IL-18 antibodies or soluble IL-18 receptors, or IL-18 binding
proteins. It has been shown that IL-12 and IL-18 have overlapping
but distinct functions and a combination of antagonists to both may
be most effective. Yet another preferred combination are
non-depleting anti-CD4 inhibitors. Yet other preferred combinations
include antagonists of the co-stimulatory pathway CD80 (B7.1) or
CD86 (B7.2) including antibodies, soluble receptors or antagonistic
ligands.
[0673] The antibodies of the invention, or antigen binding portions
thereof, may also be combined with agents, such as methotrexate,
6-MP, azathioprine sulphasalazine, mesalazine, olsalazine
chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate
(intramuscular and oral), azathioprine, cochicine, corticosteroids
(oral, inhaled and local injection), beta-2 adrenoreceptor agonists
(salbutamol, terbutaline, salmeteral), xanthines (theophylline,
aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium
and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate
mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adensosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signalling by
proinflammatory cytokines such as TNF.alpha. or IL-1 (e.g. IRAK,
NIK, IKK, p38 or MAP kinase inhibitors), IL-ID converting enzyme
inhibitors (e.g., Vx740), anti-P7s, p-selectin glycoprotein ligand
(PSGL), TNF.alpha. converting enzyme (TACE) inhibitors, T-cell
signalling inhibitors such as kinase inhibitors, metalloproteinase
inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines,
angiotensin converting enzyme inhibitors, soluble cytokine
receptors and derivatives thereof (e.g. soluble p55 or p75 TNF
receptors and the derivatives p75TNFRIgG (Enbrel.TM.) and
p55TNFRIgG (Lenercept), sIL-1RI, sIL-1RII, sIL-6R, soluble IL-13
receptor (sIL-13)) and antiinflammatory cytokines (e.g. IL-4,
IL-10, IL-11, IL-13 and TGF.beta.). Preferred combinations include
methotrexate or leflunomide and in moderate or severe rheumatoid
arthritis cases, cyclosporine.
[0674] Non-limiting examples of therapeutic agents for inflammatory
bowel disease with which an antibody, or antibody portion, of the
invention can be combined include the following: budenoside;
epidermal growth factor; corticosteroids; cyclosporin,
sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine;
metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine;
balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor
antagonists; anti-IL-1.beta. monoclonal antibodies; anti-IL-6
monoclonal antibodies; growth factors; elastase inhibitors;
pyridinyl-imidazole compounds; antibodies to or antagonists of
other human cytokines or growth factors, for example, TNF, LT,
IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF,
FGF, and PDGF. Antibodies of the invention, or antigen binding
portions thereof, can be combined with antibodies to cell surface
molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45,
CD69, CD90 or their ligands. The antibodies of the invention, or
antigen binding portions thereof, may also be combined with agents,
such as methotrexate, cyclosporin, FK506, rapamycin, mycophenolate
mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adenosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signalling by
proinflammatory cytokines such as TNF.alpha. or IL-1 (e.g. IRAK,
NIK, IKK, p38 or MAP kinase inhibitors), IL-1, converting enzyme
inhibitors (e.g., Vx740), anti-P7s, p-selectin glycoprotein ligand
(PSGL), TNF.alpha. converting enzyme inhibitors, T-cell signalling
inhibitors such as kinase inhibitors, metalloproteinase inhibitors,
sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin
converting enzyme inhibitors, soluble cytokine receptors and
derivatives thereof (e.g. soluble p55 or p75 TNF receptors,
sIL-1RI, sIL-1RII, sIL-6R, soluble IL-13 receptor (sIL-13)) and
antiinflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13 and
TGF.beta.).
[0675] Preferred examples of therapeutic agents for Crohn's disease
in which an antibody or an antigen binding portion can be combined
include the following: TNF antagonists, for example, anti-TNF
antibodies, D2E7 (U.S. application Ser. No. 08/599,226, filed Feb.
9, 1996), cA2 (Remicade.TM.), CDP 571, anti-TNF antibody fragments
(e.g., CDP870), TNFR-Ig constructs (p75TNFRIgG (Enbrel.TM.) and
p55TNFRIgG (Lenercept)), anti-P7s, p-selectin glycoprotein ligand
(PSGL), soluble IL-13 receptor (sIL-13), and PDE4 inhibitors.
Antibodies of the invention or antigen binding portions thereof,
can be combined with corticosteroids, for example, budenoside and
dexamethasone. Antibodies of the invention or antigen binding
portions thereof, may also be combined with agents such as
sulfasalazine, 5-aminosalicylic acid and olsalazine, and agents
which interfere with synthesis or action of proinflammatory
cytokines such as IL-1, for example, IL-1.beta. converting enzyme
inhibitors (e.g., Vx740) and IL-Ira. Antibodies of the invention or
antigen binding portion thereof may also be used with T cell
signaling inhibitors, for example, tyrosine kinase inhibitors
6-mercaptopurines. Antibodies of the invention or antigen binding
portions thereof, can be combined with IL-11.
[0676] Non-limiting examples of therapeutic agents for multiple
sclerosis with which an antibody, or antibody portion, of the
invention can be combined include the following: corticosteroids;
prednisolone; methylprednisolone; azathioprine; cyclophosphamide;
cyclosporine; methotrexate; 4-aminopyridine; tizanidine;
interferon-.beta.1a (Avonex; Biogen); interferon-.beta.1b
(Betaseron; Chiron/Berlex); Copolymer 1 (Cop-1; Copaxone; Teva
Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous
immunoglobulin; clabribine; antibodies to or antagonists of other
human cytokines or growth factors, for example, TNF, LT, IL-1,
IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF,
and PDGF. Antibodies of the invention, or antigen binding portions
thereof, can be combined with antibodies to cell surface molecules
such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69,
CD80, CD86, CD90 or their ligands. The antibodies of the invention,
or antigen binding portions thereof, may also be combined with
agents, such as methotrexate, cyclosporine, FK506, rapamycin,
mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adensosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signalling by
proinflammatory cytokines such as TNF.alpha. or IL-1 (e.g. IRAK,
NIK, IKK, p38 or MAP kinase inhibitors), IL-1.beta. converting
enzyme inhibitors (e.g., Vx740), anti-P7s, p-selectin glycoprotein
ligand (PSGL), TACE inhibitors, T-cell signalling inhibitors such
as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine,
azathioprine, 6-mercaptopurines, angiotensin converting enzyme
inhibitors, soluble cytokine receptors and derivatives thereof
(e.g. soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R,
soluble IL-13 receptor (sIL-13)) and antiinflammatory cytokines
(e.g. IL-4, IL-10, IL-13 and TGF.beta.).
[0677] Preferred examples of therapeutic agents for multiple
sclerosis in which the antibody or antigen binding portion thereof
can be combined to include interferon-0, for example, IFN.beta.1a
and IFN.beta.1b; copaxone, corticosteroids, IL-1 inhibitors, TNF
inhibitors, and antibodies to CD40 ligand and CD80.
[0678] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of an antibody or antibody portion of the
invention. A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result. A therapeutically effective amount
of the antibody or antibody portion may vary according to factors
such as the disease state, age, sex, and weight of the individual,
and the ability of the antibody or antibody portion to elicit a
desired response in the individual. A therapeutically effective
amount is also one in which any toxic or detrimental effects of the
antibody or antibody portion are outweighed by the therapeutically
beneficial effects. A "prophylactically effective amount" refers to
an amount effective, at dosages and for periods of time necessary,
to achieve the desired prophylactic result. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount.
[0679] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
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 mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the active compound and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0680] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody or antibody
portion of the invention is 0.01-20 mg/kg, more preferably 1-10
mg/kg, even more preferably 0.3-1 mg/kg. It is to be noted that
dosage values may vary with the type and severity of the condition
to be alleviated. It is to be further understood that for any
particular subject, specific dosage regimens should be adjusted
over time according to the individual need and the professional
judgment of the person administering or supervising the
administration of the compositions, and that dosage ranges set
forth herein are exemplary only and are not intended to limit the
scope or practice of the claimed composition.
VII. Uses of the Antibodies of the Invention
[0681] Given their ability to bind to hIL-12, the anti-hIL-12
antibodies, or portions thereof, of the invention can be used to
detect hIL-12 (e.g., in a biological sample, such as serum or
plasma), using a conventional immunoassay, such as an enzyme linked
immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue
immunohistochemistry. The invention provides a method for detecting
hIL-12 in a biological sample comprising contacting a biological
sample with an antibody, or antibody portion, of the invention and
detecting either the antibody (or antibody portion) bound to hIL-12
or unbound antibody (or antibody portion), to thereby detect hIL-12
in the biological sample. The antibody is directly or indirectly
labeled with a detectable substance to facilitate detection of the
bound or unbound antibody. Suitable detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials and radioactive materials. Examples of
suitable enzymes include horseradish peroxidase, alkaline
phosphatase, .beta.-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; and examples of suitable
radioactive material include .sup.125I, .sup.131I, .sup.35S or
.sup.3H.
[0682] Alternative to labeling the antibody, hIL-12 can be assayed
in biological fluids by a competition immunoassay utilizing rhIL-12
standards labeled with a detectable substance and an unlabeled
anti-hIL-12 antibody. In this assay, the biological sample, the
labeled rhIL-12 standards and the anti-hIL-12 antibody are combined
and the amount of labeled rhIL-12 standard bound to the unlabeled
antibody is determined. The amount of hIL-12 in the biological
sample is inversely proportional to the amount of labeled rhIL-12
standard bound to the anti-hIL-12 antibody.
[0683] The Y61 and J695 antibodies of the invention can also be
used to detect IL-12 from species other than humans, in particular
IL-12 from primates. For example, Y61 can be used to detect IL-12
in the cynomolgus monkey and the rhesus monkey. J695 can be used to
detect IL-12 in the cynomolgus monkey, rhesus monkey, and baboon.
However, neither antibody cross reacts with mouse or rat IL-12 (see
Example 3, subsection F).
[0684] The antibodies and antibody portions of the invention are
capable of neutralizing hIL-12 activity in vitro (see Example 3)
and in vivo (see Example 4). Accordingly, the antibodies and
antibody portions of the invention can be used to inhibit IL-12
activity, e.g., in a cell culture containing hIL-12, in human
subjects or in other mammalian subjects having IL-12 with which an
antibody of the invention cross-reacts (e.g. primates such as
baboon, cynomolgus and rhesus). In a preferred embodiment, the
invention provides an isolated human antibody, or antigen-binding
portion thereof, that neutralizes the activity of human IL-12, and
at least one additional primate IL-12 selected from the group
consisting of baboon IL-12, marmoset IL-12, chimpanzee IL-12,
cynomolgus IL-12 and rhesus IL-12, but which does not neutralize
the activity of the mouse IL-12. Preferably, the IL-12 is human
IL-12. For example, in a cell culture containing, or suspected of
containing hIL-12, an antibody or antibody portion of the invention
can be added to the culture medium to inhibit hIL-12 activity in
the culture.
[0685] In another embodiment, the invention provides a method for
inhibiting IL-12 activity in a subject suffering from a disorder in
which IL-12 activity is detrimental. IL-12 has been implicated in
the pathophysiology of a wide variety of disorders (Windhagen et
al., (1995) J. Exp. Med. 182: 1985-1996; Morita et al. (1998)
Arthritis and Rheumatism. 41: 306-314; Bucht et al., (1996) Clin.
Exp. Immunol. 103: 347-367; Fais et al. (1994) J. Interferon Res.
14:235-238; Parronchi et al., (1997) Am. J. Path. 150:823-832;
Monteleone et al., (1997) Gastroenterology. 112:1169-1178, and
Berrebi et al., (1998) Am. J. Path 152:667-672; Parronchi et al
(1997) Am. J. Path. 150:823-832). The invention provides methods
for inhibiting IL-12 activity in a subject suffering from such a
disorder, which method comprises administering to the subject an
antibody or antibody portion of the invention such that IL-12
activity in the subject is inhibited. Preferably, the IL-12 is
human IL-12 and the subject is a human subject. Alternatively, the
subject can be a mammal expressing a IL-12 with which an antibody
of the invention cross-reacts. Still further the subject can be a
mammal into which has been introduced hIL-12 (e.g., by
administration of hIL-12 or by expression of an hIL-12 transgene).
An antibody of the invention can be administered to a human subject
for therapeutic purposes (discussed further below). Moreover, an
antibody of the invention can be administered to a non-human mammal
expressing a IL-12 with which the antibody cross-reacts for
veterinary purposes or as an animal model of human disease.
Regarding the latter, such animal models may be useful for
evaluating the therapeutic efficacy of antibodies of the invention
(e.g., testing of dosages and time courses of administration).
[0686] As used herein, the phrase "a disorder in which IL-12
activity is detrimental" is intended to include diseases and other
disorders in which the presence of IL-12 in a subject suffering
from the disorder has been shown to be or is suspected of being
either responsible for the pathophysiology of the disorder or a
factor that contributes to a worsening of the disorder.
Accordingly, a disorder in which IL-12 activity is detrimental is a
disorder in which inhibition of IL-12 activity is expected to
alleviate the symptoms and/or progression of the disorder. Such
disorders may be evidenced, for example, by an increase in the
concentration of IL-12 in a biological fluid of a subject suffering
from the disorder (e.g., an increase in the concentration of IL-12
in serum, plasma, synovial fluid, etc. of the subject), which can
be detected, for example, using an anti-IL-12 antibody as described
above. There are numerous examples of disorders in which IL-12
activity is detrimental. In one embodiment, the antibodies or
antigen binding portions thereof, can be used in therapy to treat
the diseases or disorders described herein. In another embodiment,
the antibodies or antigen binding portions thereof, can be used for
the manufacture of a medicine for treating the diseases or
disorders described herein. The use of the antibodies and antibody
portions of the invention in the treatment of a few non-limiting
specific disorders is discussed further below:
A. Rheumatoid Arthritis:
[0687] Interleukin-12 has been implicated in playing a role in
inflammatory diseases such as rheumatoid arthritis. Inducible
IL-12p40 message has been detected in synovia from rheumatoid
arthritis patients and IL-12 has been shown to be present in the
synovial fluids from patients with rheumatoid arthritis (see e.g.,
Morita et al., (1998) Arthritis and Rheumatism 41: 306-314). IL-12
positive cells have been found to be present in the sublining layer
of the rheumatoid arthritis synovium. The human antibodies, and
antibody portions of the invention can be used to treat, for
example, rheumatoid arthritis, juvenile rheumatoid arthritis, Lyme
arthritis, rheumatoid spondylitis, osteoarthritis and gouty
arthritis. Typically, the antibody, or antibody portion, is
administered systemically, although for certain disorders, local
administration of the antibody or antibody portion may be
beneficial. An antibody, or antibody portion, of the invention also
can be administered with one or more additional therapeutic agents
useful in the treatment of autoimmune diseases.
[0688] In the collagen induced arthritis (CIA) murine model for
rheumatoid arthritis, treatment of mice with an anti-IL-12 mAb (rat
anti-mouse IL-12 monoclonal antibody, C17.15) prior to arthritis
profoundly suppressed the onset, and reduced the incidence and
severity of disease. Treatment with the anti-IL-12 mAb early after
onset of arthritis reduced severity, but later treatment of the
mice with the anti-IL-12 mAb after the onset of disease had minimal
effect on disease severity.
B. Crohn's Disease
[0689] Interleukin-12 also plays a role in the inflammatory bowel
disease, Crohn's disease. Increased expression of IFN-.gamma. and
IL-12 occurs in the intestinal mucosa of patients with Crohn's
disease (see e.g., Fais et al., (1994) J. Interferon Res. 14:
235-238; Parronchi et al., (1997) Amer. J. Pathol. 150: 823-832;
Monteleone et al., (1997) Gastroenterology 112: 1169-1178; Berrebi
et al., (1998) Amer. J. Pathol. 152: 667-672). Anti-IL-12
antibodies have been shown to suppress disease in mouse models of
colitis, e.g., TNBS induced colitis IL-2 knockout mice, and
recently in IL-10 knock-out mice. Accordingly, the antibodies, and
antibody portions, of the invention, can be used in the treatment
of inflammatory bowel diseases.
C. Multiple Sclerosis
[0690] Interleukin-12 has been implicated as a key mediator of
multiple sclerosis. Expression of the inducible IL-12 p40 message
or IL-12 itself can be demonstrated in lesions of patients with
multiple sclerosis (Windhagen et al., (1995) J. Exp. Med. 182:
1985-1996, Drulovic et al., (1997) J. Neurol. Sci. 147: 145-150).
Chronic progressive patients with multiple sclerosis have elevated
circulating levels of IL-12. Investigations with T-cells and
antigen presenting cells (APCs) from patients with multiple
sclerosis revealed a self-perpetuating series of immune
interactions as the basis of progressive multiple sclerosis leading
to a Th1-type immune response. Increased secretion of IFN-.gamma.
from the T cells led to increased IL-12 production by APCs, which
perpetuated the cycle leading to a chronic state of a Th1-type
immune activation and disease (Balashov et al., (1997) Proc. Natl.
Acad. Sci. 94: 599-603). The role of IL-12 in multiple sclerosis
has been investigated using mouse and rat experimental allergic
encephalomyelitis (EAE) models of multiple sclerosis. In a
relapsing-remitting EAE model of multiple sclerosis in mice,
pretreatment with anti-IL-12 mAb delayed paralysis and reduced
clinical scores. Treatment with anti-IL-12 mAb at the peak of
paralysis or during the subsequent remission period reduced
clinical scores. Accordingly, the antibodies or antigen binding
portions thereof of the invention may serve to alleviate symptoms
associated with multiple sclerosis in humans.
D. Insulin-Dependent Diabetes Mellitus
[0691] Interleukin-12 has been implicated as an important mediator
of insulin-dependent diabetes mellitus (IDDM). IDDM was induced in
NOD mice by administration of IL-12, and anti-IL-12 antibodies were
protective in an adoptive transfer model of IDDM. Early onset IDDM
patients often experience a so-called "honeymoon period" during
which some residual islet cell function is maintained. These
residual islet cells produce insulin and regulate blood glucose
levels better than administered insulin. Treatment of these early
onset patients with an anti-IL-12 antibody may prevent further
destruction of islet cells, thereby maintaining an endogenous
source of insulin.
E. Psoriasis
[0692] Interleukin-12 has been implicated as a key mediator in
psoriasis. Psoriasis involves acute and chronic skin lesions that
are associated with a TH1-type cytokine expression profile. (Hamid
et al. (1996) J. Allergy Clin. Immunol. 1:225-231; Turka et al.
(1995) Mol. Med. 1:690-699). IL-12 p35 and p40 mRNAs were detected
in diseased human skin samples. Accordingly, the antibodies or
antigen binding portions thereof of the invention may serve to
alleviate chronic skin disorders such psoriasis.
[0693] The present invention is further illustrated by the
following examples which should not be construed as limiting in any
way. The contents of all cited references, including literature
references, issued patents, and published patent applications, as
cited throughout this application are hereby expressly incorporated
by reference. It should further be understood that the contents of
all the tables attached hereto (see Appendix A) are incorporated by
reference. TABLE-US-00002 TABLE 1 VH3 Family Germline Amino Acid
Sequences Numbering according to Kabat (Joe9 VH included for
comparison) CDR H2 CDR H1 555 666 11111111112222222223 33333
33334444444444 5552225555555666666 66667777777777888222888888899999
SEQ ID NO: germline VH 123456789012345678921234567890 12345
67890123456789 012ABC3456789012345 67890123456789012ABC345678901234
594 dp-29 EVQLVESGGGLVQPGGSLRLSCAASGFTFS DHYMD WVRQAPGKGLEWVG
RTRNKANSYTTEYAASVKG RFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR 595 DP-30
EVQLVESGGGLVQPGGSLRLSCAASGFTFS DHYMS WVRQAQGKGLELVG
LIRNKANSYTTEYAASVKG RLTISREDSKNTLYLQMSSLKTEDLAVYYCAR 596 HC15-7
EVQLVESGGGLVQPGGSLRLSCAASGFTFS DHYMS WVRQAQGKGLELVG
LIRNKANSYTTEYAASVKG RLTISREDSKNTMYLQMSNLKTEDLAVYYCAR 597 VHD26
EVQLLESGGGLVQPGGSLRLSCAASGFTFS DHYMS WVRQAQGKGLELVG
LIRNKANSYTTEYAASVKG RLTISREDSKNTLYLQMSSLKTEDLAVYYCAR 598 DP-31
EVQLVESGGGLVQPGRSLRLSCAASGFTFD DYAMH WVRQAPGKGLEWVS
GISW..NSGSIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCAK 599 DP-32
EVQLVESGGGVVRPGGSLRLSCAASGFTFD DYGMS WVRQAPGKGLEWVS
GINW..NGGSTGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYHCAR 600 DP-33
EVQLVESGGVVVQPGGSLRLSCAASGFTFD DYTMH WVRQAPGKGLEWVS
LISW..DGGSTYYADSVKG RFTISRDNSKNSLYLQMNSLRTEDTALYYCAK 601 dp-35
QVQLVESGGGLVKPGGSLRLSCAASGFTFS DYYMS WIRQAPGKGLEWVS
YI..SSSGSTIYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 602 VH3-8
QVQLLESGGGLVKPGGSLRLSCAASGFTFS DYYNS WIRQAPGKGLEWVS
YI..SSSSSYTNYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 603 yac-9
EVQLVESGGGLVQPGGSLKLSCAASGFTFS GSAMH WVRQASGKGLEWVG
RIRSKANSYATAYAASVKG RFTISRDDSKNTAYLQMNSLKTEDTAVYYCTR 604 dp-38
EVQLVESGGGLVKPGGSLRLSCAASGFTFS NAWMS WVRQAPGKGLEWVG
RIKSKTDGGTTDYAAPVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT 605 LSG2
EVQLVESGGGLVKPGGSLRLSCAASGFTFS NAWMS WVRQAPGKGLEWVG
RIESKTDGGTTDYAAPVKG RFTISRDDSKNTYLQMNSLKTEDTAVYYCTT 606 LSG3
EVQLVESGGGLVKPGGSLRLSCAASGFTFS NAWMS WVRQAPGKGLEWVG
RIKSKTDGGTTDYAAPVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT 607 LSG4
EVQLVESGGGLVKPGGSLRLSCAASGFTFS NAWMS WVRQAPGKGLEWVG
RIKSKTDGGTTNYAAPVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT 608 LSG6
EVQLVESGGGLVKPGGSLRLSCAASGFTFS NAWMN WVRQAPGKGLEWVG
RIKSKTDGGTTDYAAPVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT 609 V3-15
EVQLVESGGALVKPGGSLRLSCAASGFTFS NAWMS WVRQAPGKGLEWVG
RIKSKTDGGTTDYAAPVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT 610 dp-39
EVQLVESGGGLVQPGGSLRLSCPASGFTFS NHYMS WVRQAPGKGLEWSVS
YI..SGDSGYTNYADSVKG RFTISRDNANNSPYLQMNSLRAEDTAVYYCVK 611 dp-40
EVQLVESGGGLVQPGGSLRLSCAASGFTFS NHYTS WVRQAPGKGLEWVS
YS..SGNSGYTNYADSVKG RFTISRDBAKNSLYLQMNSLRAEDTAVYYCVK 612 dp-59
EVQLVESGGGLVQPGGSLRLSCAASGFTFS NSDMN WVHQAPGKGLEWVS
GV..SWNGSRTHYADSVKG RFIISRDNSRNTLYLQTNSLRAEDTAVYYCVR 613 v3-16p
EVQLVESGGGLVQPGGSLRLSCAASGFTFS NSDMN WARKAPGKGLEWVS
GV..SWNGSRTHYVDSVKR RFIISRDNSRNSLYLQKNRRRAEDMAVYYCVR 614 v3-19p
TVQLVESGGGLVEPGGSLRLSCAASGFTFS NSDMN WVRQAPGKGLEWVS
GV..SWNGSRTHYADSVKG RFIISRDNSRNFLYQQMNSLRPEDMAVYYCVR 615 v3-13
EVHLVESGGGLVQPGGALRLSCAASGFTFS NYDMH WVRQATGKGLEWVS
AN..GTAG.DTYYPGSVKG RFTISRENAKNSLYLQMNSLRAGDTAVYYCAR 616 DP-42
EVQLVETGGGLIQPGGSLRLSCAASGFTVS SNYMS WVRQAPGKGLEWVS
VI.Y..SGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 617 dp-44
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFS SYAMH WVRQAPGKGLEWVS
AI...GTGGGTYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDMAVYYCAR 618 DP-45
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFS SYAMH WVRQAPGKGLEWVS
AI...GTGGGTYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDMAVYYCAR 619 dp-47
EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS
AI..SGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 620 f1m
EVQLVESGGGLVQPGGSLRLSCSASGFTFS SYAMH WVRQAPGKGLEYVS
AI..SSNGGSTYYADSVKG RFTISRDNSKNTLYVQMSSLRAEDTAVYYCVK 621 P1
EVQLVESGGGLVQPGGSLRLSCSASGFTGS SYAMH WVRQAPGKGLEYVS
AI..SSNGGSTYYADSVKG RFTLSRDNSKNTLYVQMSSLRAEDTAVYYCVK 622 v3-64
EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYAMH WVRQAPGKGLEYVS
AI..SSNGGSTYYANSVKG RFTISRDNSKNTLYLQMGSLRAEDMAVYYCAR 623 vh26
EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS
AI..SGSGGSTYYGDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 624 B25
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA
VI..SYDGSNKYYTDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 625 b32e
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 626 B37
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMSSLRAEDTAVYYCAR 627 B43
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 628 B48
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 629 B52
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 630 B54
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 631 cos-8
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 632 dp-46
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 633 F2M
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEYVS
AI..SSNGGSTYYADSVKG RFTISRDNSKNTLYLQMSSLRAEDTAVYYCAK 634 F3
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEYVS
AI..SSNGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 635 F7
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 636 hv3005
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 637 P2
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 638 dp-48
EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYDMH WVRQATGKGLEWVS
AI..GTAG.DTYYPGSVKG RFTISRENAKNSLYLQMNSLRAGDTAVYYCAR 639 dp-58
EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYEMN WVRQAPGKGLEWSVS
YI..SSSGSTIYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 640 B1
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRLRARLCITVRE 641 B13
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 642 B18
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 643 B26
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 644 B28E
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 645 B29E
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 646 B29M
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 647 B30
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..WYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 648 B32M
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 649 cos-3
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
FI..RYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 650 dp-49
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 651 dp-50
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..WYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 652 P6
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..WYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 653 P9E
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVRK--- 654 v3-30
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..SYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR 655 v3-33
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
VI..WYDGSNKYYADSVKG RFTISRDNSTNTLFLQMNSLRAEDTAVYYCAR 656 dp-51
EVQLVESGGGLVQPGRSLRLSCAASGFTFS SYSMN WVRQAPGKGLEWVS
YI..SSSSSTIYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 657 dp-77
EVQLVESGGGLVQPGRSLRLSCAASGFTFS SYSMN WVRQAPGKGLEWVS
SI..SSSSSYIYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 658 HHG4
EVQLVESGGGLVQPGRSLRLSCAASGFTFS SYSMN WVRQAPGKGLEWVS
SI..SSSSSYIYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 659 v3-21
EVQLVESGGGLVQPGRSLRLSCAASGFTFS SYSMN WVRQAPGKGLEWVS
SI..SSSSSYIYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 660 v3-48
EVQLVESGGGLVQPGRSLRLSCAASGFTFS SYSMN WVRQAPGKGLEWVS
YI..SSSSSTIYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 661 DP-52
EDQLVESGGGLVQPGRSLRLSCAASGFTFS SYVLH WVRRAPGKGPEWVS
AIG...TGDTYYADSVMG RFTISRDNAKKSLYLQMNSLIAEDNAVYYCAR 662 cos-6
EVQLVESGGGLVQPGRSLRLSCAASGFTFS SYWMH WVRQAPGKGLVWVS
RI..NSDGSSTSYADSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAR 663 dp-53
EVQLVESGGGLVQPGRSLRLSCAASGFTFS SYWMH WVRQAPGKGLVWVS
RI..NSDGSSTSYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 664 dp-54
EVQLVESGGGLVQPGRSLRLSCAASGFTFS SYWMS WVRQAPGKGLEWVA
NI..KQDGSEKYYVDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 665 dp-87
EVQLVESGGGLVQPGRSLRLSCAASGFTFS SYWMH WVRQAPGKGLVWVS
RI..NSDGSSTSYADSMKG QFTISRDNAKNTLYLQMNSLRAEDMAVYYCTR 666 VH3-11
EVQLVESGGGLVQPGRSLRLSCAASGFTFS SYWMS WVRQAPGKGLEWVA
NI..KQDGSEKYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 667 JOE9 VE
QVQLVQSGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA
FI..RYDGSNKYYADSVKG RFTISRDNSKNTLYLQMKSLRAEDTAVYYCTT V.lamda.1
Family Germline Amino Acid Sequences Numbering according to Kabat.
(Joe9 VL included for comparison) 222 99 1111111111222
2227772233333 333334444444444 5555555
55566666666667777777777888888888 899999955 SEQ. ID. NO: *gene VL
123456789012 456ABD8901234 567890123456789 0123456
78901234567890123456789012345678 9012345AB
668 1b DPL5 QSVLTQPPSVSAAPGQKVTISC SGSSSNIGNNY.VS WYQQLPGTAPKLLIY
DNNKRPS GIPDRFSGSKSGTSATLGITGLQTGDEADYYC GTWDSSLSA 669 1d DPL4
QSVLTQPPSVSAAPGQKVTISC SGSSSDMGNYA.VS WYQQLPGTAPKLLIY ENNKRPS
GIPDRFSGSKSGTSATLGITGLWPEDEADYYC LAWDTSPRA 670 1c DPL2
QSVLTQPPSVSAAPGQKVTISC SGSSSNIGSNT.VN WYQQLPGTAPKLLIY SNNQRPS
GVPDRFSGSKSGTSASLAISGLQSGDEADYYC AAWDDSLNG 671 1g DPL3
QSVLTQPPSVSAAPGQKVTISC SGSSSNIGSNY.VY WYQQLPGTAPKLLIY RNNQRPS
GVPDRFSGSKSGTSASLAISGLQSGDEADYYC AAWDDSLNG 672 1a DPL1
QSVLTQPPSVSAAPGQKVTISC SGSSSNIGNN.AVN WYQQLPGTAPKLLIY YDDLLPS
GVSDRFSGSKSGTSASLAISGLRSEDEADYYC AAWDDSLNG 673 1f DPL9
QSVLTQPPSVSAAPGQKVTISC TGSSSNIGAGYVVH WYQQLPGKAPKLLIY GNSNRPS
GVPDQFSGSKSGTSASLAISGLQSEDEADYYC KAWDNSLNA 674 1e DPL8
QSVLTQPPSVSAAPGQKVTISC TGSSSNIGAGYVVH WYQQLPGKAPKLLIY GNSNRPS
GVPDRFSGSKSGTSASLAITGLQAEDEADYYC QSYDSSLSG 675 JOE9 VL
SYVLTQPPSVSGTPGQRVTISC SGGRSNIGSNT.VK WYQQLPGKAPKLLIY GNDQRPS
GVPDRFSGSKSGTSASLAITGVQAEDEADYYC QSYDSSLSG *Williams, JMB, 1996,
264, 220-232
[0694] TABLE-US-00003 TABLE 2 H3 L3 SEQ SEQ ID ID RB assay PHA
assay IFN gamma Clone NO: H3 NO: L3 koff IC50 (M) IC50 (M) IC50 (M)
Joe9 wt 77 SGSYDY 110 QSYDSSLRGSRV 1.00E-01 1.50E-06 1.00E-06 Joe
9wt IgG1 77 SGSYDY 110 QSYDSSLRGSRV 5.00E-07 70-1 78 HGSHDN 110
Joe9 wt 1.34e-2 2.00E-07 70-1 IgG1 78 HGSMDN 110 Joe9 wt 2.00E-07
70-2 79 HGSYDY 110 Joe9 wt 3.30E-02 3-5.0E-7 70-7 80 RRRSNY 110
Joe9 wt 1.29E-01 3-5.0E-7 70-13 81 SGSIDY 110 Joe9 wt 7.20E-02
3-5.0E-7 78-34 77 wt 111 QSYDRGFTGSRV 1.64e-2 2.00E-07 6.00E-07
78-25 77 wt 112 QSYDSSLRGSRV 5.00E-02 78-28 77 wt 112 QSYDSSLRGSRV
4.66E-02 78-35 77 wt 113 QSYDSSLTGSRV 4.99E-02 4.00E-07 79-1 77 wt
114 QSYDSSLWGSRV 2.00E-07 6.00E-07 101-14 79 70-2 111 78-34
7.52E-03 101-9 79 70-2. 113 78-35 8.54E-03 101-19 81 70-13 111
78-34 4.56E-02 101-8 81 70-13 111 78-34 1.01E-02 101-4 81 70-13 113
78-35 9.76E-03 101-5 81 70-13 113 78-35 4.45E-02 101-11 (12) 78
70-1 111 78-34 4.5e-3 3.00E-08 101-11 IgG1 78 70-1 111 78-34
1.60E-09 26-1 (2,3) 78 70-1 114 79-1 7.4e-3 6.00E-08 136-9 82
HGSHDD 115 QTYDISESGSRV 3.20E-03 136-10 82 HGSHDD 116 QSYDRGFTGSRV
1.40E-03 2.00E-09 136-14 83 HGSHDN 117 QTYDRGFTGSRV 1.10E-03
3.00E-10 1.00E-07 136-15 83 HGSHDN 118 QTYDKGFTGSSV 7.4e-4 1.00E-10
2.00E-09 136-15 83 HGSHDN 118 QTYDKGFTGSSV 4.60E-04 6.00E-09
germline 136-16 83 HGSHDN 119 QSYDRRFTGSRV 6.10E-04 3.00E-10
5.00E-09 136-17 83 HGSHDN 120 QSYPWNFTGSRV 2.90E-05 2.00E-09
7.00E-09 136-18 83 HGSHDN 121 QSYDRGFTGSRV 1.10E-03 8.00E-10 136-21
83 HGSRDN 122 QSYDNGFTGSRV 4.20E-04 2.00E-09 136-24 83 HGSHDN 123
QSYDNAVTASKV 8.90E-04 1.00E-09 101-11 84 TT HGSHDN WGOG 124
QSYDRGFTGSRV 4.5 .times. 10 - 3 2 .times. 10 - 9 2.00E-08 136-15M1
85 AK ...... .... 124 QSYDRGFTGSRV 4.00E-10 149-4 86 .. ...... .S..
124 ............ 1.37 .times. 10 - 3 8 .times. 10 - 11 3.00E-09
149-5 87 .. .....T .... 125 QSYDSSLWGTRV 1.02 .times. 10 - 3 1.2
.times. 10 - 10 3.00E-09 149-6 84 .. ...... .... 124 ............
2.73 .times. 10 - 3 6 .times. 10 - 10 2.00E-09 149-7 84 .. ......
.... 126 .....D...... 1.13 .times. 10 - 3 9 .times. 10 - 10
3.00E-09 149-8 88 K. ...... .... 2.33 .times. 10 - 3 3 .times. 10 -
9 149-9 89 K. ...... ..H. 127 ...E......M. 3.54 .times. 10 - 3 1.8
.times. 10 - 10 149-11 90 .. ...... .S.. 128 ....N....A.. 1.43
.times. 10 - 2 2 .times. 10 - 10 4.00E-09 149-12 84 .. ...... ....
3.73 .times. 10 - 3 neutralising 149-13 84 .. ...... .... 2.22
.times. 10 - 3 5 .times. 10 - 10 149-14 91. .. .R..N. .... 1.5
.times. 10 - 10 6.00E-09 92 TT HGSHDN 124 QSYDRGFTGSRV 156-1 93 ..
.....T 126 .....D...... 5.00E-03 156-2 93 .. .....T 129
.....R...... 156-3 93 .. .....T 128 ....N....A.. 9.00E-03 156-4 93
.. .....T 127 ...E.....SM. 156-5 93 .. .....T 130 .T..K.....S.
156-6 92 .. ...... 126 .....D...... 3.00E-03 156-7 92 .. ...... 129
.....R...... 156-8 92 .. ...... 128 ....N....A.. 156-9 92 .. ......
127 ...E.....SM. 156-10 92 .. ...... 130 .T..K.....S. 156-11 94 .K
...... 126 .....D...... 156-12 94 .K ...... 129 .....R...... 156-13
94 .K ...... 128 ....N....A.. 156-14 94 .K ...... 127 ...E.....SM.
156-15 94 .K ...... 130 .T..K.....S. 156-16 93 .. .....T 124
............ 156-17 92 .. ...... 125 ....SSLW.T.. 6.00E-03 156-18
93 .. .....T 125 ....SSLW.T.. 92 TT HGSHDN 124 QSYDRGFTGSRV 103-1
95 .. Q.R... 124 ............ 2.9 .times. 10 - 3 103-2 96 K. R.R...
130 .T..K.....S. 7.3 .times. 10 - 4 7.00E-11 1.00E-09 103-3 97 ..
.....K 124 ............ 2.5 .times. 10 - 3 103-6 131 .....D...T..
4.5 .times. 10 - 4 103-7 98 .. .....D 131 .....D...T.. 3.7 .times.
10 - 4 1.40E-10 1.00E-09 103-8 99 K. ...... 130 .T..K.....S. 3.3
.times. 10 - 4 6.00E-11 1.50E-09 103-14 & 9 100 KT HGSHDN 132
QSYDRGFTGSMV 6.7e-4 4.00E-11 1.20E-09 103-8 & 2 100 KT HGSHDN
133 QTYDKGFTGSSV 5.3e-4 1.50E-09 103-4 101 TT HGSHDN 134
QSYDRGFTGARV 1.6e-4 8.60E-11 9.00E-10 103-152 101 TT HGSHDN 135
QSYERGFTGARV 8.60E-11 102 TT SGSYDY 136 QSYDRGFTGSRVF 170-1 102 ..
...... 137 .........FK.. 2.35E-03 170-2 102 .. ...... 138
.......VSAY.. 8.80E-04 170-3 102 .. ...... 139 ......L.VTK..
1.11E-03 170-4 102 .. ...... 140 ......Y.A.... 8.11E-04 170-7 102
.. ...... 141 ..........K.. 5.30E-04 170-11 102 .. ...... 142
......L..F... 4.40E-04 170-13 102 .. ...... 143 .........YK..
1.59E-03 170-15 102 .. ...... 144 ......L..Y.L. 4.43E-03 170-19 103
.. H..H.N 145 ........DYK.. 1.00E-03 170-21 104 .. H..Q.N 146
.........P.L. 3.89E-03 170-22 102 .. ...... 147 ......L......
5.60E-04 170-23 103 .. H..H.N 148 .........A..W 1.00E-03 2.00E-10
170-24 104 .. H..Q.N 149 .........Y... 2.80E-04 5.00E-10 170-35 105
A. H..Q.N 136 ............. 1.00E-05 170-38 150 .........P...
2.10E-04 170-39 151 ......M.S.... 2.79E-03 170-36 83 HGSHDN 152
QSYDRDSTGSRVF 4.00E-04 2.00E-10 170-25 106 HGSQDT 153 QSYDSSLRGSRVF
5.00E-04 5.00E-11 106 SGSYDY 136 QSYDRGFTGSRVF 73-B1 107 SGSYDY 154
H...SD....... 3.25E-03 >1E-8 73-B2 107 SGSYDY 155 H.SES........
2.07E-03 73-B6 107 SSSYDY 156 H...NR....... 2.51E-03 >1E-8 73-C1
107 SGSYDY 157 H...SR....... 2.71E-03 >1E-8 73-C2 107 SGSYDY 158
....SE....... 3.79E-03 73-C6 107 SGSYDY 159 ....T........ 3.96E-03
73-D1 107 SGSYDY 160 H...S........ 3.99E-03 73-D2 107 SGSYDY 161
....T........ 3.56E-03 73-D4 107 SGSYDY 162 H...TK....... 5.36E-03
73-D5 107 SGSYDY 163 H.S.S........ 3.57E-03 73-E3 107 SGSYDY 164
....SD....... 4.98E-03 73-E6 107 SGSYDY 165 H..ES........ 4.17E-03
73-F3 107 SGSYDY 166 ....APWS..... 7.08E-03 73-F5 107 SGSYDY 167
...DSD....K.. 3.74E-03 73-G2 107 SGSYDY 168 HTN.S........ 3.98E-03
73-G3 107 SGSYDY 169 H...TR....... 3.50E-03 73-G4 107 SGSYDY 170
....MR....... 6.58E-03 73-G5 107 SGSYDY 171 H.S.SDS...... 6.01E-03
73-G6 107 SGSYDY 172 ...NTD....... 6.30E-03 73-H2 107 SGSYDY 173
....S........ 5.93E-03 73-F6 107 SGSYDY 174 H...M........ 5.87E-03
73-H3 107 SGSYDY 175 H...N........ 6.85E-03 73-C5 107 SGSYDY 176
H.H..D....... 4.84E-03 73-B7 108 HGSQDN 177 QSYDSSLRGSRV 2.50E-03
7.00E-09
136 QSYDRGFTGSRVF M2 A2 83 HGSHDN 178 ......IH..... 4.00E-02 M2 A4
83 HGSHDN 179 ....S..P..... 8.49E-03 M2 A5 83 HGSHDN 180
....I.S...... 4.01E-02 M2 B1 83 HGSHDN 181 ....S.L...... 7.97E-03
M2 B3 83 HGSHDN 182 ....I.M...... 4.60E-02 M2 B4 83 HGSHDN 183
....I.L...... 4.42E-02 M2 B5 83 HGSHDN 184 ....S.V...... 8.38E-03
M2 B6 83 HGSHDN 185 ......L.A.... 2.81E-02 M2 C2 83 HGSHDN 181
....S.L...... 4 85E-02 M2 C3 83 HGSHDN 186 ....T.L...... 4.62E-02
M2 C4 83 HGSHDN 181 ....S.L...... 8.16E-03 M2 C5 83 HGSHDN 187
....TAL...... 4.71E-02 M2 D1 83 HGSHDN 188 ....IR....... 3.71E-02
M2 D2 83 HGSHDN 189 ....IRS...... 3.85E-02 M2 D3 83 HGSHDN 190
....NRL...... 3.33E-02 M2 D4 83 HGSHDN 191 ...ETS....... 5.81E-02
M2 D5 83 HGSHDN 192 ....SSS...... 5.18E-02 M2 D6 83 HGSHDN 193
....S...A.... 5.01E-02 M2 E1 83 HGSHDN 194 .T..K.....S.. 5.32E-02
M2 E2 83 HGSHDN 195 ....N........ 4.77E-02 M2 E6 83 HGSHDN 196
....T...K.... 9.77E-03 M2 F1 83 HGSHDN 197 ....SDV...... 6.16E-02
M2 H5 83 HGSHDN 198 ....A........ 9.90E-03 124 QSYDRGFTGSRV A5 83
HGSHDN 199 ......THPSML 1.12E-03 A12 83 HGSHDN 200 ......TTPRPM
1.43E-03 A4 83 HGSHDN 201 ......RNPALT 1.47E-03 A6 83 HGSHDN 202
......THPWLH 1.87E-03 A10 83 HGSHDN 203 ......NSPATV 1.87E-03 A11
83 HGSHDN 204 ......TFPSPQ 2.07E-03 C2 83 HGSHDN 205 ......LNPSAT
2.23E-03 A8 83 HGSHDN 206 ......KSNKML 2.37E-03 B8 83 HGSHDN 207
......HTAHLY 2.40E-03 C6 83 HGSHDN 208 ......QTPSIT 2.42E-03 A3 83
HGSHDN 209 ......YPRNIL 2.51E-03 B11 83 HGSHDN 210 ......ITPGLA
2.95E-03 B5 83 HGSHDN 211 ......QPHAVL 3.04E-03 C10 83 HGSHDN 212
......NSPIPT 3.10E-03 C4 83 HGSHDN 213 ......TPNNSF 3.23E-03 C3 83
HGSHDN 214 ....S.VDPGPY 3.34E-03 B2 83 HGSHDN 215 ......RPRHAL
3.61E-03 A2 83 HGSHDN 216 ......PYHPIR 3.80E-03 C5 83 HGSHDN 217
......PHTQPT 3.91E-03 A7 83 HGSHDN 218 ......HNNFSP 3.95E-03 C9 83
HGSHDN 219 ......PTHLPH 3.97E-03 B3 83 HGSHDN 220 ......TPSYPT
4.12E-03 C8 83 HGSHDN 221 ....S.TSNLLP 5.36E-03 B7 83 HGSHDN 222
......DSNHDL 5.45E-03 A1 83 HGSHDN 223 ......LPRLTH 5.66E-03 C7 83
HGSHDN 224 ......IPTSYL 5.83E-03 C12 83 HGSHDN 225 ......LRVQAP
5.85E-03 B10 83 HGSHDN 226 ......LSDSPL 6.04E-03 B6 83 HGSHDN 227
....S.SLRRIL 7.58E-03 A9 83 HGSHDN 228 ......PARTSP 7.98E-03 B9 83
HGSHDN 229 ......RAAHPQ 8.66E-03 124 QSYDRGFTGSRV 177-D7 83 HGSHDN
230 ......TQPABI 4.07E-04 177-G6 83 HGSHDN 231 ......THPTMI
5.50E-04 177-D9 83 HGSHDN 232 ......RIPABT 6.32E-04 177-C6 83
HGSHDN 233 ......THPVPA 7.94E-04 177-H5 83 HGSHDN 234 ......SBPIPA
1.32E-03 177-H9 83 HGSHDN 235 ......THPVPA 1.58E-03 177-H10 83
HGSHDN 236 ......THPTMY 3.44E-03 144-F1 83 HGSHDN 237 ......HHYTTF
5.80E-04 43-E3 83 HGSHDN 238 ......SHPAAE 8.00E-04 43-E9 83 HGSHDN
239 ......TIPSIE 8.00E-04 43-G2 83 HGSHDN 240 ......SSPAIM 7.00E-04
43-G3 83 HGSHDN 241 ......IWPNLN 9.00E-04 31-A6 83 HGSHDN 242
......THPNLN 5.00E-04 31-B5 83 HGSHDN 243 ......THPSIS 5.00E-04 124
QSYDRGFTGSRV Y17 83 HGSHDN 244 QSYDRGSAPMIN 8.90E-05 4.50E-10
>1E-8 Y19 83 HGSHDN 245 QSYDRGHHPAMS 2.26E-04 3.00E-11 >1E-8
Y38 83 HGSHDN 246 ......THPSIT 5.08E-04 5.50E-11 2.60E-09 Y45 83
HGSHDN 247 ......TDPAIV 6.17E-04 4.00E-11 4.30E-09 Y61 83 HGSHDN
248 ......THPALL 2.75e-4 4E-11 1.40E-10 Y61 IgG 83 HGSHDN 248
......THPALL 1.50E-04 1.60E-11 1.30E-10 Y61 IgG 83 HGSHDN 248
......THPALL 1.50E-04 1.60E-11 1.30E-10 1.60E-10 germline Y139 83
HGSHDN 249 ......SHPALT 5.92E-04 3E-11 4.50E-10 Y139 IgG1 83 HGSHDN
249 ......SHPALT 1.00E-09 Y174 83 HGSHDN 250 ......TTPAPE 7.55E-04
6E-11 2.00E-09 Y177 83 HGSHDN 251 ......SHPTLI 6.61E-04 5E-11
1.00E-09 A5 83 HGSHDN 252 ......THPSML 4.50E-04 6.60E-11 A12 83
HGSHDN 253 ......TTPRPM 5.57E-04 2.50E-10 D9 83 HGSHDN 254
......RLPAQT 8.21E-04 3.5E-09 >> G6 83 HGSHDN 255
......THPLTI 5.08E-04 1E-10 1.00E-09 G6 IgG1 83 HGSHDN 255
......THPLTI 1.00E-09 C6 83 HGSHDN 256 QSYDRGQTPSIT 1.07E-03
3.5E-10 1.00E-08 Y55 83 HGSHDN 257 QSYDRGTHFQMY 1.06E-03 1.40E-10
>1E-8 A4 83 HGSHDN 258 QSYDRGRNPALT 6.30E-04 2.50E-10 A03 83
HGSHDN 259 QSYDRGTHPLTM 3.04E-04 3.00E-11 4.00E-10 A03 IgG1 83
HGSHDN 260 QSYDRGTHPLTM 3.04e-4 2.90E-11 3.80E-10 A03 IgG 83 HGSHDN
260 QSYDRGTHPLTM 2.50E-04 3.50E-11 1.75E-10 germline 83 99-B11 83
HGSHDN 261 QSYDSGYTGSRV 5.40E-03 99-C11 83 HGSHDN 262 QSYDSGFTGSRV
5.70E-03 99-H4 83 HGSHDN 263 QSYDSRFTGSRV 4.80E-03 99-E9 83 HGSHDN
262 QSYDSGFTGSRV 5.40E-03 99-H7 83 HGSHDN 264 QSYPDGTPASRV 3.30E-03
99-H11 83 HGSHDN 265 QSYSTHMPISRV 4.90E-03 99-F6 83 HGSHDN 266
QSYDSGSTGSRV 4.90E-03 99-F7 83 HGSHDN 267 QSYPNSYPISRV 4.80E-03
99-F8 83 HGSHDN 268 QSYIRAPQQV 3.70E-03 99-F11 83 HGSHDN 262
QSYDSGFTGSRV 5.40E-03 99-G7 83 HGSHDN 269 QSYLKSRAFSRV 4.80E-03
99-G11 83 HGSHDN 270 QSYDSRFTGSRV 4.30E-03 124 QSYDRGFTGSRV
L3.3R3M-B1 83 HGSHDN 271 ......FTGSMV 5.46E+00 L3.3R3M-B3 83 HGSHDN
272 ......FTGSMV 5.51E+00 L3.3R3M-C6 83 HGSHDN 273 ......FTGFDG
6.17E+00 L3.3R3M-F9 83 HGSHDN 274 ......TAPALS 4.99E+00 L3.3R3M-G8
83 HGSHDN 275 ......SYPALR 5.55E+00 L3.3R3M-H6 83 HGSHDN 276
......NWPNSN 5.69E+00 L3.3R3M-H10 83 HGSHDN 277 ......TAPSLL
5.35E+00 L3.3R3M-A3 83 HGSHDN 278 ......FTGSMV 5.37E+00 L3.3R3M-F8
83 HGSHDN 279 ......TTPRIR 4.99E+00 L3.3R3M-G1 83 HGSHDN 280
......FTGSMV 4.21E+00 L3.3R3M-G7 83 HGSHDN 281 ......FTGSMV
4.24E+00 L3.3R3M-H11 83 HGSHDN 282 ......MIPALT 3.95E+00 Y61-L94N
109 CKT HGSHDN 283 QSYDRNTHPALL 8.00E-11 Y61-L94F 109 CKT HGSHDN
284 QSYDRFTHPALL 6.00E-11 Y61-L94Y 109 CKT HGSHDN 285 QSYDRYTHPALL
2.00E-11 2.00E-11 Y61-L94Y IgG 109 CKT HGSHDN 285 QSYDRYTHPALL
1.25E-04 6.00E-11 5.00E-11 4.00E-11 Y61-L50Y 109 CKT HGSHDN 286
QSYDRGTHPALL 2.00E-11 2.00E-11
Y61-L50Y* IgG 109 CKT HGSHDN 286 QSYDRGTHPALL 6.98E-05 2.00E-11
3.00E-11 Y61-L50Y- 109 CKT HGSHDN 286 QSYDRGTHPALL 2.99E-05
6.00E-11 2.00E-11 H31E** IgG Y61-L50Y- 109 CKT HGSHDN 287
QSYDRYTHPALL 4.64E-05 1.00E-11 1.00E-11 H31E- 94Y** IgG J695 (Y61-
109 CKT HGSHDN 287 QSYDRYTHPALL 5.14E-05 5.00E-11 1.00E-11 5.00E-12
L94Y-L50Y IgG*) *CDR L2: L50G to Y **CDR L2: L50G to Y; CDR H1;
H31S to E
[0695] TABLE-US-00004 TABLE 3 CDR H1 CDR H2 Kabat Number 27 28 29
30 31 32 33 34 35 50 51 52 52A 53 54 55 56 57 58 59 60 61 62 63 64
65 Y61 VH F T F S S Y G M H F I R Y D G S N K Y Y A D S V K G
Contact Positions X X X X x X X X X X X X Hypermutation X X X X X X
Positions CDR H3 CDR L1 Kabat number 95 96 97 98 101 102 24 25 26
27 27A 27B 28 29 30 31 32 33 34 Y61 VL H G S H D N S G G R S N I G
S N T V K Contact Positions X X X X X X X X x Hypermutation
Positions X X X CDR L2 CDR L3 Kabat number 50 51 52 53 54 55 56 89
90 91 92 93 94 95 95A 95B 95C 96 97 Y61 VL G N D Q R P S Q S Y D R
G T H P A L L Contact Positions X x X x x X X X x Hypermutation
Positions X X x contact and/or hypermutation position X contact
and/or hypermutation position mutated in Y61
[0696] TABLE-US-00005 TABLE 4 Neutralization Activity in the
Presence of Excess Free IL-12 p40 PHA assay IC50 PHA assay IC50 (M)
PHA assay IC50 (M) SEQ ID NO: Clone (M) p70:p40 1:0 p70:p40 1:20
p70:p40 1:50 VH: 47 136-15 2.00E-09 5.00E-09 4.00E-09 VL: 48 VH: 51
149-5 6.50E-09 7.00E-09 4.00E-09 VL: 52 VH: 53 149-6 9.00E-10
1.00E-09 1.00E-09 VL: 54 VH: 84 149-7 3.50E-09 2.50E-09 4.00E-09
VL: 126 VH: 23 Y61 IgG 1.80E-10 1.80E-10 VL: 24 VH: 65 AO3 IgG1
2.50E-10 2.20E-10 VL: 66 VH: 31 J695 1.00E-11 3.50E-11 VL: 32
EXAMPLES
Example 1
Isolation of Anti-IL-12 Antibodies
A. Screening for IL-12 Binding Antibodies
[0697] Antibodies to hIL-12 were isolated by screening three
separate scFv phage display libraries prepared using human VL and
VH cDNAs from mRNA derived from human tonsils (referred to as scFv
1), tonsil and peripheral blood lymphocytes (PBL) (referred to as
scFv 2), and bone marrow-derived lymphocytes (referred to as BMDL).
Construction of the library and methods for selection are described
in Vaughan et al. (1996) Nature Biotech. 14: 309-314.
[0698] The libraries were screened using the antigens, human IL-12
p70 subunit, human IL-12 p40 subunit, chimaeric IL-12 (mouse
p40/human p35), mouse IL-12, biotinylated human IL-12 and
biotinylated chimaeric IL-12. IL-12 specific antibodies were
selected by coating the antigen onto immunotubes using standard
procedures (Marks et al., (1991) J. Mol. Biol. 222: 581-597). The
scFv library 2 was screened using either IL-12, or
biotinylated-IL-12, and generated a significant number of IL-12
specific binders. Five different clonotypes were selected,
determined by BstN1 enzymatic digestion patterns, and confirmed by
DNA sequencing. The main clonotypes were VHDP58NVLDPL11,
VHDP77NVLDPK31, VHDP47/VL and VHDP77/VLDPK31, all of which
recognized the p40 subunit of IL-12.
[0699] Screening of the BMDL library with IL-12 p70 generated 3
different clonotypes. Two of these were found to be cross-reactive
clones. The dominant clone was sequenced and consisted of
VHDP35/VLDP. This clone recognizes the p40 subunit of IL-12.
Screening of the scFv library 1, using IL-12 p70, did not produce
specific IL-12 antibodies.
[0700] In order to identify IL-12 antibodies which preferentially
bind to the p70 heterodimer or the p35 subunit of IL-12, rather
than the p40 subunit, the combined scFv 1+2 library, and the BMDL
library were used. To select IL-12 antibodies that recognized the
p70 heterodimer or p35 subunit, phage libraries were preincubated
and selected in the presence of free p40. Sequencing of isolated
clones revealed 9 different antibody lineages. Subunit preferences
were further analyzed by `micro-Friguet` titration. The supernatant
containing scFv was titrated on biotin-captured IL-12 in an ELISA
and the ED.sub.50 determined. The concentration of scFv producing
50% ED was preincubated with increasing concentrations of free p70
or p40 (inhibitors). A decrease in the ELISA signal on biotin-IL-12
coated plates was measured and plotted against the concentration of
free p70 or p40. This provided the IC.sub.50 for each clone with
respect to p70 and p40. If the titrations for both subunits
overlaps, then the scFv binds to both p40 and p70. Any variation
from this gives the degree of preference of p70 over p40.
B. Affinity Maturation of Antibody Lineage Specific for IL-12 (Joe
9)
[0701] The clones were tested for their ability to inhibit IL-12
binding to its receptor in an IL-12 receptor binding assay
(referred to as RBA), and for their ability to inhibit IL-12
induced proliferation of PHA stimulated human blast cells (PHA
assay), described in Example 3. Clone Joe 9 had the lowest
IC.sub.50 value in both the RBA and the PHA assay, with an
IC.sub.50 value of 1.times.10.sup.-6 M in both assays. In addition
the heavy chain variable region (VH) of Joe 9 had the least number
of changes compared to the closest germline sequence COS-3,
identified from the VBASE database. Table 1 (see Appendix A) shows
the V.sub.H3 family of germline sequences, of which COS-3 is a
member, as well as members of V.sub..lamda.1 family of germline
sequences. Therefore, Joe 9 was selected for affinity maturation.
The amino acids sequences of VH and VL of the Joe9 wild type (Joe9
wt) antibody are shown in FIG. 1A-1D.
[0702] In order to increase the affinity of Joe 9, various
mutations of the complementarity determining region 3 (CDR3) of
both the heavy and light chains were made. The CDR3 variants were
created by site-directed PCR mutagenesis using degenerate
oligonucleotides specific for either the heavy chain CDR3 (referred
to as "H3") or the light chain CDR3 (referred to as "L3"), with an
average of three base substitutions in each CDR3 (referred to as
"spike"). PCR mutagenesis of the heavy chain CDR3 was performed
using the degenerate heavy chain oligonucleotide containing a
random mixture of all four nucleotides,
5'TGTCCCTTGGCCCCA(G)(T)(A)(G)(T)(C)(A)(T)(A)(G)(C)(T)(C)(C)(C)(A)(C)(T)
GGTCGTACAGTAATA 3' (SEQ ID NO: 580), and oligonucleotide pUC
Reverse Tag GAC ACC TCG ATC AGC GGA TAA CAA TTTCAC ACA GG (SEQ ID
NO: 581) to generate a repertoire of heavy chain CDR3 mutants. The
parent light chain was amplified using Joe 9 reverse
oligonucleotide (5'TGG GGC CAA GGG ACA3' (SEQ ID NO:582) and the
fdteteseq 24+21 oligonucleotide (5'-ATT CGT CCT ATA CCG TTC TAC TTT
GTC GTC TTT CCA GAC GTT AGT-3' (SEQ ID NO: 583).
[0703] Complementarity between the two PCR products was used to
drive annealing of the two fragments in a PCR assembly reaction and
the full length recombined scFv library was amplified with pUC
Reverse Tag (SEQ ID NO: 581) and fdTag 5'-ATT CGT CCT ATA CCG
TTC-3' (SEQ ID NO: 584). PCR mutagenesis of the light chain was
performed using the light chain oligonucleotide containing a
mixture of all four nucleotides
5'GGTCCCAGTTCCGAAGACCCTCGAACC(C)(C)(T)(C)(A)(G)(G)(C)(T)
(G)(C)(T)(G)(T)(C)ATATGACTGGCAGTAATAGTCAGC 3' (SEQ ID NO: 585), and
Joe 9 reverse oligonucleotide 5'TGG GGC CAA GGG ACA3' (SEQ ID NO:
586) to produce a repertoire of light chain CDR3 mutants. The
parent heavy chain was amplified with pUC Reverse Tag (SEQ ID NO:
581) and HuJH3FOR oligonucleotide 5'TGAAGAGACGGTGACCATTGTCCC3' (SEQ
ID NO: 587). Complementarity between the two PCR products was used
to drive annealing of the two fragments in a PCR assembly reaction
and the full length recombined scFv library was amplified with
Reverse Tag GAC ACC TCG ATC AGC G (SEQ ID NO: 588) and HuJX 2-3 FOR
NOT oligonucleotide 5'GAG TCA TTC TCG ACT TGC GGC CGC ACC TAG GAC
GGT CAG CTT GGT CCC 3' (SEQ ID NO: 589).
[0704] Heavy chain CDR3 mutants were selected using 1 nM
biotinylated IL-12, and washed for 1 h at room temperature in PBS
containing free IL-12 or p40 at a concentration of 7 nM. Clones
were analyzed by phage ELISA and those that bound to IL-12 were
tested in BIAcore kinetic binding studies using a low density IL-12
chip (see procedure for BIAcore analysis in Example 5). Generally,
BIAcore analysis measures real-time binding interactions between
ligand (recombinant human IL-12 immobilized on a biosensor matrix)
and analyte (antibodies in solution) by surface plasmon resonance
(SPR) using the BIAcore system (Pharmacia Biosensor, Piscataway,
N.J.). The system utilizes the optical properties of SPR to detect
alterations in protein concentrations within a dextran biosensor
matrix. Proteins are covalently bound to the dextran matrix at
known concentrations. Antibodies are injected through the dextran
matrix and specific binding between injected antibodies and
immobilized ligand results in an increased matrix protein
concentration and resultant change in the SPR signal. These changes
in SPR signal are recorded as resonance units (RU) and are
displayed with respect to time along the y-axis of a sensorgram. To
determine the off rate (k.sub.off), on rate (k.sub.on), association
rate (Ka) and dissociation rate (Kd) constants, BIAcore kinetic
evaluation software (version 2.1) was used. Clones that
demonstrated an improvement in the k.sub.off rate were analyzed by
neutralization assays which included inhibition by antibody of
IL-12 binding to its receptor (RBA assay), inhibition of
IL-12-induced proliferation in PHA stimulated human blast cells
(PHA assay), and inhibition of IL-12-induced interferon gamma
production by human blast cells (IFN gamma assay). A summary of the
dissociation rates and/or IC.sub.50 values from neutralization
assays of heavy chain CDR3 spiked clones 70-1 through 70-13 is
presented in Table 2 (see Appendix A). Clone 70-1 displayed a
k.sub.off rate that was better than the parent Joe 9 clone, and had
the lowest IC.sub.50 value of 2.0.times.10.sup.-7 M. Therefore
clone 70-1 was selected for conversion to complete IgG1.
[0705] Light chain CDR3 mutants were selected using 1 nM
biotin-IL-12 and washed with PBS containing 7 nM free p40. Clones
were screened in phage ELISA and those that bound to IL-12 were
tested in BIAcore binding analysis using low density IL-12 chips.
Clones that displayed an off rate which was better than the parent
Joe 9 clone were tested in neutralization assays which measured
either, inhibition of IL-12 receptor binding, or inhibition of PHA
blast cell proliferation. A summary of the dissociation rates
and/or IC.sub.50 values from neutralization assays of light chain
CDR3 mutant clones, 78-34 through 79-1, is presented in Table 2
(see Appendix A).
[0706] Based on the k.sub.off rate, clones 78-34 and 78-35
displayed an improved k.sub.off rate compared to the parent Joe 9.
Both of these clones were selected for combination analysis with
heavy chain mutants.
C. Combination Clones
[0707] Mutant light and heavy chain clones that exhibited the best
binding characteristics were used for combination and assembly of
scFvs. Mutant clones with improved potency characteristics were
combined by PCR overlap extension and pull-through of the mutated
VH and VL segments as described above. Clones 101-14 through 26-1,
shown in Table 2 (see Appendix A), were produced from the
combination of heavy chain mutants (70-2, 70-13 and 70-1) with
light chain mutants (78-34, 78-35 and 79-1). The k.sub.off rates
and/or IC.sub.50 values from neutralization assays for these clones
are presented in Table 2.
[0708] BIAcore binding analysis identified clone 101-11, produced
from the combination of the heavy chain CDR3 mutant clone 70-1 with
the light chain CDR3 mutant clone 78-34, as having an off rate of
0.0045 si. This k.sub.off rate was a significant improvement
compared to the k.sub.off rates for either the heavy chain CDR3
mutant clone 70-1 (0.0134.sup.s-1), or for the light chain CDR3
mutant clone 78-34 (0.0164.sup.s-1) alone. Furthermore, clone
101-11 showed a significant improvement in neutralization assays.
Accordingly, clone 101-11 was selected for affinity maturation as
described below.
D. Affinity Maturation of Clone 101-11
[0709] Further affinity maturation of clone 101-11 consisted of
repeat cycles of PCR mutagenesis of both the heavy and light chain
CDR3s of 101-11 using spiked oligonucleotide primers. The clones
were selected with decreasing concentrations of biotinylated IL-12
(bio-IL-12). The binding characteristics of the mutated clones was
assessed by BIAcore binding analysis and RBA, PHA neutralization
assays. The k.sub.off rates and/or IC.sub.50 values for clones
136-9 through 170-25 are presented in Table 2 (see Appendix A).
Clone 103-14 demonstrated an improved IC.sub.50 value in both the
receptor binding assay and the PHA blast assay. Clone 103-14 also
demonstrated a low k.sub.off rate, and accordingly was selected for
further affinity maturation.
E. Generation and Selection of Randomized Libraries of Clone 103-14
Light CDR3
[0710] The light chain CDR3 of clone 103-14 (QSYDRGFTGSMV (SEQ ED
NO: 590)) was systematically randomized in 3 segments using 3
different libraries as outlined below, where X is encoded by a
randomized codon of sequence NNS with N being any nucleotide and S
being either deoxycytosine or deoxyguanidine. TABLE-US-00006 L3.1 =
XXXXXXFTGSMV (SEQ ID NO: 591) L3.2 = QSYXXXXXXSMV (SEQ ID NO: 592)
L3.3 = QSYDRGXXXXXX (SEQ ID NO: 593)
[0711] Randomized mutagenesis of all three light chain CDRs
(referred to as L3.1, L3.2, and L3.3) of clone 103-14 was
performed. The heavy chain CDR3 (referred to as H3) of clone 103-14
was not mutated. Four randomized libraries based on clone 103-14
(H3 and L3.1, L3.2 & L3.3) were constructed and subjected to a
large variety of selection conditions that involved using limiting
antigen concentration and the presence or absence of excess free
antigen (p40 and p70). The outputs from selections (clones 73-B1
through 99-G11) were screened primarily by BIAcore, and on occasion
with RBA and are shown in Table 2 (see Appendix A).
[0712] Random mutagenesis of the light chain CDR of 103-14
generated clone Y61, which exhibited a significant improvement in
IC.sub.50 value compared to the parent clone 103-14. Y61 was
selected for conversion to a whole IgG1. Whole Y61-IgG1 has an
IC.sub.50 value of approximately 130 pM determined by the PHA
assay. The IC.sub.50 value was not affected by a 50 fold molar
excess of free p40, demonstrating that free p40 did not cross-react
with Y61 anti-IL-12 antibody to thereby decrease the antibody
binding to the heterodimer. The full length sequences of Y61 heavy
chain variable region and light chain variable region are shown
below.
Y61 Heavy Chain Variable Region Peptide Sequence
[0713] TABLE-US-00007 (SEQ ID NO: 23) CDR H1
QVQLVESGGGVVQPGRSLRLSCAASFTFS SYGMH WVRQAPGKGLEWVA CDR H2
FIRYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCKT CDR H3 HGSHDN
WGQGTMVTVSS
Y61 Light Chain Variable Region Peptide Sequence
[0714] TABLE-US-00008 (SEQ ID NO: 24) CDR L1 QSVLTQPPSVSGAPGQRVTISC
SGGRSNIGSNTVK WYQQLPGTAPKLL IY CDR L2 GNDQRPS
GVPDRFSGSKSGTSASLAITGLQAEDEADYYC CDR L3 QSYDRGTHPALL
FGTGTKVTVLG
CDR residues are assigned according to the Kabat definitions.
Example 2
Mutation of Y61 at Hypermutation and Contact Positions
[0715] Typically selection of recombinant antibodies with improved
affinities can be carried out using phage display methods. This is
accomplished by randomly mutating combinations of CDR residues to
generate large libraries containing single-chain antibodies of
different sequences. Typically, antibodies with improved affinities
are selected based on their ability to reach an equilibrium in an
antibody-antigen reaction. However, when Y61 scFV was expressed on
phage surface and incubated with IL-12, selection conditions could
not be found that would allow the system to reach normal
antibody-antigen equilibrium. The scFV-phage remained bound to
IL-12, presumably due to a non-specific interaction, since purified
Y61 scFv exhibits normal dissociation kinetics. Since the usual
methods of phage-display affinity maturation to Y61 (i.e. library
generation and selections by mutagenesis of multiple CDR residues)
could not be utilized, a new strategy was developed in which
individual CDR positions were mutated.
[0716] This strategy involves selection of appropriate CDR
positions for mutation and is based on identification and selection
of amino acids that are preferred selective mutagenesis positions,
contact positions, and/or hypermutation positions. Contact
positions are defined as residues that have a high probability of
contact with an antigen when the antigen interacts with the
antibody, while hypermutation positions are defined as residues
considered to have a high probability for somatic hypermutation
during in vivo affinity maturation of the antibody. Preferred
selective mutagenesis positions are CDR positions that are both
contact and hypermutation positions. The Y61 antibody was already
optimized in the CDR3 regions using the procedure described in
Example 1, therefore it was difficult to further improve the area
which lies at the center of the antibody binding site using
phage-display selection methods. Greater improvements in activity
were obtained by mutation of potential contact positions outside
the CDR3 regions by either removing a detrimental antigen-antibody
contact or, engineering a new contact.
[0717] Amino acids residues of Y61 which were considered contact
points with antigen, and those CDR positions which are sites of
somatic hypermutations during in vivo affinity maturation, are
shown in Table 3 (see Appendix A). For Y61 affinity maturation, 15
residues outside CDR3, 3 residues within the L3 loop, and 5
residues in the H3 loop were selected for PCR mutagenesis.
[0718] Y61 scFv gene was cloned into the pUC119(Sfi) plasmid vector
for mutagenesis. Oligonucleotides were designed and synthesized
with randomized codons to mutate each selected position. Following
PCR mutagenesis, a small number of clones (.about.24) were
sequenced and expressed in a host cell, for example, in a
bacterial, yeast or mammalian host cell. The expressed antibody was
purified and the k.sub.off measured using the BIAcore system.
Clones with improved off-rates, as compared to Y61, were then
tested in neutralization assays. This procedure was repeated for
other CDR positions. Individual mutations shown to have improved
neutralization activity were combined to generate an antibody with
even greater neutralization potency.
[0719] The Y61 CDR positions that were mutated in order to improve
neutralization potency, and the respective amino-acid substitutions
at each position are shown in FIGS. 2A-2H. Off-rates, as determined
by BIAcore analysis, are given. These off rates are also shown in
the histograms to the right of each table.
[0720] Results of these substitutions at positions H30, H32, H33,
H50, H53, H54, H58, H95, H97, H101, L50, L92, L93, demonstrated
that all amino-acid substitutions examined resulted in antibodies
with poorer off-rates than Y61. At positions H52, L32, and L50,
only a one amino acid substitution was found to improve the
off-rate of Y61, all other changes adversely affected activity. For
L50, this single Gly-Tyr change significantly (5-10 times) improved
the neutralization potency of Y61. The results demonstrated the
importance of these positions to Y61 activity, and suggest that in
most cases phage-display was able to select for the optimal
residues. However, at positions H31, H56, L30, and L94, several
substitutions were found to improve Y61 off-rate, suggesting that
these positions were also important for antigen binding, although
the phage display approach did not allow selection of the optimal
residues.
[0721] Selective mutation of contact and hypermutation positions of
Y61 identified amino acid residue L50 in the light chain CDR2, and
residue L94 of the light chain CDR3, which improved the
neutralization ability of Y61. A combination of these mutations
produced an additive effect, generating an antibody, J695, that
exhibited a significant increase in neutralization ability. The
full length sequence of J695 heavy and light chain variable region
sequences is shown below.
J695 Heavy Chain Variable Region Peptide Sequence
[0722] TABLE-US-00009 (SEQ ID NO: 31) CDR H1
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWV A CDR H2
FIRYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCKT CDR H3 HGSHDN
WGQGTMVTVSS
J695 Light Chain Variable Region Peptide Sequence
[0723] TABLE-US-00010 (SEQ ID NO: 32) CDR L1 QSVLTQPPSVSGAPGQRVTISC
SGSRSNIGSNTVK WYQQLPGTAPKLL IY CDR L2 YNDQRPS
GVPDRFSGSKSGTSASLAITGLQAEDEADYYC CDR L3 QSYDRYTHPALL
FGTGTKVTVLG
CDR residues are assigned according to the Kabat definitions.
[0724] A summary of the heavy and light chain variable region
sequence alignments showing the lineage development of clones that
were on the path from Joe9 to J695 is shown in FIGS. 1A-1D. The
CDRs and residue numbering are according to Kabat.
Example 3
Functional Activity of Anti-hIL-12 Antibodies
[0725] To examine the functional activity of the human anti-human
IL-12 antibodies of the invention, the antibodies were used in
several assays that measure the ability of an antibody to inhibit
IL-12 activity.
A. Preparation of Human PHA-Activated Lymphoblasts
[0726] Human peripheral blood mononuclear cells (PBMC) were
isolated from a leukopac collected from a healthy donor by
Ficoll-Hypaque gradient centrifugation for 45 minutes at 1500 rpm
as described in Current Protocols in Immunology, Unit 7.1. PBMC at
the interface of the aqueous blood solution and the lymphocyte
separation medium were collected and washed three times with
phosphate-buffered saline (PBS) by centrifugation for 15 minutes at
1500 rpm to remove Ficoll-Paque particles.
[0727] The PBMC were then activated to form lymphoblasts as
described in Current Protocols in Immunology, Unit 6.16. The washed
PBMC were resuspended at 0.5-1.times.10.sup.6 cells/ml in RPMI
complete medium (RPMI 1640 medium, 10% fetal bovine serum (FBS),
100 U/ml penicillin, 100 .mu.g/ml streptomycin), supplemented with
0.2% (v/v) PHA-P (Difco, Detroit, Mich.) and cultured for four days
at 37.degree. C. in a 5% CO.sub.2 atmosphere. After four days, cell
cultures were split 1:1 by volume in RPMI complete medium, plus
0.2% (v/v) PHA-P and 50 U/ml recombinant human IL-2. Recombinant
human IL-2 was produced by transfection of an expression vector
carrying the human IL-2 cDNA into COS cells (see Kaufman et al.,
(1991) Nucleic Acids Res. 19, 4484-4490), and purified as described
in PCT/US96/01382. Cell cultures were then incubated for an
additional one to three days. PHA blast cells were harvested,
washed twice with RPMI complete medium and frozen in 95% FBS, 5%
DMSO at 10.times.10.sup.6 cells/ml.
[0728] PHA blast cells to be used for the IL-12 receptor binding
assay (see section B) were collected after one day culture in the
presence of IL-2, whereas PHA blast cells to be used for the PHA
blast proliferation assay (see section C) and the interferon-gamma
induction assay (see section D) were collected after three day
culture in the presence of IL-2.
B. IL-12 Receptor Binding Assay
[0729] The ability of anti-IL-12 antibodies to inhibit binding of
radiolabelled IL-12 to IL-12 receptors on PHA blasts were analyzed
as follows. Various concentrations of anti-IL-12 antibody were
preincubated for 1 hour at 37.degree. C. with 50-100 pM
.sup.125I-hIL-12 (iodinated hIL-12 was prepared using the
Bolton-Hunter labeling method to a specific activity of 20-40mCi/mg
from NEN-Dupont) in binding buffer (RPMI 1640, 5% FBS, 25 mM Hepes
pH 7.4). PHA blast cells isolated as described above, were washed
once and resuspended in binding buffer to a cell density of
2.times.10.sup.7 cells/ml. PHA blasts (1.times.10.sup.6 cells) were
added to the antibody .sup.125I-hIL-12 mixture and incubated for
two hours at room temperature. Cell bound radioactivity was
separated from free .sup.125I-hIL-12 by centrifugation of the assay
mixture for 30 seconds at room temperature, aspiration of the
liquid and a wash with 0.1 ml binding buffer, followed by
centrifugation at 4.degree. C. for 4 min at 10,000.times.g. The
cell pellet was examined for cell bound radioactivity using a gamma
counter. Total binding was determined in the absence of antibody
and non-specific binding was determined by inclusion of 25 nM
unlabeled IL-12 in the assay. Incubations were carried out in
duplicate.
[0730] In the IL-12 receptor binding assay using the Y61 and J695
human anti-IL-12 antibodies, both antibodies demonstrated a
comparable inhibition of IL-12 receptor binding. Y61 inhibited
IL-12 receptor binding with an IC.sub.50 value of approximately
1.6.times.10.sup.-11 M, while J695 had an IC.sub.50 value of
approximately 1.1.times.10.sup.-11 M.
C. Human PHA Blast Proliferation Assay
[0731] Anti-IL-12 antibodies were evaluated for their ability to
inhibit PHA blast proliferation (which proliferation is stimulated
by IL-12). Serial dilutions of anti-IL-12 antibody were
preincubated for 1 hour at 37.degree. C., 5% CO.sub.2 with 230
pg/ml hIL-12 in 100 ml RPMI complete medium in a microtiter plate
(U-bottom, 96-well, Costar, Cambridge, Mass.). PHA blast cells
isolated as described above, were washed once and resuspended in
RPMI complete medium to a cell density of 3.times.10.sup.5
cells/ml. PHA blasts (100 ml, 3.times.10.sup.4 cells) were added to
the antibody/hIL-12 mixture, incubated for 3 days at 37.degree. C.,
5% CO.sub.2 and labeled for 4-6 hours with 0.5 mCi/well
(3H)-Thymidine (Amersham, Arlington Heights, Ill.). The culture
contents were harvested onto glass fiber filters by means of a cell
harvester (Tomtec, Orange, Conn.) and (3H)-Thymidine incorporation
into cellular DNA was measured by liquid scintillation counting.
All samples were assayed in duplicate.
[0732] The results of neutralization in the presence of varying
concentrations of p70:p40 (i.e. the ratio of IL-12 heterodimer to
free p40 subunit) is shown in Table 4 (see Appendix A).
[0733] Analysis of the Y61 human anti-IL-12 antibody in the PHA
blast proliferation assay demonstrated that the antibody inhibited
PHA blast proliferation with an IC.sub.50 value of approximately
1.8.times.10.sup.-10 M in the presence of IL-12 p70 alone, without
any excess p40 (p70:p40 ratio of 1:0). In the presence of a 50-fold
excess of free p40 (p70:p40 at a ratio of 1:50), the Y61 antibody
inhibited PHA blast proliferation with an IC.sub.50 value of
approximately 1.8.times.10.sup.-10 M. This result demonstrates that
the ability of Y61 to inhibit blast proliferation is not
compromised by the presence of excess p40.
[0734] The human anti-IL-12 antibody, J695 inhibited PHA blast
proliferation with an IC.sub.50 value of approximately
1.0.times.10.sup.-11 M in the presence of p70:p40 at a ratio of
1:0. In the presence of a p70:p40 ratio of 1:50, this antibody
inhibited PHA blast proliferation with an IC.sub.50 value of
approximately 5.8.+-.2.8.times.10.sup.-12 M (n=2), demonstrating
that the excess p40 had only a slight inhibitory effect on the
antibody. Overall results demonstrate the improved neutralization
activity of J695 in comparison with Y61 due to the mutations at L50
and L94.
D. Interferon-Gamma Induction Assay
[0735] The ability of anti-IL-12 antibodies to inhibit the
production of IFN.gamma. by PHA blasts (which production is
stimulated by IL-12) was analyzed as follows. Various
concentrations of anti-IL-12 antibody were preincubated for 1 hour
at 37.degree. C., 5% CO.sub.2 with 200-400 pg/ml hIL-12 in 100 ml
RPMI complete medium in a microtiter plate (U-bottom, 96-well,
Costar). PHA blast cells isolated as described above, were washed
once and resuspended in RPMI complete medium to a cell density of
1.times.07 cells/ml. PHA blasts (100 .mu.l of 1.times.10.sup.6
cells) were added to the antibody/hIL-12 mixture and incubated for
18 hours at 37.degree. C. and 5% CO.sub.2. After incubation, 150
.mu.l of cell free supernatant was withdrawn from each well and the
level of human IFN.gamma. produced was measured by ELISA (Endogen
Interferon gamma ELISA, Endogen, Cambridge, Mass.). Each
supernatant was assayed in duplicate.
[0736] Analysis of human anti-hIL-12 antibody, Y61 in this assay
demonstrated that Y61 inhibited human IFN.gamma. production with an
IC.sub.50 value of approximately 1.6.times.10.sup.-10 M, while the
human anti-IL-12 antibody, J695, inhibited human IFN.gamma.
production with an IC.sub.50 value of approximately
5.0.+-.2.3.times.10.sup.-12 M (n=3). The result demonstrates the
substantial improvement in the affinity of J695 as a result of the
modifications at L50 and L94.
E. Induction of Non-Human IL-12 from Isolated PBMC
[0737] To examine the cross-reactivity of the human anti-hIL-12
antibodies with IL-12 from other species, non-human IL-12 was
produced as follows. PBMC were separated from fresh heparinized
blood by density gradient centrifugation as described above using
lymphoprep (Nycomed, Oslo, Norway) for cynomolgus monkey, baboon,
and dog, PBMC, Accu-paque (Accurate Chemical & Sci. Corp.,
Westbury, N.Y.) for dog PBMC or Lympholyte-rat (Accurate Chemical
& Sci. Corp., Westbury, N.Y.) for rat PBMC.
[0738] The PBMC were then induced to produce IL-12 as described
(D'Andrea et al., (1992) J. Exp. Med. 176, 1387-1398, Villinger et
al., (1995) J. Immunol. 155, 3946-3954, Buettner et al., (1998)
Cytokine 10, 241-248). The washed PBMC were resuspended at
1.times.10.sup.6 cells/ml in RPMI complete medium, supplemented
with 0.0075% (wt/vol) of SAC (Pansorbin; Calbiochem-Behring Co., La
Jolla, Calif.) or 1-5 mg/ml ConA (Sigma Chemical Co., St. Louis,
Mo.) plus 0.0075% SAC and incubated for 18 hours at 37.degree. C.
in a 5% CO.sub.2 atmosphere. Cell-free and SAC-free medium was
collected by centrifugation and filtering through 0.2 mm
filters.
[0739] IL-12 from the rhesus monkey was obtained as recombinant
rhesus IL-12 from Emory University School of Medicine, Atlanta,
Ga.
F. Murine 2D6 Cell Proliferation Assay
[0740] The murine T cell clone 2D6 proliferates in response to
murine IL-2, IL-4, IL-7 and IL-12 (Maruo et al., (1997) J.
Leukocyte Biol. 61, 346-352). A significant proliferation was also
detected in response to rat PBMC supernatants containing rat IL-12.
The cells do not respond to dog, cynomolgus, baboon or human IL-12.
Murine 2D6 cells were propagated in RPMI complete medium
supplemented with 50 mM beta-mercaptoethanol (.beta.ME) and 30
ng/ml murine IL-12. One day prior to the assay, the murine IL-12
was washed out and the cells were incubated overnight in RPMI
complete medium plus .beta.ME.
[0741] Serial dilutions of anti-IL-12 antibody were preincubated
for 1 hour at 37.degree. C., 5% CO.sub.2 with 40 pg/ml murine IL-12
in 100 ml RPMI complete medium plus PME in a microtiter plate
(U-bottom, 96-well, Costar). 2D6 cells were washed once and
resuspended in RPMI complete medium containin.gamma. PME to a cell
density of 1.times.10.sup.5 cells/ml. 2D6 cells (100 .mu.l,
1.times.10.sup.4 cells) were added to the antibody/hIL-12 mixture,
incubated for 3 days at 37.degree. C., 5% CO.sub.2 and labeled for
4-6 hours with 0.5 mCi/well (3H)-Thymidine. The culture contents
were harvested and counted by liquid scintillation counting. All
samples were assayed in duplicate.
G. Species Cross-Reactivity of J695 with Non-Human IL-12
[0742] Species cross-reactivity of J695 with non-human IL-12 was
analyzed using PBMC's isolated from several non-human species. The
presence of non-human IL-12 activity in the rat, dog, cynomolgus
and baboon PBMC supernatants was confirmed using several bioassays
described above, such as the murine 2D6 cell proliferation assay,
the human PHA blast proliferation assay and the interferon-gamma
induction assay by blocking the non-human PBMC induced responses
with rabbit and/or sheep polyclonal antibodies to murine and/or
human IL-12. Cross-reactivity of the human anti-hIL-12 antibodies
Y61 and J695 with non-human IL-12 in PBMC supernatants or purified
murine and rhesus IL-12 was then assessed in the same bioassay(s)
by determining the J695 antibody concentration at which 50%
inhibition of the response was observed. The species
cross-reactivity results are summarized in Table 5. The results
demonstrate that Y61 and J695 are each able to recognize IL-12 from
monkeys (e.g, cynomolgus and rhesus IL-12 for Y61, and cynomolgus,
rhesus and baboon for J695) and that J695 is approximately 35 fold
less active on dog IL-12; neither Y61 nor J695 cross reacts with
mouse or rat IL-12.
H. Human Cytokine Specificity of J695
[0743] The specificity of J695 was tested in a competition ELISA in
which a panel of human cytokines was tested for their ability to
interfere with the binding of soluble J695 to immobilized human
IL-12. The panel of human cytokines included IL-1.alpha. and
IL-1.beta. (Genzyme, Boston, Mass.), IL-2 (Endogen), IL-4, IL-10,
IL-17, IFN-gamma, and TGF-.beta.1 (R&D, Minneapolis, Minn.)
IL-8 (Calbiochem), PDGF, IGF-I, and IGF-II (Boehringer Mannheim
Corp., Indianapolis, Ind.), TNF.alpha. and lymphotoxin, IL-6,
soluble IL-6 receptor, IL-11, IL-12 p70, IL-12 p40, M-CSF, and LIF.
EBI-3, an IL-12 p40 related protein that is induced by Epstein-Barr
virus infection in B lymphocytes (Devergne et TABLE-US-00011 TABLE
5 Species Cross Reactivity Data IC.sub.50 (M) Antibody Mouse IL-12
Rat IL-12 Dog IL-12 Cyno IL-12 Rhesus IL-12 Baboon IL-12 Human
IL-12 Name Specificity Purified PBMC sup PBMC sup PBMC sup Purified
PBMC sup Purified C17.15 rat-.alpha.muIL12 3.0 .times. 10.sup.-11
R03B03 rabbit-.alpha.muIL12 1.5 .times. 10.sup.-10 6.0 .times.
10.sup.-10 C8.6.2 mouse-.alpha.huIL12 1.2 .times. 10.sup.-10 1.0
.times. 10.sup.-10 2.0 .times. 10.sup.-10 5.0 .times. 10.sup.-11
Y61 human-.alpha.huIL12 Non-neutralizing 2.2 .times. 10.sup.-10 1.0
.times. 10.sup.-10 1.7 .times. 10.sup.-10 J695 human-.alpha.huIL12
Non-neutralizing Non-neutralizing 3.5 .times. 10.sup.-10 1.0
.times. 10.sup.-11 1.0 .times. 10.sup.-11 1.5 .times. 10.sup.-11
5.0 .times. 10.sup.-12
al., (1996) J. Virol. 70, 1143-1153) was expressed as a human
IgG-Fc chimera (EBI-3/Fc) Single-stranded salmon sperm DNA (Sigma)
was also tested.
[0744] Flat-bottom ELISA immunoassay microtiter plates (96 well,
high binding, Costar) were coated overnight at 4.degree. C. with
0.1 ml human IL-12 (2 .mu.g/ml in 0.1 M carbonate coating buffer (4
volumes 0.1 M NaHCO.sub.3 plus 8.5 volumes 0.1 M NaHCO.sub.3)). The
plates were washed twice with PBS containing 0.05% Tween 20
(PBS-T), blocked with 200 .mu.l of 1 mg/ml bovine serum albumin
(BSA, Sigma) in PBS-T for 1 hour at room temperature, and again
washed twice with PBS-T. Samples (100 .mu.l) containing IL-12
antibody J695 (100 ng/ml) and each cytokine (2 nM) in PBS-T
containing 50 .mu.g/ml BSA (PBS-T/BSA) were added and incubated for
2 h at room temperature. The plates were washed 4 times and
incubated for 1 h at room temperature with 100 .mu.l mouse
anti-human lambda-HRP (1:500 in PBS-T/BSA, Southern Biotech. Ass.
Inc., Birmingham, Ala.). The plates were washed 4 times and
developed with ABTS (Kirkegaard & Perry Lab., Gaithersburg,
Md.) for 20-30 minutes in the dark. The OD.sub.450 nm was read
using a microplate reader (Molecular Devices, Menlo Park, Calif.).
Percent binding was determined relative to J695 binding to the
IL-12 coated plate in the absence of any soluble cytokine.
[0745] The results demonstrated that J695 binding to immobilized
human IL-12 was blocked only by human IL-12 p70 and to a lesser
extent, by human IL-12 p40 and not by any of the other cytokines
tested.
I. Binding to a Novel IL-12 Molecule
[0746] An alternative IL-12 heterodimer has been described, in
which the p35 subunit is replaced by a novel p19 molecule. P19 was
identified using 3D homology searching for IL-6/IL-12 family
members, and is synthesized by activated dendritic cells. P19 binds
to p40 to form a p19/p40 dimer, which has IL-12-like activity, but
is not as potent as the p35/p40 heterodimer in IFN.gamma.
induction. Antibodies which recognize p40 alone, but preferably in
the context of a p70 molecule (e.g., J695 and Y61, see Example 3H)
are expected to also neutralize both the p35/p40 molecules and the
p19/p40 molecules.
Example 4
In Vivo Activity of Anti-hIL-12 Antibodies
[0747] The in vivo effects of IL-12 antibodies on IL-12 induced
responses were examined in a model modified from one used by Bree
et al. to study the effect of human IL-12 on peripheral hematology
in cynomolgus monkey Bree et al., (1994) Biochem Biophys Res. Comm.
204: 1150-1157. In those previous studies, administration of human
IL-12 at 1 .mu.g/kg/day for a period of 5 days resulted in a
decrease in white blood cell count (WBC), especially in the
lymphocyte and monocyte subsets after 24 hours. A decrease in the
platelet count was observed at 72 hours. Levels of plasma
neopterin, a marker of monocyte activation in response to
IFN-.gamma., began to elevate at 24 hours and were the highest at
72 hours.
[0748] In the first study with human anti-hIL-12 antibodies,
fifteen healthy cynomolgus monkeys with an average weight of 5 kg,
were sedated and divided into 5 groups (n=3). Group 1 received an
intravenous (IV) administration of 10 mg/kg human intravenous
immunoglobulin (IVIG, Miles, Eckhart, Ind., purified using protein
A Sepharose). Group 2 received an intravenous administration of 1
mg/kg C8.6.2 (neutralizing mouse anti-human IL-12 monoclonal
antibody). Group 3 received an intravenous administration of 10
mg/kg C8.6.2. Group 4 received an intravenous administration of 1
mg/kg Y61 (human anti-human IL-12 antibody, purified from CHO cell
conditioned medium). Group 5 received an intravenous administration
of 10 mg/kg Y61.
[0749] One hour after the antibody administration all animals
received a single subcutaneous (SC) injection of human IL-12 (1
.mu.g/kg). Blood samples were taken at the following time points:
baseline, 8, 24, 48, 96 and 216 hours, and analyzed for complete
blood cell counts with differentials and serum chemistry. Serum
human IL-12, C8.6.2 antibody, Y61 antibody, monkey IFN-gamma,
monkey IL-10, monkey IL-6 and plasma neopterin levels were also
measured.
[0750] Animals treated with IL-12 plus IVIG control antibody (Group
1) showed many of the expected hematological changes, including
decreases in WBC, platelets, lymphocyte count and monocyte count.
These decreases were not seen or were less pronounced in the
animals treated with either the C8.6.2 or Y61 antibody at 1 or 10
mg/kg (Groups 2-5).
[0751] Serum or plasma samples were analyzed by ELISA specific for
monkey IFN-gamma and monkey IL-10 (Biosource International,
Camarillo, Calif.), monkey IL-6 (Endogen) and plasma neopterin (ICN
Pharmaceuticals, Orangeburg, N.Y.). IFN-gamma, IL-10 or IL-6 were
not detected in any of the IL-12 treated animals including the
control animals treated with IL-12 plus IVIG. This was probably due
to the low level exposure to IL-12 (only 1 dose of 1 .mu.g/kg).
Nevertheless, plasma neopterin levels increased about three fold in
the IL-12 plus IVIG treated animals but did not change in all
C8.6.2 or Y61 treated animals, including the lower dose (1 mg/kg)
Y61 treated animals, indicating that Y61 was effective in vivo in
blocking this sensitive response to IL-12.
[0752] In a second study, in vivo activity and pharmacodynamics
(PD) of J695 in cynomolgous monkeys were studied by administering
exogenous rhIL-12 and determining if J695 could block or reduce the
responses normally associated with rhIL-12 administration. Male
cynomolgus monkeys (n=3 per group) were administered a single dose
of 0.05, 0.2, or 1.0 mg/kg J695 or 1 mg/kg intravenous
immunoglobulin (IVIG) as a bolus intravenous (IV) injection via a
saphenous vein or subcutaneously (SC) in the dorsal skin. One hour
following the administration of J695 or IVIG, all animals received
a single SC dose of 1 .mu.g/kg rhIL-12 in the dorsal skin. Blood
samples were collected via the femoral vein up to 28 days after
J695 administration. Serum was acquired from each blood sample and
assayed for IL-12, J695, IFN-.gamma., and anti-J695 antibodies by
ELISA. Neopterin was assayed by reverse-phase high performance
liquid chromatography.
[0753] The levels of neopterin, normalized with respect to the
levels of neopterin that were measured before administration of
J695 or rhIL-12, are shown in FIG. 3. To compare the suppression of
neopterin between groups, the area under the curve (AUC) normalized
for neopterin levels was calculated for each animal (Table 6).
Neopterin exposure (AUC) was suppressed in a dose-dependent manner
between approximately 71 and 93% in the IV groups and between 71
and 100% in SC groups, relative to the IVIG control groups. These
results suggest that the dose of J695 necessary for 50% inhibition
of the neopterin response (ED.sub.50) was less than 0.05 mg/kg when
administered by either the IV or SC route. TABLE-US-00012 TABLE 6
Dose-Dependent Suppression of IL-12 Induced Neopterin by J695 in
Cynomolgus Monkeys % Reduction of AUC of Neopterin AUC Route of
dosing IVIG J695 Dose IVIG Dose Normalized Compared with or J695
and rhIL-12 (mg/kg) (mg/kg) Neopterin Levels Control Single IV
injection -- 1.0 1745 .+-. 845 0 followed 1 hr later by a 0.05 --
502 .+-. 135 71.3 dose of 1 .mu.g/kg human 0.2 -- 199 .+-. 316 88.6
IL-12 given SC 1.0 -- 128 .+-. 292 92.7 Single SC injection -- 1.0
1480 .+-. 604 0 followed 1 hour later 0.05 -- 426 .+-. 108 71.2 by
a dose of 1 .mu.g/kg 0.2 -- 395 .+-. 45.9 73.3 human IL-12 given SC
1.0 -- 0 .+-. 109 100
[0754] Treatment with J695 also prevented or reduced the changes in
hematology normally associated with rhIL-12 administration
(leukopenia and thrombocytopenia). At 24 hours after rhIL-12
administration lymphocyte counts were reduced by approximately 50%
when compared to baseline values in the control IV and SC IVIG
treated groups. Administration of J695 either SC or IV at all three
dose levels prevented this reduction, resulting in lymphocyte
counts at 24 hours approximately the same as baseline values. At 48
hours after IL-12 administration, platelet counts in the groups
treated with W and SC IVIG were reduced by approximately 25% when
compared to baseline values.
[0755] An example dose schedule targeted to maintain serum levels
above the 90% effect level would be 1 mg/kg IV and SC given
approximately every other week, or 0.3 mg/kg given approximately
every week, assuming slight accumulation during repeated dosing.
This study demonstrates that antibody can be given safely to
monkeys at such dosages. In independent toxicity studies, it was
further found that up to 100 mg/kg of the antibody can be given
safely to monkeys.
[0756] J695 was also effective in preventing IFN-.gamma. production
in mice treated with a chimeric IL-12, a molecule which combines
the murine p35 subunit with the human IL-12 p40 subunit. In
contrast to human IL-12 which is biologically inactive in mice,
this chimeric IL-12 retains biological function in mice, including
induction of IFN-.gamma.. In addition, the human p40 subunit allows
the molecule to be bound and neutralized by J695. Chimeric IL-12 at
a dose of 0.05 mg/kg i.p. was administered to female C3H/HeJ mice
(10/experimental group) in five daily doses on days 0, 1, 2, 3, and
4. J695 was given on days 0, 2 and 4 at doses of 0.05, 0.01, 0.002,
0.0004, 0.00008, and 0.000016 mg/kg i.p., 30' prior to the IL-12
injections. The control hulgGl.gamma. was given IP at a dose of
0.05 mg/kg on days 0, 2, and 4. The mice were bled on day 5, and
serum IFN-.gamma. levels were determined by ELISA. The results
demonstrated that J695 caused dose-dependent inhibition of
IFN-.gamma. production with an ED.sub.50 of approximately 0.001
mg/kg. Collectively, these results demonstrate that J695 is a
potent inhibitor of 11-12 activity in vivo.
Example 5
Kinetic Analysis of Binding of Human Antibodies to Recombinant
Human IL-12 (rhIL-12)
[0757] Real-time binding interactions between captured ligand
(human anti-rhIL-12 antibody J695, captured on a biosensor matrix)
and analyte (rhIL12 in solution) were measured by surface plasmon
resonance (SPR) using the BIAcore system (Biacore AB, Uppsala,
Sweden). The system utilizes the optical properties of SPR to
detect alterations in protein concentration within a dextran
biosensor matrix. Proteins are covalently bound to the dextran
matrix at known concentrations. Antibodies are injected through the
dextran matrix and specific binding between injected antibodies and
immobilized ligand results in an increased matrix protein
concentration and resultant change in the SPR signal. These changes
in SPR signal are recorded as resonance units (RU) and are
displayed with respect to time along the y-axis of a
sensorgram.
[0758] To facilitate immobilization of goat anti-human IgG
(Southern Biotechnology Associates, Cat. No. 2040-01, Birmingham,
Ala.) on the biosensor matrix, goat anti-human IgG is covalently
linked via free amine groups to the dextran matrix by first
activating carboxyl groups on the matrix with 100 mM
N-hydroxysuccinimide (NHS) and 400 mM
N-Ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride
(EDC). Next, goat anti-human IgG is injected across the activated
matrix. Thirty-five microliters of goat anti-human IgG (25
.mu.g/ml), diluted in sodium acetate, pH 4.5, is injected across
the activated biosensor and free amines on the protein are bound
directly to the activated carboxyl groups. Unreacted matrix
EDC-esters are deactivated by an injection of 1 M ethanolamine.
Standard amine coupling kits were commercially available (Biacore
AB, Cat. No. BR-1000-50, Uppsala, Sweden).
[0759] J695 was diluted in HBS running buffer (Biacore AB, Cat. No.
BR-1001-88, Uppsala, Sweden) to be captured on the matrix via goat
anti-human IgG. To determine the capacity of rhIL12-specific
antibodies to bind immobilized goat anti-human IgG, a binding assay
was conducted as follows. Aliquots of J695 (25 .mu.g/ml; 25 .mu.l
aliquots) were injected through the goat anti-human IgG polyclonal
antibody coupled dextran matrix at a flow rate of 5 .mu.l/min.
Before injection of the protein and immediately afterward, HBS
buffer alone flowed through each flow cell. The net difference in
signal between the baseline and the point corresponding to
approximately 30 seconds after completion of J695 injection was
taken to represent the amount of IgG1 J695 bound (approximately
1200 RU's). Direct rhIL12 specific antibody binding to soluble
rhIL12 was measured. Cytokines were diluted in HBS running buffer
and 50 .mu.l aliquots were injected through the immobilized protein
matrices at a flow rate of 5 .mu.l/min. The concentrations of
rhIL-12 employed were 10, 20, 25, 40, 50, 80, 100, 150 and 200 nM.
Prior to injection of rhIL-12, and immediately afterwards, HBS
buffer alone flowed through each flow cell. The net difference in
baseline signal and signal after completion of cytokine injection
was taken to represent the binding value of the particular sample.
Biosensor matrices were regenerated using 100 mM HCl before
injection of the next sample. To determine the dissociation
constant (off-rate), association constant (on-rate), BIAcore
kinetic evaluation software (version 2.1) was used.
[0760] Representative results of CHO derived J695 binding to
rhIL-12 as compared to the COS derived J695, are shown in Table 7.
TABLE-US-00013 TABLE 7 Binding of CHO or COS derived J695 to
rhIL-12. Ab, Source rhIL12, nM rhIL12 bound, RU's bound, RU's
rhIL12/AB CHO 200 1112 1613 1.48 CHO 150 1033 1525 1.45 CHO 100 994
1490 1.43 CHO 80 955 1457 1.40 CHO 50 912 1434 1.36 CHO 40 877 1413
1.33 CHO 25 818 1398 1.25 CHO 20 773 1382 1.20 CHO 10 627 1371 0.98
COS 200 1172 1690 1.49 COS 150 1084 1586 1.46 COS 100 1024 1524
1.44 COS 80 985 1489 1.42 COS 50 932 1457 1.37 COS 40 894 1431 1.34
COS 25 833 1409 1.27 COS 20 783 1394 1.20 COS 10 642 1377 1.00
[0761] Molecular kinetic interactions between captured J695 and
soluble rhIL-12 were quantitatively analyzed using BIAcore
technology. Several independent experiments were performed and the
results were analyzed by the available BIAcore mathematical
analysis software to derive kinetic rate constants, as shown in
Table 8. TABLE-US-00014 TABLE 8 Apparent kinetic rate and affinity
constants of J695 for rhIL-12. On-rate (M-1s-1), Off-rate (s-1), Kd
(M), Antibody Source Avg. Avg. Avg. J695 CHO 3.52E+05 4.72E-05
1.34E-10 J695 COS 3.40E+05 2.61E-05 9.74E-11
[0762] There was a small difference between the calculated apparent
constant (Kd) for the interaction between CHO derived J695
(Kd=1.34.sup.-10M.sup.-1) and COS derived J695
(Kd=9.74.times.10.sup.-11 M.sup.-1) antibodies. The apparent
dissociation constant (Kd) between J695 and rhIL12 was estimated
from the observed rate constants by the formula:
Kd=off-rate/on-rate.
[0763] To determine the apparent association and dissociation rate
constant for the interaction between J695 and rhIL-12, several
binding reactions were performed using a fixed amount of J695 (2
.mu.g/ml) and varying concentrations of rhIL-12. Real-time binding
interaction sensorgrams between captured J695 and soluble rhIL12
showed that both forms of antibody were very similar for both the
association and dissociation phase.
[0764] To further evaluate the capacity of captured IgG1 J695 mAb
to bind soluble recombinant cytokine, a direct BIAcore method was
used. In this method, goat anti-human IgG (25 .mu.g/ml) coupled
carboxymethyl dextran sensor surface was coated with IgG1 J695 (2
.mu.g/ml) and recombinant cytokine was then added. When soluble
rhIL12 was injected across a biosensor surface captured with CHO or
COS derived IgG1 J695, the amount of signal increased as the
concentration of cytokine in the solution increased. No binding was
observed with rmIL12 (R&D Systems, Cat. No. 419-ML,
Minneapolis, Minn.) or rh IL12 any concentration tested up to 1000
nM. These results support the conclusion that IgG1 J695 antibodies
recognize a distinct determinant on rhIL-12.
[0765] Table 9 shows the results of an experiment using BIAcore to
demonstrate human IgG1 J695 mAb binding to only soluble rhIL12 and
none of the other recombinant cytokines. TABLE-US-00015 TABLE 9
Epitope mapping of J695 using BIAcore technology. Soluble analyte
Captured ligand COS J695 Captured ligand CHO J695 rec. human
Positive Positive IL12 rec. murine Negative Negative IL12
Example 6
Further Studies of J695 Affinity for IL-12
[0766] Molecular kinetic interactions between J695 antibody and
human IL-12 were quantitatively analyzed using BIAcore plasmon
resonance technology, and apparent kinetic rate constants were
derived.
[0767] BIAcore technology was used to measure the binding of
soluble rhIL-12 to solid phase captured J695. A goat anti-human IgG
antibody was immobilized on the biosensor chips, then a fixed
amount of J695 was injected and captured on the surface. Varying
concentrations of rhIL-12 were applied, and the binding of IL-12 at
different concentrations to J695 was measured as a function of
time. Apparent dissociation and association rate constants were
calculated, assuming zero-order dissociation and first order
association kinetics, as well as a simple one-to-one molecular
interaction between J695 and IL-12. Three independent experiments
were performed, and the values shown are averages for the three
experiments. From these measurements, the apparent dissociation
(k.sub.d) and association (k.sub.a) rate constants were derived and
used to calculate a K.sub.d value for the interaction (see Table
10). The results indicated that J695 has a high affinity for
rhIL-12. TABLE-US-00016 TABLE 10 Kinetic Parameters for the
Interaction Between J695 and Human IL-12 Kinetic Parameter Value
k.sub.d 3.71 .+-. 0.40 .times. 10.sup.-5 s.sup.-1 k.sub.a 3.81 .+-.
0.48 .times. 10.sup.5 M.sup.-1s.sup.-1 k.sub.d 9.74 .times.
10.sup.-11 M (14 ng/mL)
Example 7
Characteristics and Neutralization Activity of C17.15, a Rat
Monoclonal Antibody to Murine Interleukin-12
[0768] To assess the relevance of IL-12 treatment studies in mouse
models of inflammation and autoimmunity using monoclonal antibodies
specific for murine IL-12 to similar approaches in human disease,
the interaction of C17.15, a rat anti-murine IL-12 monoclonal
antibody with murine IL-12, was examined. The ability of C17.15 to
neutralize murine IL-12 activity in a PHA blast proliferation
assay, and to block murine IL-12 binding to cell surface receptors,
was assessed, as were the kinetics of the C17.15-murine IL-12
binding interaction.
[0769] In a human PHA blast proliferation assay (See Example 3),
serial dilutions of C17.15 or rat IgG2a (a control antibody) were
preincubated with 230 pg/mL murine IL-12 for 1 hr at 37.degree. C.
PHA-stimulated blast cells were added to the antibody-IL-12
mixtures and incubated for 3 days at 37.degree. C. The cells were
subsequently labeled for 6 h with 1 .mu.Ci/well
[.sup.3H]-thymidine. The cultures were harvested and
[.sup.3H]-thymidine incorporation was measured. Background
non-specific proliferation was measured in the absence of added
murine IL-12. All samples were assayed in duplicate. The IC.sub.50
(M) of C17.15 for recombinant murine IL-12 in this assay was found
to be 1.4.times.10.sup.-11, as compared to the IC.sub.50 value of
5.8.times.10.sup.-12 observed for J695 for recombinant human IL-12
under the same conditions (see Table 11). TABLE-US-00017 TABLE 11
Comparison of the properties of anti-human IL-12 monoclonal
antibody J695 and the rat anti-mouse IL-12 monoclonal antibody
C17.15 Receptor Biomolecular Interaction Assay Binding PHA blast
k.sub.a, on-rate k.sub.d, off-rate Assay Assay Antibody Epitope
(M.sup.-1s.sup.-1) (s.sup.-1) K.sub.d (M) IC.sub.50 (M) IC.sub.50
(M) J695 Hu p40 3.81 .times. 10.sup.5 3.71 .times. 10.sup.-5 9.74
.times. 10.sup.-11 1.1 .times. 10.sup.-11 5.8 .times. 10.sup.-12
C17.15 Mu p40 3.80 .times. 10.sup.5 1.84 .times. 10.sup.-4 4.80
.times. 10.sup.-10 1.5 .times. 10.sup.-10 1.4 .times.
10.sup.-11
[0770] The ability of C17.15 to inhibit the binding of murine IL-12
to cellular receptors was also measured. Serial dilutions of C17.15
were pre-incubated for 1 hr at 37.degree. C. with 100 pM
[.sup.125I]-murine IL-12 in binding buffer. The 2D6 cells
(2.times.10.sup.6) were added to the antibody/[.sup.125I]-murine
IL-12 mixture and incubated for 2 hours at room temperature.
Cell-bound radioactivity was separated from free [.sup.125I]-IL-12,
and the remaining cell-bound radioactivity was determined. Total
binding of the labeled murine IL-12 to receptors on 2D6 cells was
determined in the absence of antibody, and non-specific binding was
determined by the inclusion of 25 nM unlabelled murine IL-12 in the
assay. Specific binding was calculated as the total binding minus
the non-specific binding. Incubations were carried out in
duplicate. The results showed that C17.15 has an IC.sub.50 (M) of
1.5.times.10.sup.-10 for inhibition of binding of murine IL-12 to
cellular receptors.
[0771] The affinity of C17.15 for recombinant murine IL-12 was
assessed by biomolecular interaction analysis. A goat anti-rat IgG
antibody was immobilized on the biosensor chips, followed by an
injection of a fixed amount of the C17.15 antibody, resulting in
capture of C17.15 on the surface of the chip. Varying
concentrations of recombinant murine IL-12 were applied to the
C17.15 surface, and the binding of murine IL-12 to the immobilized
C17.15 was measured as a function of time. Apparent dissociation
and association rate constants were calculated, assuming a zero
order dissociation and first order association kinetics as well as
a simple one to one molecular interaction between the immobilized
C17.15 and murine IL-12. From these measurements, the apparent
dissociation (k.sub.d, off-rate) and association (k.sub.a, on-rate)
rate constants were calculated. These results were used to
calculate a K.sub.d value for the interaction. An on-rate of
3.8.times.10.sup.5 M.sup.-1s.sup.-1, an off-rate of
1.84.times.10.sup.4 s.sup.-1, and a K.sub.d of 4.8.times.10.sup.-10
was observed for the recombinant murine IL-12-C17.15
interaction.
[0772] The observed activities of C17.15 in neutralizing murine
IL-12 activity and binding to cell surface receptors, as well as
the kinetics of binding of C17.15 to murine IL-12 correlate with
similar measurements for the J695-rhIL-12 interaction. This
indicates that the modes of action of the rat anti-mouse IL-12
antibody C17.15 and anti-human IL-12 antibody J695 are nearly
identical based upon on-rate, off-rate, K.sub.d, IC.sub.50, and the
PHA blast assay. Therefore, C17.15 was used as a homologous
antibody to J695 in murine models of inflammation and autoimmune
disease to study the effects of IL-12 blockade on the initiation or
progression of disease in these model animals (see Example 8).
Example 8
Treatment of Autoimmune or Inflammation-Based Diseases in Mice by
.alpha.-Murine IL-12 Antibody Administration
A. Suppression of Collagen-Induced Arthritis in Mice by the
.alpha.-Il-12 Antibody C17.15
[0773] A correlation between IL-12 levels and rheumatoid arthritis
(RA) has been demonstrated. For example, elevated levels of IL-12
p70 have been detected in the synovia of RA patients compared with
healthy controls (Morita et al (1998) Arthritis and Rheumatism. 41:
306-314). Therefore, the ability of C17.15, a rat anti-mouse IL-12
antibody, to suppress collagen-induced arthritis in mice was
assessed.
[0774] Male DBA/1 mice (10/group) were immunized with type II
collagen on Day 0 and treated with C17.15, or control rat IgG, at
10 mg/kg intraperitoneally on alternate days from Day--1 (1 day
prior to collagen immunization) to Day 12. The animals were
monitored clinically for the development of arthritis in the paws
until Day 90. The arthritis was graded as: 0--normal; 1--arthritis
localized to one joint; 2--more than one joint involved but not
whole paw; 3--whole paw involved; 4--deformity of paw; 5--ankylosis
of involved joints. The arthritis score of a mouse was the sum of
the arthritic grades in each individual paw of the mouse (max=20).
The results are expressed as mean .+-.SEM in each group.
[0775] The results, as shown in FIG. 4, indicate that an arthritic
score was measurable in the C17.15-treated mice only after day 50
post-treatment, and that the peak mean arthritic score obtained
with the C17.15-treated mice was at least 5-fold lower than that
measured in the IgG-treated mice. This demonstrated that the rat
anti-mouse IL-12 antibody C17.15 prevented the development of
collagen-induced arthritis in mice.
B. Suppression of Colitis in Mice by the Rat .alpha.-Murine IL-12
Antibody C17.15
[0776] IL-12 has also been demonstrated to play a role in the
development/pathology of colitis. For example, anti-IL-12
antibodies have been shown to suppress disease in mouse models of
colitis, e.g., TNBS induced colitis IL-2 knockout mice (Simpson et
al. (1998) J. Exp. Med. 187(8): 1225-34). Similarly, anti-IL-2
antibodies have been demonstrated to suppress colitis formation in
IL-10 knock-out mice. The ability of the rat anti-mouse IL-12
antibody, C17.15, to suppress TNBS colitis in mice was assessed in
two studies (Davidson et al. (1998) J. Immunol. 161(6):
3143-9).
[0777] In the first study, colitis was induced in pathogen free SJL
mice by the administration of a 150 mL 50% ethanol solution
containing 2.0 mg TNBS delivered via a pediatric umbilical artery
catheter into the rectum. Control animals were treated with a 150
.mu.L 50% ethanol solution only. A single dose of 0.75, 0.5, 0.25,
or 0.1 mg C17.15 or 0.75 mg control rat IgG2a was given
intravenously via the tail vein at day 11, and the therapeutic
effect of the treatment was assessed by weighing the animals on
days 11 and 17, and histological scoring at day 17. The weight of
the mice treated with C17.15 increased within 48 hours of antibody
treatment and normalized on day 6 after treatment. The effect of
treatment with C17.15 was confirmed histologically. Further,
assessments of IFN-.gamma. secretion by CD4.sup.+ T-cells from
spleen and colon of the treated mice, as well as IL-12 levels from
spleen or colon-derived macrophages from the treated mice were also
made (see Table 12).
[0778] In the second study, the dosing was optimized and the mice
were treated with a total dose of 0.1 mg or 0.5 mg C17.15 or 0.1 mg
control IgG2a, respectively, split between days 12 and 14. It was
found that the administration of C17.15 in a single dose at the
dosage of 0.1 mg/mouse or 0.25 mg/mouse led to only partial
improvement in TNBS-induced colitis and did not result in a
significant reduction in the CD4.sup.+ T cell production of
IFN-.gamma. in vitro, but did result in a significant decrease in
secretion of IL-12, compared to untreated controls. At a single
dose of 0.5 mg/mouse or greater a response was observed. Taking the
lowest dose of antibody tested and administering it in two divided
injections (at days 12 and 14) improved the dosing regimen,
indicating that multiple low doses can be more effective than a
single bolus dose. The data obtained are shown in Table 12.
TABLE-US-00018 TABLE 12 Anti-mouse Il-12 mAb C17.15 Suppresses
Established Colitis in Mice Weight IFN-.gamma. spleen IL-12 spleen
Disease Treatment (g) CD4.sup.+ cells macrophages Induction Day 0
Day 11 Day 11 Day 17 (U/mL) (pg/ml) TNBS + Ethanol Control IgG2a
16.0 15.26 3326 300 0.75 mg TNBS + Ethanol C17.15 0.75 mg 16.0
20.21 1732 0 TNBS + Ethanol C17.15 0.5 mg 16.36 19.94 1723 0 TNBS +
Ethanol C17.15 0.25 mg 16.28 17.7 3618 7 TNBS + Ethanol C17.15 0.1
mg 16.2 17.98 3489 22 Ethanol control -- 20.76 21.16 1135 0
[0779] Administration of C17.15 monoclonal anti-IL-12 in two
divided doses spaced one day apart totaling 0.1 mg/mouse or 0.05
mg/mouse led to complete reversal of colitis as assessed by wasting
and macroscopic appearance of the colon. In addition, this dose
schedule led to significant down-regulation of lamina propria
T-cell production of IFN-.gamma. and macrophage production of
IL-12, so that the latter were comparable to levels seen in control
ethanol-treated mice without TNBS-colitis. Thus, C17.15
administration to mouse models for TNBS colitis reversed the
progression of the disease in a dose-dependent manner.
C. Suppression of Experimental Autoimmune Encephalomyelitis (EAE)
in Mice by .alpha.-IL-12 Antibodies
[0780] It is commonly believed that IL-12 plays a role in the
pathogenesis of multiple sclerosis (MS). The inducible IL-12 p40
message has been shown to be expressed in acute plaques of MS
patients but not in inflammatory brain infarct lesions (Windhagen,
A. et al. (1995) J. Exp. Med. 182: 1985-1996). T cells from MS
patients (but not control T cells) stimulate IL-12 production from
antigen-presenting cells through unregulated CD40L expression
(Balashov, K. E. et al. (1997) Proc. Natl. Acad. Sci. USA 94:
599-603). MS patients have enhanced IFN-.gamma. secretion that can
be blocked with .alpha.-IL-12 antibodies in vitro (Balashov, K. E.
et al. (1997) Proc. Natl. Acad. Sci. USA 94: 599-603). Elevated
levels of serum IL-12 are detected in MS patients, but not in other
neurological diseases (Nicoletti, F. et al. (1996) J. Neuroimmunol.
70: 87-90). Increased IL-12 production has been shown to correlate
with disease activity in MS patients (Cormabella, M. et al. (1998)
J. Clin. Invest. 102: 671-678). The role of IL-12 in the
pathogenesis of a murine model of multiple sclerosis, experimental
autoimmune encephalomyelitis (EAE), has been studied (Leonard, J.
P. et al. (1995) J. Exp. Med. 181: 281-386; Banedjee, S. et al.
(1998) Arthritis Rheum. (1998) 41: S33; and Segal, B. M. et al.
(1998) J. Exp. Med. 187: 537-546). The disease in this model is
known to be induced by T cells of the TH.sub.1 subset. Therefore,
the ability of .alpha.-IL-12 antibodies to prevent the onset of
acute EAE was assessed.
[0781] An .alpha.-IL-12 antibody was found to be able to inhibit
the onset of acute EAE, to suppress the disease after onset, and to
decrease the severity of relapses in mice immunized with the
autoantigen, myelin basic protein (Banerjee, S. et al. (1998)
Arthritis Rheum. (1998) 41: S33). The beneficial effects of
.alpha.-IL-12 antibody treatment in the mice persisted for over two
months after stopping treatment. It has also been demonstrated that
anti-IL-12 antibodies suppress the disease in mice that are
recipients of encephalitogenic T cells by adoptive transfer
(Leonard, J. P. et al. (1995) J. Exp. Med. 181: 281-386).
Example 9
Clinical Pharmacology of J695
[0782] In a double blind, crossover study, 64 healthy, human male
subjects were administered ascending doses of J695 or placebo.
Measurement of complement fragment C3a prior to and 0.25 h after
dosing did not demonstrate activation of the complement system. CRP
and fibrinogen levels were only increased in subjects in whom
symptoms of concurrent infections were observed.
[0783] All subjects survived and the overall tolerability of J695
was very good. In no case did treatment have to be stopped because
of adverse events (AEs). The most commonly observed AEs were
headache and common cold/bronchitis, neither of which were
categorized as severe.
[0784] One of the study subjects, a 33-year-old single male, was
suffering from psoriasis guttata at the start of the study.
According to the randomized study design, this subject by chance
received 5 mg/kg J695 by SC administration. Ten days prior to
administration of the antibody, the subject showed only small
discrete papular lesions on the arms and legs. At the time of the
antibody administration, the subject displayed increased reddening,
thickness of the erythematous plaques, and increased
hyperkaratosis. One week after J695 administration, the subject
reported an improvement in skin condition, including flattening of
the lesions and a decrease in scaling. Shortly after the second
administration of J695 (5 mg/kg IV), the subject's skin was totally
cleared of psoriatic lesions, in the absence of any local
treatment. Erythematous plaques covered with white scales
reappeared concomitant with the expected clearance of J695 after
the second administration of antibody.
Example 10
Comparison of J695 Produced by Two CHO Cell Lines
[0785] For recombinant expression of J695, a recombinant expression
vector encoding both the antibody heavy chain and the antibody
light chain is introduced into dhfr-CHO cells (Urlaub, G. and
Chasin, L. A. (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220) by
calcium phosphate-mediated transfection. Within the recombinant
expression vector, the antibody heavy and light chain genes are
each operatively linked to enhancer/promoter regulatory elements
(e.g., derived from SV40, CMV, adenovirus and the like, such as a
CMV enhancer/AdMLP promoter regulatory element or an SV40
enhancer/AdMLP promoter regulatory element) to drive high levels of
transcription of the genes. The recombinant expression vector also
carries a DHFR gene, which allows for selection of CHO cells that
have been transfected with the vector using methotrexate
selection/amplification.
[0786] One hundred and fifty micrograms of an expression vector
encoding the peptide sequences of the human antibody J695 were
dissolved in 2.7 ml water in a 50 ml conical tube. Three hundred
.mu.L of 2.5 M CaCl.sub.2 were added and this DNA mixture was added
dropwise to 3 ml of 2.times.HEPES buffered saline in a 50 ml
conical tube. After vortexing for 5 sec and incubating at room
temperature for 20 min, 1 mL was distributed evenly over each plate
(still in F12 medium), and the plates were incubated at 37.degree.
C. for 4 h. Liquid was removed by aspiration and 2 ml of 10% DMSO
in F12 were added to each plate. The DMSO shock continued for 1
min, after which the DMSO was diluted by the addition of 5 ml PBS
to each plate. Plates were washed twice in PBS, followed by the
addition of 10 ml of alpha MEM, supplemented with H/T and 5% FBS
(selective for cells expressing DHFR) and overnight incubation at
37.degree. C. Cells were seeded into 96-well plates at a density of
100 cells per well, and plates were incubated at 37.degree. C., 5%
CO.sub.2 for two weeks, with one change of medium per week.
[0787] Five days after the final medium change, culture
supernatants were diluted 1:50 and tested using an ELISA specific
for human IgG gamma chain. The clones yielding the highest ELISA
signal were transferred from the 96-well plates to 12-well plates
in 1.5 ml/well of Alpha MEM+5% dialyzed serum. After 3 days,
another ELISA specific for human IgG gamma chain was performed, and
the 12 clones with the greatest activity were split into the alpha
MEM+5% dialyzed serum and 20 nM MTX. Cell line 031898 218 grew in
the presence of 20 nM MTX without any apparent cell death or
reduction in growth rate, produced 1.8 .mu.g/ml hIgG in a three-day
assay. T-25 cultures of 031898 218, growing in medium containing
MTX, produced an average of 11.9 .mu.g/ml of J695. The line,
designated ALP903, was adapted to growth in suspension under
serum-free conditions, where it produced 7.5 pg J695/cell/24 h.
[0788] ALP903 cells, after initial selection in alpha MEM/5% FBS/20
nM MTX medium, were passed again in 20 nM MTX. The cells were
cultured under 100 nM MTX selection, followed by passaging in 500
nM MTX twice in the next 30 days. At that time the culture was
producing 32 .mu.g J695/mL/24 h. The culture was subcloned by
limiting dilution. Subclone 218-22 produced 16.5 .mu.g/mL in a
96-well plate in 2 days and 50.3 .mu.g/mL of J695 in a 12-well dish
in 2 days. Clone 218-22 was cultured in alpha MEM/5% dialyzed
FBS/500 nM MTX for 38 days, followed by adaptation to serum-free
spinner culture, as above. The average cell-specific productivity
of the serum-free suspension culture, designated ALP 905, was 58
pg/cell/24 h.
[0789] The first cell line used to produce J695 (ALP 903) resulted
in lower yields of the antibody from culture than a second cell
line, ALP 905. To assure that the ALP 905-produced J695 was
functionally identical to that produced from ALP 903, both batches
of antibodies were assessed for IL-12 affinity, for the ability to
block IL-12 binding to cellular receptors, for the ability to
inhibit IFN-.gamma. induction by IL-12, and for the ability to
inhibit IL-12-mediated PHA blast proliferation.
[0790] The affinities of J695 batches ALP 903 and ALP 905 for IL-12
were determined by measuring the kinetic rate constants of binding
to IL-12 by surface plasmon resonance studies (BIAcore analyses).
The off-rate constant (k.sub.d) and the on-rate constant (k.sub.a)
of antibody batches ALP903 and ALP905 for binding to rhIL-12 were
determined in three experiments (as described in Example 3). The
affinity, K.sub.d, of binding to IL-12 was calculated by dividing
the off-rate constant by the on-rate constant. K.sub.d was
calculated for each separate experiment and then averaged. The
results showed that the determined kinetic parameters and affinity
of binding to rhIL-12 were very similar for J695 batches ALP 903
and ALP 905: the calculated K.sub.d was
1.19.+-.0.22.times.10.sup.-10 M for batch ALP 903 and
1.49.+-.0.47.times.10.sup.-10 M for batch ALP 905 (see Table
13).
[0791] The ability of J695 derived from both ALP 903 and ALP 905 to
block binding of rhIL-12 to IL-12 receptors on human PHA-activated
T-lymphoblasts was assessed (see Example 3). Each sample of J695
was tested at a starting concentration of 1.times.10.sup.-8 with
10-fold serial dilutions. The antibody was preincubated for 1 hour
at 37.degree. C. with 50 pM [.sup.125I]-human IL-12 in binding
buffer. PHA blast cells were added to the
antibody/[.sup.125I]-human IL-12 mixture and incubated for 2 h at
room temperature. Cell bound radioactivity was separated from free
[.sup.125I]-IL-12 by centrifugation and washing steps, and %
inhibition was calculated. The IC.sub.50 values for J695 were
determined from the inhibition curves using 4-parameter curve
fitting and were confirmed by two independent experiments.
Incubations were carried out in duplicate. The results for the two
batches of J695 were very similar (see Table 13).
[0792] The ability of J695 from both ALP 903 and ALP 905 cells to
inhibit rhIL-12-induced IFN-.gamma. production by human
PHA-activated lymphoblasts in vitro was assessed. Serial dilutions
of J695 were preincubated with 200 pg/mL rhIL-12 for 1 h at
37.degree. C. PHA lymphoblast cells were added and incubated for 18
hours at 37.degree. C. After incubation, cell free supernatant was
withdrawn and the level of human IFN-.gamma. determined by ELISA.
The IC.sub.50 values from the inhibition curves were plotted
against the antibody concentration using 4-parameter curve fitting.
The results demonstrate that the ability of the two batches to
inhibit IFN-.gamma. production is very similar.
[0793] The in vitro PHA blast cell proliferation assay was used to
measure the neutralization capacity of ALP 903 and ALP 905 J695 for
rhIL-12. Serial dilutions of J695 of each type were preincubated
with 230 pg/mL human IL-12 for 1 h at 37.degree. C. Next PHA blast
cells were added and incubated for 3 days at 37.degree. C. The
cells were then labeled for 6 hours with 1 .gamma.Ci/well
[.sup.3H]-thymidine. The cultures were harvested and
[.sup.3H]-thymidine incorporation measured. Non-specific
proliferation (background) was measured in the absence of rhIL-12.
The IC.sub.50 values for ALP 903 and ALP 905 J695 were found to be
very similar and are set forth in Table 13.
[0794] The activity of the J695 antibodies in neutralizing rhIL-12
activity, in blocking IL-12 binding to cell surface receptors, and
in binding to rhIL-12 did not significantly differ from batch ALP
903 to batch ALP 905, and thus the antibodies produced from these
two different cell types were equivalent. TABLE-US-00019 TABLE 13
Comparison of the Properties of J695 lots ALP 903 and ALP 905 PHA
blast IFN-.gamma. k.sub.a, On-rate k.sub.d, Off-rate RB assay Assay
IC.sub.50 Assay IC.sub.50 Antibody (M.sup.-1, s.sup.-1) (s.sup.-1)
K.sub.d(M) IC.sub.50 (M) (M) (M) J695 3.75 .times. 10.sup.5 4.46
.times. 10.sup.-5 1.19 .times. 10.sup.-10 3.4 .times. 10.sup.-11
5.5 .times. 10.sup.-12 5.8 .times. 10.sup.-12 ALP 903 J695 3.91
.times. 10.sup.5 5.59 .times. 10.sup.-5 1.49 .times. 10.sup.-10 3.0
.times. 10.sup.-11 4.4 .times. 10.sup.-12 4.3 .times. 10.sup.-12
ALP 905
Example 11
Anti-rhIL12 Monoclonal Antibody Epitope Map Determination by
Biacore
[0795] Real-time biospecific interaction analysis (BIA) based on
surface plasmon resonance technology was used to map the epitope
specificity patterns of J695 and 4 other monoclonal antibodies
against soluble recombinant human IL12. The technique does not
require labeling of either antibodies or antigen. Antibodies
directed against separate and distinct epitopes will bind
simultaneously to the antigen, whereas antibodies directed against
closely related epitopes will interfere with each other's binding.
Furthermore, if the second antibody fails to bind, the epitopes
defined by the two antibodies may be identical or overlapping, or
binding of the first antibody may prevent binding of the second
antibody through allosteric inhibition caused by a conformational
alteration in the target molecule.
[0796] An epitope mapping assay using Biacore was performed. First,
carboxyl groups on the dextran matrix were activated with 100 mM
N-hydroxysuccinimide (NHS) and 400 mM
N-Ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride
(EDC), across four different flow cells. Next, Antibody 1 was
injected across the activated matrix. Approximately fifty
microliters of anti-rhIL12 antibody (25 .mu.g/ml), diluted in
sodium acetate, pH 4.5, was injected across the activated biosensor
and free amines on the protein are bound directly to the activated
carboxyl groups. Typically, 5000 resonance units were immobilized.
Unreacted matrix EDC-esters were deactivated by an injection of 1 M
ethanolamine. Standard amine coupling kits were commercially
available (Biacore AB, Cat. No. BR-1000-50, Uppsala, Sweden). SPR
measurements were performed using CM biosensor chip (Biacore AB,
Cat No BR-1000-14, Uppsala, Sweden). All antibodies and antigens to
be analyzed on the biosensor surface were diluted in HBS-EP running
buffer (Biacore AB, Cat No BR-1001-88, Uppsala, Sweden).
[0797] Next, rhuIL12 (100 nM) was injected across covalently
immobilized antibody on the CM5 biosensor surface at a flow rate of
25 .mu.l/min. Before injection of the antigen and immediately
afterward, HBS-EP buffer alone flowed through each flow cell.
Excess soluble Antibody 2 (25 .mu.g/ml) was then injected across
captured rhuIL12 (5 minute contact time). Before injection of
Antibody 2 and immediately afterward, HBS-EP buffer alone flowed
through each flow cell. The net difference in the signals between
the baseline and the point corresponding to approximately 30
seconds after completion of Mab injection was taken to represent
the final binding value. Again, the response was measured in
Resonance Units. Biosensor matrices were regenerated using 10 mM
HCl (5 minute contact time) before injection of the next
sample.
[0798] Antibodies and antigens supplied by Abbott Bioresearch
Center and/or commercial vendors were as follows: Human J695; Mouse
C8.6.2 Mab; Human 1D4.7 Mab; Human C340 Mab; Mouse 7G3 Mab; Human
IgG1 control (1.0 mg/ml, Sigma Catalog No. 1-3889). Each of the
antibodies used in this study binds to an epitope of the p40
subunit of human IL-12. The antibodies ID4.7 and 7G3 are described
in U.S. Provisional Patent Application No. 60/695,679, filed Jun.
29, 2005, and U.S. patent application Ser. No. 11/478,096, filed
Jun. 29, 2006. The antibody C340 is described in U.S. Pat. No.
7,063,964 and U.S. Pat. No. 6,902,734. The entire contents of each
of the foregoing patents and patent applications are hereby
incorporated herein by reference. The antibody C8.6.2 is a subclone
of, and thus has the same characteristics as, the antibody C8.6,
which is described in A. D'Andrea et al., 1992 J. Exp. Med.
176:1387-1398, the entire contents of which are hereby incorporated
herein by reference. Recombinant human interleukin 12 (rhIL12,
commercially available by Wyeth).
[0799] The reactivity patterns for the monoclonal antibodies tested
are displayed in Table 14. In these experiments, J695 and 7G3
monoclonal antibodies could bind recombinant human IL-12
concurrently. In this case, simultaneous binding with recombinant
human IL-12 confirms that the antibodies occupy different sites.
Sensorgrams obtained from mapping experiments revealed that 1D4.7
and 7G3 monoclonal antibodies recognize independent epitopes.
Another pair of monoclonal antibodies showed simultaneous binding
in the order [C340]-[rhIL12]-[7G3]. Again, a positive result in
this sequence indicated distinct and independent epitopes for these
two antibodies. Test antibodies gave negative results when used in
the order [C8.6.2]-[rhIL12]-[C8.6.2], [C340]-[rhIL12]-[C8.6.2] and
[C340]-[rhIL12]-[1D4.7]. The lack of binding of the second antibody
is presumably due to occupation of the epitope. In a negative
control, omission of recombinant human IL-12 from the normal
Biacore assay sequence reduced the response from the second
antibody to background levels. Further, murine and human IgG
matched isotype controls gave negative results, regardless of
orientation. TABLE-US-00020 TABLE 14 Summary of pair-wise epitope
mapping of IL-12 antibodies by Biacore Directly immobilized
ant-human IL-12 Mab (approximately 5000 RU's) Sample FC 1 FC 2 FC 3
FC 4 1 2.degree. Soluble Mouse IgG1 J695 Human IgG1 Human IgG1
anti-human IL-12 Mab C8.6.2 C340 1D4.7 2 Mouse IgG C8.6.2 - slow
rhIL-12 - - displacement 3 J695 rapid rhIL-12 - rapid rIL-12 rapid
rhIL-12 displacement displacement displacement 4 Human IgG1 C340 -
slow rhIL-12 - - displacement 5 Human IgG1 1D4.7 - slow rhIL-12 - -
displacement 6 Mouse IgG1 7G3 + + + + If both anti-human IL-12
antibodies can bind simultaneously to the target molecule
(rhIL-12), they define separate and independent epitopes. If the
second antibody fails to bind, then the epitopes defined by the two
antibodies may be identical or overlapping, or binding of the first
antibody may prevent binding of the second through a conformational
alteration in the target molecule. Soluble antigen = recombinant
IL-12 (p40/p35).
[0800] The results described in this Example demonstrate that
Biacore can be used to characterize monoclonal antibody epitopes.
Each antibody and antigen was injected over a long time period,
until a plateau was reached in the SPR signal. Examination of the
sensorgrams showed that all human anti-rhIL12 monoclonal antibodies
bound specifically to rhIL12 when they were directly immobilized
across carboxymethyl dextran surface. The results presented in this
Example demonstrate that the antibodies that can bind
simultaneously to IL-12 are binding to non-overlapping epitopes. In
particular, 7G3 binds to a different epitope that is not shared by
J695, C8.6.2, C340 or 1D4.7. The results further demonstrate that
the antibodies tested that cannot bind simultaneously to IL-12 are
binding to overlapping epitopes (i.e., no binding of antibody 2
observed when IL-12 is bound to antibody 1). In particular, C8.6.2,
C340 and 1D4.7 bind to overlapping epitopes as binding of one of
these antibodies to IL-12 does not allow binding of the second
antibody in all cases. Finally, the results demonstrate that the
antibodies that cannot bind simultaneously to IL-12 are prevented
from binding due to an allosteric interaction that prevents the
antibodies from binding simultaneously (i.e., antibody 2 releases
IL-12 from antibody 1). In particular, J695 cannot bind rhuIL-12
simultaneously with either C8.6.2, C340 or 1D4.7. The inability of
the antibodies to bind IL-12 simultaneously does not indicate a
simple competition between the antibodies for the same binding
site, but rather that a conformational alteration in the target
molecule IL-12 occurs upon binding. This unusual allosteric
mechanism is evident in the observation that soluble J695 rapidly
displaced IL-12 bound to immobilized C8.6.2, C340 or 1D4.7.
Conversely, C8.6.2, C340 and 1D4.7 slowly displaced IL-12 bound to
immobilized J695.
Example 12
Identification of Antibodies Capable of Altering the Conformational
Structure of an Interleukin Containing a p40 Subunit
[0801] Antibodies are identified that bind to the p40 subunit of an
interleukin, e.g., IL-12 or IL-23, and are capable of altering its
conformational structure by using real-time biospecific interaction
analysis (BIA). The experimental protocol used is essentially as
described above in Example 11.
[0802] Specifically, carboxyl groups on a dextran matrix are
activated, typically with 100 mM N-hydroxysuccinimide (NHS) and 400
mM N-Ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride
(EDC). Next, a first antibody that specifically binds to the p40
subunit of an interleukin, e.g., IL-12 or IL-23, is then injected
across the activated matrix. In particular, the first antibody
injected across the activated matrix is an antibody that binds to
an epitope of the p40 subunit of IL-12 to which the antibodies
C8.6.2, C340 or 1D4.7 bind. Preferably, the first antibody is
either C8.6.2, C340 or 1D4.7. Approximately fifty microliters of
the first antibody (e.g., at 25 .mu.g/ml, diluted in sodium
acetate, pH 4.5) is then injected across the activated biosensor
and free amines on the protein are bound directly to the activated
carboxyl groups. Typically, 5000 resonance units are immobilized.
Unreacted matrix EDC-esters are deactivated by an injection of 1 M
ethanolamine. SPR measurements are performed using CM biosensor
chip (Biacore AB, Cat No BR-1000-14, Uppsala, Sweden) as described
above in Example 11. All antibodies and antigens to be analyzed on
the biosensor surface are diluted in HBS-EP running buffer (Biacore
AB, Cat No BR-1001-88, Uppsala, Sweden).
[0803] Next, the p40 subunit of the interleukin, e.g., IL-12 or
IL-23 (e.g., 100 nM) is injected across the covalently immobilized
antibody on the CM5 biosensor surface, typically at a flow rate of
approximately 25 .mu.l/min. The p40 subunit of the interleukin,
e.g., IL-12 or IL-23, can be injected, for example, either as an
isolated single subunit (or fragment thereof) or alternatively can
be injected in a heterodimeric form, wherein the heterodimer
comprises the p40 subunit and a second subunit, e.g., the
heterodimer p40/p35 (IL-12), or an alternate heterodimer comprising
the p40 subunit of IL-12 and a p19 subunit (p40/p19; IL-23). Before
injection of the antigen and immediately afterward, HBS-EP buffer
alone is flowed through each flow cell. Following this, an excess
of a soluble test antibody (e.g., at a concentration of 25
.mu.g/ml) is injected across the captured p40 subunit of the
interleukin (e.g., IL-12 or IL-23), typically for a contact time of
approximately 5 minutes. Before injection of the test antibody and
immediately afterward, HBS-EP buffer alone is again flowed through
each flow cell. The net difference in the signals between the
baseline and the point corresponding to approximately 30 seconds
after completion of antibody injection is taken to represent the
final binding value and is measured in Resonance Units. Antibodies
which rapidly displace the p40 subunit of the interleukin (e.g.,
IL-12 or IL-23) from the first antibody immobilized on the CM5
biosensor surface are identified as antibodies that can alter the
conformational structure of the interleukin, e.g., the p40 subunit
of the interleukin.
[0804] This conformational alteration in the structure of the
interleukin ((e.g., IL-12 or IL-23) may be confirmed by any of the
well known techniques in the art for monitoring 3-dimensional
structures of proteins. For example X-ray crystallography or
Circular Dichroism may be used.
EQUIVALENTS
[0805] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims
Sequence CWU 0
0
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 675 <210>
SEQ ID NO 1 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 1 could be either His or Ser
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
4 could be either Tyr or His <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 6 could be either Tyr, Asn or Thr
<400> SEQUENCE: 1 Xaa Gly Ser Xaa Asp Xaa 1 5 <210> SEQ
ID NO 2 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 2 could be either Ser or Thr
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
4 could be either Asp or Glu <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 5 could be either Ser, Arg or Lys
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
6 could be either Ser, Gly or Tyr <220> FEATURE: <223>
OTHER INFORMATION: Xaa at position 7 could be either Leu, Phe, Thr
or Ser <220> FEATURE: <223> OTHER INFORMATION: Xaa at
position 8 could be either Arg, Ser, Thr, Trp or His <220>
FEATURE: <223> OTHER INFORMATION: Xaa at position 9 could be
either Gly or Pro <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 10 could be either Ser, Thr, Ala or
Leu <220> FEATURE: <223> OTHER INFORMATION: Xaa at
position 11 could be either Arg, Ser, Met, Thr or Leu <220>
FEATURE: <223> OTHER INFORMATION: Xaa at position 12 could be
either Val, Ile, Thr, Met or Leu <400> SEQUENCE: 2 Gln Xaa
Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 <210> SEQ ID
NO 3 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 3 Phe Ile Arg Tyr Asp
Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 4 <211> LENGTH: 7 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <220> FEATURE: <223>
OTHER INFORMATION: Xaa at position 1 could be either Gly or Tyr
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
3 could be either Asp or Ser <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 4 could be either Gln or Asn
<400> SEQUENCE: 4 Xaa Asn Xaa Xaa Arg Pro Ser 1 5 <210>
SEQ ID NO 5 <211> LENGTH: 9 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: Xaa represents either Ser or Glu <400> SEQUENCE:
5 Phe Thr Phe Ser Xaa Tyr Gly Met His 1 5 <210> SEQ ID NO 6
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
Xaa at position 1 could be either Ser or Thr <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 3 could be either
Ser or Gly <220> FEATURE: <223> OTHER INFORMATION: Xaa
at position 4 could be either Arg or Ser <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 8 could be either
Gly or Val <220> FEATURE: <223> OTHER INFORMATION: Xaa
at position 9 could be either Ser or Ala <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 10 could be either
Asn, Gly or Tyr <220> FEATURE: <223> OTHER INFORMATION:
Xaa at position 11 could be either Thr or Asp <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 13 could be either
Lys or His <400> SEQUENCE: 6 Xaa Gly Xaa Xaa Ser Asn Ile Xaa
Xaa Xaa Xaa Val Xaa 1 5 10 <210> SEQ ID NO 7 <211>
LENGTH: 115 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <220> FEATURE: <223> OTHER INFORMATION: Xaa at
position 6 could be either Gln or Glu <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 16 could be either
Arg or Gly <220> FEATURE: <223> OTHER INFORMATION: Xaa
at position 31 could be either Ser or Glu <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 84 could be either
Lys or Asn <220> FEATURE: <223> OTHER INFORMATION: Xaa
at position 97 could be either Thr, Ala or Lys <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 98 could be either
Thr or Lys <220> FEATURE: <223> OTHER INFORMATION: Xaa
at position 99 could be either Ser or His <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 102 could be either
Tyr or His <220> FEATURE: <223> OTHER INFORMATION: Xaa
at position 104 could be either Tyr, Asn or Thr <400>
SEQUENCE: 7 Gln Val Gln Leu Val Xaa Ser Gly Gly Gly Val Val Gln Pro
Gly Xaa 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Xaa Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Asx 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Xaa
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Xaa Xaa
Xaa Gly Ser Xaa Asp Xaa Trp Gly Gln Gly Thr Met Val Thr 100 105 110
Val Ser Ser 115 <210> SEQ ID NO 8 <211> LENGTH: 112
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
1 could be either Ser or Gln <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 2 could be either Tyr or Ser
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
13 could be either Thr or Ala <220> FEATURE: <223>
OTHER INFORMATION: Xaa at position 23 and 91 could be either Ser or
Thr <220> FEATURE: <223> OTHER INFORMATION: Xaa at
position 25 could be either Gly or Ser <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 26 could be either
Arg or Ser <220> FEATURE: <223> OTHER INFORMATION: Xaa
at position 30 could be either Gly or Val <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 31 could be either
Ser or Ala <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 35 could be either
Lys or His <220> FEATURE: <223> OTHER INFORMATION: Xaa
at position 51 could be either Gly or Lys <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 54 could be either
Gln or Asn <220> FEATURE: <223> OTHER INFORMATION: Xaa
at position 79 could be either Val or Leu <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 93 could be either
Asp or Glu <220> FEATURE: <223> OTHER INFORMATION: Xaa
at position 94 could be either Ser, Arg or Lys <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 95 could be either
Ser, Gly or Tyr <220> FEATURE: <223> OTHER INFORMATION:
Xaa at position 96 could be either Leu, Phe, Thr or Ser <220>
FEATURE: <223> OTHER INFORMATION: Xaa at position 97 could be
either Arg, Ser, Thr, Trp or His <220> FEATURE: <223>
OTHER INFORMATION: Xaa at position 98 could be either Gly or Pro
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
99 could be either Ser, Thr, Ala or Leu <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 100 could be either
Arg, Ser, Met, Thr or Leu <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 101 could be either Val, Ile, Thr, Met
or Leu <220> FEATURE: <223> OTHER INFORMATION: Xaa at
position 32 could be either Asn, Gly or Tyr <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 33 could be either
Thr or Asp <220> FEATURE: <223> OTHER INFORMATION: Xaa
at position 53 could be either Asp or Ser <400> SEQUENCE: 8
Xaa Xaa Val Leu Thr Gln Pro Pro Ser Val Ser Gly Xaa Pro Gly Gln 1 5
10 15 Arg Val Thr Ile Ser Cys Xaa Gly Xaa Xaa Ser Asn Ile Xaa Xaa
Xaa 20 25 30 Xaa Val Xaa Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
Lys Leu Leu 35 40 45 Ile Tyr Xaa Asn Xaa Xaa Arg Pro Ser Gly Val
Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser
Leu Ala Ile Thr Gly Xaa Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr
Tyr Cys Gln Xaa Tyr Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa
Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210>
SEQ ID NO 9 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 2 could be either Gly, Val, Cys or His
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
3 could be either Ser or Thr <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 4 could be either His, Thr, Val, Arg,
<220> FEATURE: <223> OTHER INFORMATION: or Ile
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
5 could be either Asp or Ser <223> OTHER INFORMATION: Xaa at
position 6 could be either Asn, Lys, Ala, Thr, Ser, Phe, Trp, or
His <400> SEQUENCE: 9 His Xaa Xaa Xaa Xaa Xaa 1 5 <210>
SEQ ID NO 10 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <220> FEATURE: <223>
OTHER INFORMATION: Xaa at position 4 could be either Asp or Ser
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
5 represents any amino acid <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 6 could be either Gly, Asp, Gln, Leu,
Phe, Arg, His, Asn or Tyr <400> SEQUENCE: 10 Gln Ser Tyr Xaa
Xaa Xaa Thr His Pro Ala Leu Leu 1 5 10 <210> SEQ ID NO 11
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
Xaa at position 1 could be either Phe, Thr or Tyr <220>
FEATURE: <223> OTHER INFORMATION: Xaa at position 3 could be
either Arg or Ala <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 5 could be either Asp, Ser, Glu or Ala
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
6 could be either Gly or Arg <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 8 represents any amino acid
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
10 could be either Tyr or Glu <400> SEQUENCE: 11 Xaa Ile Xaa
Tyr Xaa Xaa Ser Xaa Lys Xaa Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 12 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 1 could be either
Gly, Tyr, Ser, Thr, Asn or Gln <400> SEQUENCE: 12 Xaa Asn Asp
Gln Arg Pro Ser 1 5 <210> SEQ ID NO 13 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
4 and 5 represents any amino acid <220> FEATURE: <223>
OTHER INFORMATION: Xaa at position 6 could be either Tyr or His
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
7 could be either Gly, Met, Ala, Asn or Ser <400> SEQUENCE:
13 Phe Thr Phe Xaa Xaa Xaa Xaa Met His 1 5 <210> SEQ ID NO 14
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
Xaa at position 9 could be either Ser, Cys, Arg, Asn, Asp or Thr
<220> FEATURE: <223> OTHER INFORMATION: Xaa at position
10 could be either Asn, Met or Ile <220> FEATURE: <223>
OTHER INFORMATION: Xaa at position 11 could be either Thr, Tyr,
Asp, His, Lys or Pro <400> SEQUENCE: 14 Ser Gly Gly Arg Ser
Asn Ile Gly Xaa Xaa Xaa Val Lys 1 5 10 <210> SEQ ID NO 15
<211> LENGTH: 114 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
Xaa at position 30 could be Ser or Glu <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 83 could be Lys or
Asn <220> FEATURE: <223> OTHER INFORMATION: Xaa at
position 5 could be either Gln or Glu <400> SEQUENCE: 15 Gln
Val Gln Val Xaa Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser 1 5 10
15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Xaa Tyr Gly
20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val Ala 35 40 45 Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala
Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr Leu
65 70 75 80 Gln Met Xaa Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Lys 85 90 95 Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr
Met Val Thr Val 100 105 110 Ser Ser <210> SEQ ID NO 16
<211> LENGTH: 112 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
Xaa at position 1 could be either Ser or Gln <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 2 could be Tyr or
Ser <220> FEATURE: <223> OTHER INFORMATION: Xaa at
position 13 could be either Thr or Ala <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 25 could be either
Gly or Ser <220> FEATURE: <223> OTHER INFORMATION: Xaa
at position 51 and 95 could be either Gly or Tyr <220>
FEATURE: <223> OTHER INFORMATION: Xaa at position 79 could be
either Val or Leu <400> SEQUENCE: 16 Xaa Xaa Val Leu Thr Gln
Pro Pro Ser Val Ser Gly Xaa Pro Gly Gln 1 5 10 15 Arg Val Thr Ile
Ser Cys Ser Gly Xaa Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val
Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45
Ile Tyr Xaa Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Xaa
Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp
Arg Xaa Thr 85 90 95 His Pro Ala Leu Leu Phe Gly Thr Gly Thr Lys
Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 17
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 17 His Gly Ser His Asp Asn 1 5
<210> SEQ ID NO 18 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 18 Gln
Ser Tyr Asp Arg Gly Thr His Pro Ala Leu Leu 1 5 10 <210> SEQ
ID NO 19 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 19 Phe Ile Arg Tyr Asp
Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 20 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 20 Gly
Asn Asp Gln Arg Pro Ser 1 5 <210> SEQ ID NO 21 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 21 Phe Thr Phe Ser Ser Tyr Gly Met His 1 5
<210> SEQ ID NO 22 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 22 Ser
Gly Gly Arg Ser Asn Ile Gly Ser Asn Thr Val Lys 1 5 10 <210>
SEQ ID NO 23 <211> LENGTH: 115 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 23 Gln Val
Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Lys Thr His Gly Ser His Asp
Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115
<210> SEQ ID NO 24 <211> LENGTH: 112 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 24 Ser
Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10
15 Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Trp Ile Gly Ser Asn
20 25 30 Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu
Ala Ile Thr Gly Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr
Cys Gln Ser Tyr Asp Arg Gly Thr 85 90 95 His Pro Ala Leu Leu Phe
Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ
ID NO 25 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 25 His Gly Ser His Asp
Asn 1 5 <210> SEQ ID NO 26 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
26 Gln Ser Tyr Asp Arg Tyr Thr His Pro Ala Leu Leu 1 5 10
<210> SEQ ID NO 27 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 27 Phe
Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly <210> SEQ ID NO 28 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
28 Tyr Asn Asp Gln Arg Pro Ser 1 5 <210> SEQ ID NO 29
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 29 Phe Thr Phe Ser Ser Tyr Gly
Met His 1 5 <210> SEQ ID NO 30 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 30
Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn Thr Val Lys 1 5 10
<210> SEQ ID NO 31 <211> LENGTH: 115 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 31 Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Lys Thr His Gly Ser His
Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115
<210> SEQ ID NO 32 <211> LENGTH: 112 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 32 Gln
Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10
15 Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn
20 25 30 Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45 Ile Tyr Tyr Asn Asp Gln Arg Pro Ser Gly Val Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu
Ala Ile Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr
Cys Gln Ser Tyr Asp Arg Tyr Thr 85 90 95 His Pro Ala Leu Leu Phe
Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ
ID NO 33 <211> LENGTH: 115 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 33 Gln Val Gln Leu Val
Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Thr Ser Gly Ser Tyr Asp Tyr Trp Gly
Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ
ID NO 34 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 34 Ser Tyr Val Leu Thr
Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Ser Ser Leu 85 90 95 Arg Gly Ser Arg Val Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 35
<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 35 Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Ser Gly Ser Tyr Asp Tyr Trp Gly Gln Gly
Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO
36 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 32 represents either Gly or Tyr
<400> SEQUENCE: 36 Gln Ser Val Leu Thr Gln Pro Pro Ser Val
Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly
Ser Ser Ser Asn Ile Gly Ala Xaa 20 25 30 Asp Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn
Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln 65 70 75 80
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85
90 95 Ser Gly Ser Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu
Gly 100 105 110 <210> SEQ ID NO 37 <211> LENGTH: 115
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 37 Gln Val Gln Leu Val Gln Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Thr Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val
Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO 38
<211> LENGTH: 112 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 38 Ser Tyr Val Leu Thr Gln Pro
Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser
Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Lys
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile
Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln
65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser
Ser Leu 85 90 95 Arg Gly Ser Arg Val Phe Gly Thr Gly Thr Lys Val
Thr Val Leu Gly 100 105 110
<210> SEQ ID NO 39 <211> LENGTH: 115 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 39 Gln
Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Thr Ser Gly Ser Tyr
Asp Tyr Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115
<210> SEQ ID NO 40 <211> LENGTH: 112 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 40 Ser
Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10
15 Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn
20 25 30 Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu
Ala Ile Thr Gly Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr
Cys Gln Ser Tyr Asp Arg Gly Phe 85 90 95 Thr Gly Ser Arg Val Phe
Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ
ID NO 41 <211> LENGTH: 115 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 41 Gln Val Gln Leu Val
Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Thr Ser Gly Ser Tyr Asp Tyr Trp Gly
Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ
ID NO 42 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 42 Ser Tyr Val Leu Thr
Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Ser Ser Leu 85 90 95 Trp Gly Ser Arg Val Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 43
<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 43 Gln Val Gln Leu Val Gln Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Thr Thr His Gly Ser His Asp Asn Trp Gly Gln Gly
Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO
44 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 44 Ser Tyr Val Leu Thr
Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Arg Gly Phe 85 90 95 Thr Gly Ser Arg Val Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 45
<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 45 Gln Val Gln Leu Val Gln Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Thr Thr His Gly Ser His Asp Asn Trp Gly Gln Gly
Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO
46 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 46 Ser Tyr Val Leu Thr
Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Ser Ser Leu 85 90 95 Trp Gly Ser Arg Val Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 47
<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 47
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Lys Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Lys Thr His Gly Ser
His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser
115 <210> SEQ ID NO 48 <211> LENGTH: 112 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
48 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly
Ser Asn 20 25 30 Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly
Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Thr Gly Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp
Tyr Tyr Cys Gln Thr Tyr Asp Lys Gly Phe 85 90 95 Thr Gly Ser Ser
Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110
<210> SEQ ID NO 49 <211> LENGTH: 115 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 49 Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Lys Thr His Gly Ser His
Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115
<210> SEQ ID NO 50 <211> LENGTH: 112 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 50 Gln
Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10
15 Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn
20 25 30 Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu
Ala Ile Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr
Cys Gln Thr Tyr Asp Lys Gly Phe 85 90 95 Thr Gly Ser Ser Val Phe
Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ
ID NO 51 <211> LENGTH: 115 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 51 Gln Val Gln Leu Val
Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Thr His Gly Ser His Asp Thr Trp Gly
Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ
ID NO 52 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 52 Ser Tyr Val Leu Thr
Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Ser Ser Leu 85 90 95 Trp Gly Thr Arg Val Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 53
<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 53 Gln Val Gln Leu Val Gln Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Thr Thr His Gly Ser His Asp Asn Trp Gly Gln Gly
Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO
54 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 54 Ser Tyr Val Leu Thr
Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Val Ser Asn 20 25 30 Thr
Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Arg Gly Phe 85 90 95 Thr Gly Ser Arg Val Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 55
<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 55 Gln Val Gln Leu Val Gln Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Thr Thr His Gly Ser His Asp Asn Trp
Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210>
SEQ ID NO 56 <211> LENGTH: 112 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 56 Ser Tyr
Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Val Ser Asn 20
25 30 Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Thr Gly Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Gln Ser Tyr Asp Arg Gly Phe 85 90 95 Thr Gly Ala Arg Val Phe Gly
Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID
NO 57 <211> LENGTH: 115 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 57 Gln Val Gln Leu Val
Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Lys Thr His Gly Ser His Asp Asn Trp Gly
Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ
ID NO 58 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 58 Ser Tyr Val Leu Thr
Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr
Asp Lys Gly Phe 85 90 95 Thr Gly Ser Ser Val Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 59
<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 59 Gln Val Gln Leu Val Gln Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly
Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO
60 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 60 Ser Tyr Val Leu Thr
Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Glu Arg Gly Phe 85 90 95 Thr Gly Ser Met Val Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 61
<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 61 Gln Val Gln Leu Val Gln Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly
Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO
62 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 62 Ser Tyr Val Leu Thr
Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Arg Gly Thr 85 90 95 His Pro Leu Thr Ile Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 63
<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 63 Gln Val Gln Leu Val Gln Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr
65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly
Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO
64 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 64 Ser Tyr Val Leu Thr
Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Arg Gly Ser 85 90 95 His Pro Ala Leu Thr Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 65
<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 65 Gln Val Gln Leu Val Gln Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly
Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO
66 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 66 Ser Tyr Val Leu Thr
Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Arg Gly Thr 85 90 95 His Pro Leu Thr Met Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 67
<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 67 Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly
Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO
68 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 68 Gln Ser Val Leu Thr
Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Arg Gly Thr 85 90 95 His Pro Leu Thr Met Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 69
<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 69 Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly
Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO
70 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 70 Gln Ser Val Leu Thr
Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Arg Gly Thr 85 90 95 His Pro Ala Leu Leu Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 71
<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 71 Gln Val Gln Leu Val Gln Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly
Thr Met Val Thr 100 105 110
Val Ser Ser 115 <210> SEQ ID NO 72 <211> LENGTH: 112
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 72 Gln Ser Val Leu Thr Gln Pro Pro Ser Val
Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly
Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Lys Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn
Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln 65 70 75 80
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Gly Thr 85
90 95 His Pro Ala Leu Leu Phe Gly Thr Gly Thr Lys Val Thr Val Leu
Gly 100 105 110 <210> SEQ ID NO 73 <211> LENGTH: 115
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 73 Gln Val Gln Leu Val Gln Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val
Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO 74
<211> LENGTH: 112 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 74 Gln Ser Val Leu Thr Gln Pro
Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser
Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Lys
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile
Tyr Tyr Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln
65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg
Gly Thr 85 90 95 His Pro Ala Leu Leu Phe Gly Thr Gly Thr Lys Val
Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 75 <211>
LENGTH: 115 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 75 Gln Val Gln Leu Val Gln Ser Gly
Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe
Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly
Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO
76 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 76 Gln Ser Val Leu Thr
Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Arg Tyr Thr 85 90 95 His Pro Ala Leu Leu Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly 100 105 110 <210> SEQ ID NO 77
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 77 Ser Gly Ser Tyr Asp Tyr 1 5
<210> SEQ ID NO 78 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 78 His
Gly Ser His Asp Asn 1 5 <210> SEQ ID NO 79 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 79 His Gly Ser Tyr Asp Tyr 1 5 <210>
SEQ ID NO 80 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 80 Arg Arg
Arg Ser Asn Tyr 1 5 <210> SEQ ID NO 81 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 81 Ser Gly Ser Ile Asp Tyr 1 5 <210>
SEQ ID NO 82 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 82 His Gly
Ser His Asp Asp 1 5 <210> SEQ ID NO 83 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 83 His Gly Ser His Asp Asn 1 5 <210>
SEQ ID NO 84 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 84 Thr Thr
His Gly Ser His Asp Asn Trp Gly Gln Gly 1 5 10 <210> SEQ ID
NO 85 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 85
Ala Lys His Gly Ser His Asp Asn Trp Gly Gln Gly 1 5 10 <210>
SEQ ID NO 86 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 86 Thr Thr
His Gly Ser His Asp Asn Trp Ser Gln Gly 1 5 10 <210> SEQ ID
NO 87 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 87 Thr Thr His Gly Ser
His Asp Thr Trp Gly Gln Gly 1 5 10 <210> SEQ ID NO 88
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 88 Lys Thr His Gly Ser His Asp
Asn Trp Gly Gln Gly 1 5 10 <210> SEQ ID NO 89 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 89 Lys Thr His Gly Ser His Asp Asn Trp Gly
His Gly 1 5 10 <210> SEQ ID NO 90 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 90 Thr Thr His Gly Ser His Asp Asn Trp Ser
Gln Gly 1 5 10 <210> SEQ ID NO 91 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 91 Thr Thr His Arg Ser His Asn Asn Trp Gly
Gln Gly 1 5 10 <210> SEQ ID NO 92 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 92 Thr Thr His Gly Ser His Asp Asn 1 5
<210> SEQ ID NO 93 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 93 Thr
Thr His Gly Ser His Asp Thr 1 5 <210> SEQ ID NO 94
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 94 Thr Lys His Gly Ser His Asp
Asn 1 5 <210> SEQ ID NO 95 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
95 Thr Thr Gln Gly Arg His Asp Asn 1 5 <210> SEQ ID NO 96
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 96 Lys Thr Arg Gly Arg His Asp
Asn 1 5 <210> SEQ ID NO 97 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
97 Thr Thr His Gly Ser His Asp Lys 1 5 <210> SEQ ID NO 98
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 98 Thr Thr His Gly Ser His Asp
Asp 1 5 <210> SEQ ID NO 99 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
99 Lys Thr His Gly Ser His Asp Asn 1 5 <210> SEQ ID NO 100
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 100 Lys Thr His Gly Ser His Asp
Asn 1 5 <210> SEQ ID NO 101 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
101 Thr Thr His Gly Ser His Asp Asn 1 5 <210> SEQ ID NO 102
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 102 Thr Thr Ser Gly Ser Tyr Asp
Tyr 1 5 <210> SEQ ID NO 103 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
103 Thr Thr His Gly Ser His Asp Asn 1 5 <210> SEQ ID NO 104
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 104 Thr Thr His Gly Ser Gln Asp
Asn 1 5 <210> SEQ ID NO 105 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
105 Ala Thr His Gly Ser Gln Asp Asn 1 5 <210> SEQ ID NO 106
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 106 His Gly Ser Gln Asp Thr 1 5
<210> SEQ ID NO 107 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 107
Ser Gly Ser Tyr Asp Tyr 1 5 <210> SEQ ID NO 108 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens
<400> SEQUENCE: 108 His Gly Ser Gln Asp Asn 1 5 <210>
SEQ ID NO 109 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 109 Cys
Lys Thr His Gly Ser His Asp Asn 1 5 <210> SEQ ID NO 110
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 110 Gln Ser Tyr Asp Ser Ser Leu
Arg Gly Ser Arg Val 1 5 10 <210> SEQ ID NO 111 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 111 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 112 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 112 Gln Ser Tyr Asp Ser Ser Leu Arg Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 113 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 113 Gln Ser Tyr Asp Ser Ser Leu Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 114 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 114 Gln Ser Tyr Asp Ser Ser Leu Trp Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 115 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 115 Gln Thr Tyr Asp Ile Ser Glu Ser Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 116 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 116 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 117 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 117 Gln Thr Tyr Asp Arg Gly Phe Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 118 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 118 Gln Thr Tyr Asp Lys Gly Phe Thr Gly Ser
Ser Val 1 5 10 <210> SEQ ID NO 119 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 119 Gln Ser Tyr Asp Arg Arg Phe Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 120 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 120 Gln Ser Tyr Asp Trp Asn Phe Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 121 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 121 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 122 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 122 Gln Ser Tyr Asp Asn Gly Phe Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 123 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 123 Gln Ser Tyr Asp Asn Ala Val Thr Ala Ser
Lys Val 1 5 10 <210> SEQ ID NO 124 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 124 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 125 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 125 Gln Ser Tyr Asp Ser Ser Leu Trp Gly Thr
Arg Val 1 5 10 <210> SEQ ID NO 126 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 126 Gln Ser Tyr Asp Arg Asp Phe Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 127 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 127 Gln Ser Tyr Glu Arg Gly Phe Thr Gly Ser
Met Val 1 5 10 <210> SEQ ID NO 128 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 128 Gln Ser Tyr Asp Asn Gly Phe Thr Gly Ala
Arg Val 1 5 10 <210> SEQ ID NO 129 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 129 Gln Ser Tyr Asp Arg Arg Phe Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 130 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 130 Gln Thr Tyr Asp Lys Gly Phe Thr Gly Ser
Ser Val 1 5 10 <210> SEQ ID NO 131 <211> LENGTH: 12
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 131 Gln
Ser Tyr Asp Arg Asp Phe Thr Gly Thr Arg Val 1 5 10 <210> SEQ
ID NO 132 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 132 Gln Ser Tyr Asp
Arg Gly Phe Tyr Gly Ser Met Val 1 5 10 <210> SEQ ID NO 133
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 133 Gln Thr Tyr Asp Lys Gly Phe
Thr Gly Ser Ser Val 1 5 10 <210> SEQ ID NO 134 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 134 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ala
Arg Val 1 5 10 <210> SEQ ID NO 135 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 135 Gln Ser Tyr Glu Arg Gly Phe Thr Gly Ala
Arg Val 1 5 10 <210> SEQ ID NO 136 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 136 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser
Arg Val Phe 1 5 10 <210> SEQ ID NO 137 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 137 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Phe
Lys Val Phe 1 5 10 <210> SEQ ID NO 138 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 138 Gln Ser Tyr Asp Arg Gly Phe Val Ser Ala
Tyr Val Phe 1 5 10 <210> SEQ ID NO 139 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 139 Gln Ser Tyr Asp Arg Gly Leu Thr Val Thr
Lys Val Phe 1 5 10 <210> SEQ ID NO 140 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 140 Gln Ser Tyr Asp Arg Gly Tyr Thr Ala Ser
Arg Val Phe 1 5 10 <210> SEQ ID NO 141 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 141 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser
Lys Val Phe 1 5 10 <210> SEQ ID NO 142 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 142 Gln Ser Tyr Asp Arg Gly Leu Thr Gly Phe
Arg Val Phe 1 5 10 <210> SEQ ID NO 143 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 143 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Tyr
Lys Val Phe 1 5 10 <210> SEQ ID NO 144 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 144 Gln Ser Tyr Asp Arg Gly Leu Thr Gly Tyr
Arg Leu Phe 1 5 10 <210> SEQ ID NO 145 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 145 Gln Ser Tyr Asp Arg Gly Phe Thr Asp Tyr
Lys Val Phe 1 5 10 <210> SEQ ID NO 146 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 146 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Pro
Arg Leu Phe 1 5 10 <210> SEQ ID NO 147 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 147 Gln Ser Tyr Asp Arg Gly Leu Thr Gly Ser
Arg Val Phe 1 5 10 <210> SEQ ID NO 148 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 148 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ala
Arg Val Trp 1 5 10 <210> SEQ ID NO 149 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 149 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Tyr
Arg Val Phe 1 5 10 <210> SEQ ID NO 150 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 150 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Pro
Arg Val Phe 1 5 10 <210> SEQ ID NO 151 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 151 Gln Ser Tyr Asp Arg Gly Met Thr Ser Ser
Arg Val Phe 1 5 10 <210> SEQ ID NO 152 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 152 Gln Ser Tyr Asp Arg Asp Ser Thr Gly Ser
Arg Val Phe 1 5 10 <210> SEQ ID NO 153 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 153 Gln Ser Tyr Asp Ser Ser Leu Arg Gly Ser
Arg Val Phe 1 5 10 <210> SEQ ID NO 154
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 154 His Ser Tyr Asp Ser Asp Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 155
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 155 His Ser Ser Glu Ser Gly Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 156
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 156 His Ser Tyr Asp Asn Arg Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 157
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 157 His Ser Tyr Asp Ser Arg Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 158
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 158 Gln Ser Tyr Asp Ser Glu Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 159
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 159 Gln Ser Tyr Asp Thr Gly Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 160
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 160 His Ser Tyr Asp Ser Gly Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 161
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 161 Gln Ser Tyr Asp Thr Gly Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 162
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 162 His Ser Tyr Asp Thr Lys Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 163
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 163 His Ser Ser Asp Ser Gly Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 164
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 164 Gln Ser Tyr Asp Ser Asp Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 165
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 165 His Ser Tyr Glu Ser Gly Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 166
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 166 Gln Ser Tyr Asp Ala Pro Trp
Ser Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 167
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 167 Gln Ser Tyr Asp Ser Asp Phe
Thr Gly Ser Lys Val Phe 1 5 10 <210> SEQ ID NO 168
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 168 His Thr Asn Asp Ser Gly Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 169
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 169 His Ser Tyr Asp Thr Arg Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 170
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 170 Gln Ser Tyr Asp Met Arg Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 171
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 171 His Ser Ser Asp Ser Asp Ser
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 172
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 172 Gln Ser Tyr Asn Thr Asp Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 173
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 173 Gln Ser Tyr Asp Ser Gly Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 174
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 174 His Ser Tyr Asp Met Gly Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 175
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 175 His Ser Tyr Asp Asn Gly Phe
Thr Gly Ser Arg Val Phe 1 5 10 <210> SEQ ID NO 176
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 176 His Ser His Asp Arg Asp Phe
Thr Gly Ser Arg Val Phe 1 5 10
<210> SEQ ID NO 177 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 177
Gln Ser Tyr Asp Ser Ser Leu Arg Gly Ser Arg Val 1 5 10 <210>
SEQ ID NO 178 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 178 Gln
Ser Tyr Asp Arg Gly Ile His Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 179 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 179 Gln
Ser Tyr Asp Ser Gly Phe Pro Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 180 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 180 Gln
Ser Tyr Asp Ile Gly Ser Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 181 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 181 Gln
Ser Tyr Asp Ser Gly Leu Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 182 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 182 Gln
Ser Tyr Asp Ile Gly Met Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 183 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 183 Gln
Ser Tyr Asp Ile Gly Leu Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 184 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 184 Gln
Ser Tyr Asp Ser Gly Val Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 185 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 185 Gln
Ser Tyr Asp Arg Gly Leu Thr Ala Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 186 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 186 Gln
Ser Tyr Asp Thr Gly Leu Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 187 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 187 Gln
Ser Tyr Asp Thr Ala Leu Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 188 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 188 Gln
Ser Tyr Asp Ile Arg Phe Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 189 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 189 Gln
Ser Tyr Asp Ile Arg Ser Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 190 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 190 Gln
Ser Tyr Asp Asn Arg Leu Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 191 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 191 Gln
Ser Tyr Glu Thr Ser Phe Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 192 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 192 Gln
Ser Tyr Asp Ser Ser Ser Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 193 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 193 Gln
Ser Tyr Asp Ser Gly Phe Thr Ala Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 194 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 194 Gln
Thr Tyr Asp Lys Gly Phe Thr Gly Ser Ser Val Phe 1 5 10 <210>
SEQ ID NO 195 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 195 Gln
Ser Tyr Asp Asn Gly Phe Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 196 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 196 Gln
Ser Tyr Asp Thr Gly Phe Thr Lys Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 197 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 197 Gln
Ser Tyr Asp Ser Asp Val Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 198 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 198 Gln
Ser Tyr Asp Ala Gly Phe Thr Gly Ser Arg Val Phe 1 5 10 <210>
SEQ ID NO 199 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 199 Gln
Ser Tyr Asp Arg Gly Thr His Pro Ser Met Leu
1 5 10 <210> SEQ ID NO 200 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
200 Gln Ser Tyr Asp Arg Gly Thr Thr Pro Arg Pro Met 1 5 10
<210> SEQ ID NO 201 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 201
Gln Ser Tyr Asp Arg Gly Arg Asn Pro Ala Leu Thr 1 5 10 <210>
SEQ ID NO 202 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 202 Gln
Ser Tyr Asp Arg Gly Thr His Pro Trp Leu His 1 5 10 <210> SEQ
ID NO 203 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 203 Gln Ser Tyr Asp
Arg Gly Asn Ser Pro Ala Thr Val 1 5 10 <210> SEQ ID NO 204
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 204 Gln Ser Tyr Asp Arg Gly Thr
Phe Pro Ser Pro Gln 1 5 10 <210> SEQ ID NO 205 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 205 Gln Ser Tyr Asp Arg Gly Leu Asn Pro Ser
Ala Thr 1 5 10 <210> SEQ ID NO 206 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 206 Gln Ser Tyr Asp Arg Gly Lys Ser Asn Lys
Met Leu 1 5 10 <210> SEQ ID NO 207 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 207 Gln Ser Tyr Asp Arg Gly His Thr Ala His
Leu Tyr 1 5 10 <210> SEQ ID NO 208 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 208 Gln Ser Tyr Asp Arg Gly Gln Thr Pro Ser
Ile Thr 1 5 10 <210> SEQ ID NO 209 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 209 Gln Ser Tyr Asp Arg Gly Tyr Pro Arg Asn
Ile Leu 1 5 10 <210> SEQ ID NO 210 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 210 Gln Ser Tyr Asp Arg Gly Ile Thr Pro Gly
Leu Ala 1 5 10 <210> SEQ ID NO 211 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 211 Gln Ser Tyr Asp Arg Gly Gln Pro His Ala
Val Leu 1 5 10 <210> SEQ ID NO 212 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 212 Gln Ser Tyr Asp Arg Gly Asn Ser Pro Ile
Pro Thr 1 5 10 <210> SEQ ID NO 213 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 213 Gln Ser Tyr Asp Arg Gly Thr Pro Asn Asn
Ser Phe 1 5 10 <210> SEQ ID NO 214 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 214 Gln Ser Tyr Asp Ser Gly Val Asp Pro Gly
Pro Tyr 1 5 10 <210> SEQ ID NO 215 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 215 Gln Ser Tyr Asp Arg Gly Arg Pro Arg His
Ala Leu 1 5 10 <210> SEQ ID NO 216 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 216 Gln Ser Tyr Asp Arg Gly Pro Tyr His Pro
Ile Arg 1 5 10 <210> SEQ ID NO 217 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 217 Gln Ser Tyr Asp Arg Gly Pro His Thr Gln
Pro Thr 1 5 10 <210> SEQ ID NO 218 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 218 Gln Ser Tyr Asp Arg Gly His Asn Asn Phe
Ser Pro 1 5 10 <210> SEQ ID NO 219 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 219 Gln Ser Tyr Asp Arg Gly Pro Thr His Leu
Pro His 1 5 10 <210> SEQ ID NO 220 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 220 Gln Ser Tyr Asp Arg Gly Thr Pro Ser Tyr
Pro Thr 1 5 10 <210> SEQ ID NO 221 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 221 Gln Ser Tyr Asp Ser Gly Thr Ser Asn Leu
Leu Pro 1 5 10 <210> SEQ ID NO 222 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 222
Gln Ser Tyr Asp Arg Gly Asp Ser Asn His Asp Leu 1 5 10 <210>
SEQ ID NO 223 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 223 Gln
Ser Tyr Asp Arg Gly Leu Pro Arg Leu Thr His 1 5 10 <210> SEQ
ID NO 224 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 224 Gln Ser Tyr Asp
Arg Gly Ile Pro Thr Ser Tyr Leu 1 5 10 <210> SEQ ID NO 225
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 225 Gln Ser Tyr Asp Arg Gly Leu
Arg Val Gln Ala Pro 1 5 10 <210> SEQ ID NO 226 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 226 Gln Ser Tyr Asp Arg Gly Leu Ser Asp Ser
Pro Leu 1 5 10 <210> SEQ ID NO 227 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 227 Gln Ser Tyr Asp Ser Gly Ser Leu Arg Arg
Ile Leu 1 5 10 <210> SEQ ID NO 228 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 228 Gln Ser Tyr Asp Arg Gly Pro Ala Arg Thr
Ser Pro 1 5 10 <210> SEQ ID NO 229 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 229 Gln Ser Tyr Asp Arg Gly Arg Ala Ala His
Pro Gln 1 5 10 <210> SEQ ID NO 230 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 230 Gln Ser Tyr Asp Arg Gly Thr Gln Pro Ala
Asx Ile 1 5 10 <210> SEQ ID NO 231 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 231 Gln Ser Tyr Asp Arg Gly Thr His Pro Thr
Met Ile 1 5 10 <210> SEQ ID NO 232 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 232 Gln Ser Tyr Asp Arg Gly Arg Ile Pro Ala
Asx Thr 1 5 10 <210> SEQ ID NO 233 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 233 Gln Ser Tyr Asp Arg Gly Thr His Pro Val
Pro Ala 1 5 10 <210> SEQ ID NO 234 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 234 Gln Ser Tyr Asp Arg Gly Ser Asx Pro Ile
Pro Ala 1 5 10 <210> SEQ ID NO 235 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 235 Gln Ser Tyr Asp Arg Gly Thr His Pro Val
Pro Ala 1 5 10 <210> SEQ ID NO 236 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 236 Gln Ser Tyr Asp Arg Gly Thr His Pro Thr
Met Tyr 1 5 10 <210> SEQ ID NO 237 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 237 Gln Ser Tyr Asp Arg Gly His His Tyr Thr
Thr Phe 1 5 10 <210> SEQ ID NO 238 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 238 Gln Ser Tyr Asp Arg Gly Ser His Pro Ala
Ala Glu 1 5 10 <210> SEQ ID NO 239 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 239 Gln Ser Tyr Asp Arg Gly Thr Ile Pro Ser
Ile Glu 1 5 10 <210> SEQ ID NO 240 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 240 Gln Ser Tyr Asp Arg Gly Ser Ser Pro Ala
Ile Met 1 5 10 <210> SEQ ID NO 241 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 241 Gln Ser Tyr Asp Arg Gly Ile Trp Pro Asn
Leu Asn 1 5 10 <210> SEQ ID NO 242 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 242 Gln Ser Tyr Asp Arg Gly Thr His Pro Asn
Leu Asn 1 5 10 <210> SEQ ID NO 243 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 243 Gln Ser Tyr Asp Arg Gly Thr His Pro Ser
Ile Ser 1 5 10 <210> SEQ ID NO 244 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 244 Gln Ser Tyr Asp Arg Gly Ser Ala Pro Met
Ile Asn 1 5 10 <210> SEQ ID NO 245 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 245 Gln Ser Tyr Asp Arg Gly His His Pro Ala
Met Ser 1 5 10 <210> SEQ ID NO 246 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 246 Gln Ser Tyr Asp Arg Gly Thr His Pro Ser
Ile Thr 1 5 10 <210> SEQ ID NO 247 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 247 Gln Ser Tyr Asp Arg Gly Thr Asp Pro Ala
Ile Val 1 5 10 <210> SEQ ID NO 248 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 248 Gln Ser Tyr Asp Arg Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 249 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 249 Gln Ser Tyr Asp Arg Gly Ser His Pro Ala
Leu Thr 1 5 10 <210> SEQ ID NO 250 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 250 Gln Ser Tyr Asp Arg Gly Thr Thr Pro Ala
Pro Glu 1 5 10 <210> SEQ ID NO 251 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 251 Gln Ser Tyr Asp Arg Gly Ser His Pro Thr
Leu Ile 1 5 10 <210> SEQ ID NO 252 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 252 Gln Ser Tyr Asp Arg Gly Thr His Pro Ser
Met Leu 1 5 10 <210> SEQ ID NO 253 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 253 Gln Ser Tyr Asp Arg Gly Thr Thr Pro Arg
Pro Met 1 5 10 <210> SEQ ID NO 254 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 254 Gln Ser Tyr Asp Arg Gly Arg Leu Pro Ala
Gln Thr 1 5 10 <210> SEQ ID NO 255 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 255 Gln Ser Tyr Asp Arg Gly Thr His Pro Leu
Thr Ile 1 5 10 <210> SEQ ID NO 256 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 256 Gln Ser Tyr Asp Arg Gly Gln Thr Pro Ser
Ile Thr 1 5 10 <210> SEQ ID NO 257 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 257 Gln Ser Tyr Asp Arg Gly Thr His Phe Gln
Met Tyr 1 5 10 <210> SEQ ID NO 258 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 258 Gln Ser Tyr Asp Arg Gly Arg Asn Pro Ala
Leu Thr 1 5 10 <210> SEQ ID NO 259 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 259 Gln Ser Tyr Asp Arg Gly Thr His Pro Leu
Thr Met 1 5 10 <210> SEQ ID NO 260 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 260 Gln Ser Tyr Asp Arg Gly Thr His Pro Leu
Thr Met 1 5 10 <210> SEQ ID NO 261 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 261 Gln Ser Tyr Asp Ser Gly Tyr Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 262 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 262 Gln Ser Tyr Asp Ser Gly Phe Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 263 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 263 Gln Ser Tyr Asp Ser Arg Phe Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 264 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 264 Gln Ser Tyr Pro Asp Gly Thr Pro Ala Ser
Arg Val 1 5 10 <210> SEQ ID NO 265 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 265 Gln Ser Tyr Ser Thr His Met Pro Ile Ser
Arg Val 1 5 10 <210> SEQ ID NO 266 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 266 Gln Ser Tyr Asp Ser Gly Ser Thr Gly Ser
Arg Val 1 5 10 <210> SEQ ID NO 267 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 267 Gln Ser Tyr Pro Asn Ser Tyr Pro Ile Ser
Arg Val 1 5 10 <210> SEQ ID NO 268 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 268 Gln Ser Tyr Ile Arg Ala Pro Gln Gln Val 1
5 10 <210> SEQ ID NO 269 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
269 Gln Ser Tyr Leu Lys Ser Arg Ala Phe Ser Arg Val 1 5 10
<210> SEQ ID NO 270 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 270
Gln Ser Tyr Asp Ser Arg Phe Thr Gly Ser Arg Val 1 5 10 <210>
SEQ ID NO 271 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 271 Gln
Ser Tyr Asp Arg Gly Phe Thr Gly Ser Met Val 1 5 10 <210> SEQ
ID NO 272 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 272 Gln Ser Tyr Asp
Arg Gly Phe Thr Gly Ser Met Val 1 5 10 <210> SEQ ID NO 273
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 273 Gln Ser Tyr Asp Arg Gly Phe
Thr Gly Phe Asp Gly 1 5 10 <210> SEQ ID NO 274 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 274 Gln Ser Tyr Asp Arg Gly Thr Ala Pro Ala
Leu Ser 1 5 10 <210> SEQ ID NO 275 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 275 Gln Ser Tyr Asp Arg Gly Ser Tyr Pro Ala
Leu Arg 1 5 10 <210> SEQ ID NO 276 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 276 Gln Ser Tyr Asp Arg Gly Asn Trp Pro Asn
Ser Asn 1 5 10 <210> SEQ ID NO 277 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 277 Gln Ser Tyr Asp Arg Gly Thr Ala Pro Ser
Leu Leu 1 5 10 <210> SEQ ID NO 278 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 278 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser
Met Val 1 5 10 <210> SEQ ID NO 279 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 279 Gln Ser Tyr Asp Arg Gly Thr Thr Pro Arg
Ile Arg 1 5 10 <210> SEQ ID NO 280 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 280 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser
Met Val 1 5 10 <210> SEQ ID NO 281 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 281 Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser
Met Val 1 5 10 <210> SEQ ID NO 282 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 282 Gln Ser Tyr Asp Arg Gly Met Ile Pro Ala
Leu Thr 1 5 10 <210> SEQ ID NO 283 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 283 Gln Ser Tyr Asp Arg Asn Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 284 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 284 Gln Ser Tyr Asp Arg Phe Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 285 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 285 Gln Ser Tyr Asp Arg Tyr Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 286 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 286 Gln Ser Tyr Asp Arg Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 287 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 287 Gln Ser Tyr Asp Arg Tyr Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 288 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 288 Phe Thr Phe Glu Ser Tyr Gly Met His 1 5
<210> SEQ ID NO 289 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 289
Phe Thr Phe Ser Ser Tyr Gly Met His 1 5 <210> SEQ ID NO 290
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 290 Phe Thr Phe Tyr Ser Tyr Gly
Met His 1 5
<210> SEQ ID NO 291 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 291
Phe Thr Phe His Ser Tyr Gly Met His 1 5 <210> SEQ ID NO 292
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 292 Phe Thr Phe Lys Ser Tyr Gly
Met His 1 5 <210> SEQ ID NO 293 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 293 Phe Thr Phe Arg Ser Tyr Gly Met His 1 5
<210> SEQ ID NO 294 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 294
Phe Thr Phe Asn Ser Tyr Gly Met His 1 5 <210> SEQ ID NO 295
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 295 Phe Thr Phe Thr Ser Tyr Gly
Met His 1 5 <210> SEQ ID NO 296 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 296 Phe Thr Phe Gly Ser Tyr Gly Met His 1 5
<210> SEQ ID NO 297 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 297
Phe Thr Phe Val Ser Tyr Gly Met His 1 5 <210> SEQ ID NO 298
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 298 Phe Thr Phe Ile Ser Tyr Gly
Met His 1 5 <210> SEQ ID NO 299 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 299 Phe Thr Phe Trp Ser Tyr Gly Met His 1 5
<210> SEQ ID NO 300 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 300
Phe Thr Phe Ser Glu Tyr Gly Met His 1 5 <210> SEQ ID NO 301
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 301 Phe Thr Phe Ser Cys Tyr Gly
Met His 1 5 <210> SEQ ID NO 302 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 302 Phe Thr Phe Ser Ser Tyr Gly Met His 1 5
<210> SEQ ID NO 303 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 303
Phe Thr Phe Ser Tyr Tyr Gly Met His 1 5 <210> SEQ ID NO 304
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 304 Phe Thr Phe Ser His Tyr Gly
Met His 1 5 <210> SEQ ID NO 305 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 305 Phe Thr Phe Ser Arg Tyr Gly Met His 1 5
<210> SEQ ID NO 306 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 306
Phe Thr Phe Ser Asn Tyr Gly Met His 1 5 <210> SEQ ID NO 307
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 307 Phe Thr Phe Ser Gln Tyr Gly
Met His 1 5 <210> SEQ ID NO 308 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 308 Phe Thr Phe Ser Thr Tyr Gly Met His 1 5
<210> SEQ ID NO 309 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 309
Phe Thr Phe Ser Ala Tyr Gly Met His 1 5 <210> SEQ ID NO 310
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 310 Phe Thr Phe Ser Ile Tyr Gly
Met His 1 5 <210> SEQ ID NO 311 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 311 Phe Thr Phe Ser Ser Glu Gly Met His 1 5
<210> SEQ ID NO 312 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 312
Phe Thr Phe Ser Ser Cys Gly Met His 1 5 <210> SEQ ID NO 313
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 313 Phe Thr Phe Ser Ser Ser Gly
Met His 1 5
<210> SEQ ID NO 314 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 314
Phe Thr Phe Ser Ser Tyr Gly Met His 1 5 <210> SEQ ID NO 315
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 315 Phe Thr Phe Ser Ser His Gly
Met His 1 5 <210> SEQ ID NO 316 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 316 Phe Thr Phe Ser Ser Arg Gly Met His 1 5
<210> SEQ ID NO 317 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 317
Phe Thr Phe Ser Ser Asn Gly Met His 1 5 <210> SEQ ID NO 318
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 318 Phe Thr Phe Ser Ser Thr Gly
Met His 1 5 <210> SEQ ID NO 319 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 319 Phe Thr Phe Ser Ser Ala Gly Met His 1 5
<210> SEQ ID NO 320 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 320
Phe Thr Phe Ser Ser Val Gly Met His 1 5 <210> SEQ ID NO 321
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 321 Phe Thr Phe Ser Ser Leu Gly
Met His 1 5 <210> SEQ ID NO 322 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 322 Phe Thr Phe Ser Ser Ile Gly Met His 1 5
<210> SEQ ID NO 323 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 323
Phe Thr Phe Ser Ser Tyr Asp Met His 1 5 <210> SEQ ID NO 324
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 324 Phe Thr Phe Ser Ser Tyr Glu
Met His 1 5 <210> SEQ ID NO 325 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 325 Phe Thr Phe Ser Ser Tyr Cys Met His 1 5
<210> SEQ ID NO 326 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 326
Phe Thr Phe Ser Ser Tyr Ser Met His 1 5 <210> SEQ ID NO 327
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 327 Phe Thr Phe Ser Ser Tyr Tyr
Met His 1 5 <210> SEQ ID NO 328 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 328 Phe Thr Phe Ser Ser Tyr Asn Met His 1 5
<210> SEQ ID NO 329 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 329
Phe Thr Phe Ser Ser Tyr Gly Met His 1 5 <210> SEQ ID NO 330
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 330 Phe Thr Phe Ser Ser Tyr Ala
Met His 1 5 <210> SEQ ID NO 331 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 331 Phe Thr Phe Ser Ser Tyr Val Met His 1 5
<210> SEQ ID NO 332 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 332
Phe Thr Phe Ser Ser Tyr Met Met His 1 5 <210> SEQ ID NO 333
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 333 Phe Thr Phe Ser Ser Tyr Ile
Met His 1 5 <210> SEQ ID NO 334 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 334 Phe Thr Phe Ser Ser Tyr Pro Met His 1 5
<210> SEQ ID NO 335 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 335
Glu Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 336 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 336 Cys Ile Arg Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 337
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 337 Tyr Ile Arg Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 338 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 338 His Ile Arg Tyr
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 339 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 339
Lys Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 340 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 340 Asn Ile Arg Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 341
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 341 Gln Ile Arg Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 342 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 342 Thr Ile Arg Tyr
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 343 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 343
Leu Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 344 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 344 Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 345
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 345 Phe Ile Glu Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 346 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 346 Phe Ile Ser Tyr
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 347 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 347
Phe Ile Tyr Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 348 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 348 Phe Ile His Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 349
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 349 Phe Ile Lys Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 350 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 350 Phe Ile Arg Tyr
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 351 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 351
Phe Ile Gln Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 352 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 352 Phe Ile Thr Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 353
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 353 Phe Ile Gly Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 354 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 354 Phe Ile Ala Tyr
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 355 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 355
Phe Ile Val Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15
Gly <210> SEQ ID NO 356 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
356 Phe Ile Leu Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15 Gly <210> SEQ ID NO 357 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 357 Phe Ile Trp Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 358
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 358 Phe Ile Arg Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 359 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 359 Phe Ile Arg Tyr
Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 360 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 360
Phe Ile Arg Tyr Ser Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 361 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 361 Phe Ile Arg Tyr Tyr Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 362
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 362 Phe Ile Arg Tyr Lys Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 363 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 363 Phe Ile Arg Tyr
Arg Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 364 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 364
Phe Ile Arg Tyr Asn Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 365 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 365 Phe Ile Arg Tyr Gln Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 366
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 366 Phe Ile Arg Tyr Thr Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 367 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 367 Phe Ile Arg Tyr
Ala Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 368 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 368
Phe Ile Arg Tyr Val Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 369 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 369 Phe Ile Arg Tyr Leu Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 370
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 370 Phe Ile Arg Tyr Ile Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 371 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 371 Phe Ile Arg Tyr
Phe Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 372 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 372
Phe Ile Arg Tyr Asp Asp Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 373 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 373 Phe Ile Arg Tyr Asp Glu Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 374
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 374 Phe Ile Arg Tyr Asp Ser Ser
Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 375 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 375
Phe Ile Arg Tyr Asp Tyr Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 376 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 376 Phe Ile Arg Tyr Asp Lys Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 377
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 377 Phe Ile Arg Tyr Asp Arg Ser
Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 378 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 378 Phe Ile Arg Tyr
Asp Asn Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 379 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 379
Phe Ile Arg Tyr Asp Gln Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 380 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 380 Phe Ile Arg Tyr Asp Thr Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 381
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 381 Phe Ile Arg Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 382 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 382 Phe Ile Arg Tyr
Asp Val Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 383 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 383
Phe Ile Arg Tyr Asp Phe Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 384 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 384 Phe Ile Arg Tyr Asp Gly Ser Ser Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 385
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 385 Phe Ile Arg Tyr Asp Gly Ser
Tyr Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 386 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 386 Phe Ile Arg Tyr
Asp Gly Ser His Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 387 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 387
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 388 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 388 Phe Ile Arg Tyr Asp Gly Ser Thr Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 389
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 389 Phe Ile Arg Tyr Asp Gly Ser
Gly Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 390 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 390 Phe Ile Arg Tyr
Asp Gly Ser Met Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 391 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 391
Phe Ile Arg Tyr Asp Gly Ser Leu Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 392 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 392 Phe Ile Arg Tyr Asp Gly Ser Ile Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 393
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 393 Phe Ile Arg Tyr Asp Gly Ser
Pro Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 394 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens
<400> SEQUENCE: 394 Phe Ile Arg Tyr Asp Gly Ser Phe Lys Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 395
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 395 Phe Ile Arg Tyr Asp Gly Ser
Asn Lys Glu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 396 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 396 Phe Ile Arg Tyr
Asp Gly Ser Asn Lys Ser Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 397 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 397
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 398 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 398 Phe Ile Arg Tyr Asp Gly Ser Asn Lys Asn
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 399
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 399 Phe Ile Arg Tyr Asp Gly Ser
Asn Lys Val Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 400 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 400 Phe Ile Arg Tyr
Asp Gly Ser Asn Lys Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
<210> SEQ ID NO 401 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 401
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Ile Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly <210> SEQ ID NO 402 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 402 Phe Ile Arg Tyr Asp Gly Ser Asn Lys Pro
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ ID NO 403
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 403 Phe Ile Arg Tyr Asp Gly Ser
Asn Lys Phe Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> SEQ
ID NO 404 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 404 Glu Gly Ser His
Asp Asn 1 5 <210> SEQ ID NO 405 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 405 Ser Gly Ser His Asp Asn 1 5 <210>
SEQ ID NO 406 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 406 His
Gly Ser His Asp Asn 1 5 <210> SEQ ID NO 407 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 407 Lys Gly Ser His Asp Asn 1 5 <210>
SEQ ID NO 408 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 408 Gln
Gly Ser His Asp Asn 1 5 <210> SEQ ID NO 409 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 409 Thr Gly Ser His Asp Asn 1 5 <210>
SEQ ID NO 410 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 410 Ala
Gly Ser His Asp Asn 1 5 <210> SEQ ID NO 411 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 411 Leu Gly Ser His Asp Asn 1 5 <210>
SEQ ID NO 412 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 412 Pro
Gly Ser His Asp Asn 1 5 <210> SEQ ID NO 413 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 413 Phe Gly Ser His Asp Asn 1 5 <210>
SEQ ID NO 414 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 414 His
Asp Ser His Asp Asn 1 5 <210> SEQ ID NO 415 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens
<400> SEQUENCE: 415 His Cys Ser His Asp Asn 1 5 <210>
SEQ ID NO 416 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 416 His
His Ser His Asp Asn 1 5 <210> SEQ ID NO 417 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 417 His Arg Ser His Asp Asn 1 5 <210>
SEQ ID NO 418 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 418 His
Thr Ser His Asp Asn 1 5 <210> SEQ ID NO 419 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 419 His Gly Ser His Asp Asn 1 5 <210>
SEQ ID NO 420 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 420 His
Val Ser His Asp Asn 1 5 <210> SEQ ID NO 421 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 421 His Met Ser His Asp Asn 1 5 <210>
SEQ ID NO 422 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 422 His
Leu Ser His Asp Asn 1 5 <210> SEQ ID NO 423 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 423 His Ile Ser His Asp Asn 1 5 <210>
SEQ ID NO 424 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 424 His
Pro Ser His Asp Asn 1 5 <210> SEQ ID NO 425 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 425 His Trp Ser His Asp Asn 1 5 <210>
SEQ ID NO 426 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 426 His
Gly Asp His Asp Asn 1 5 <210> SEQ ID NO 427 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 427 His Gly Ser His Asp Asn 1 5 <210>
SEQ ID NO 428 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 428 His
Gly Tyr His Asp Asn 1 5 <210> SEQ ID NO 429 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 429 His Gly His His Asp Asn 1 5 <210>
SEQ ID NO 430 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 430 His
Gly Arg His Asp Asn 1 5 <210> SEQ ID NO 431 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 431 His Gly Asn His Asp Asn 1 5 <210>
SEQ ID NO 432 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 432 His
Gly Thr His Asp Asn 1 5 <210> SEQ ID NO 433 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 433 His Gly Gly His Asp Asn 1 5 <210>
SEQ ID NO 434 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 434 His
Gly Ala His Asp Asn 1 5 <210> SEQ ID NO 435 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 435 His Gly Ile His Asp Asn 1 5 <210>
SEQ ID NO 436 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 436 His
Gly Pro His Asp Asn 1 5 <210> SEQ ID NO 437 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 437 His Gly Trp His Asp Asn 1 5 <210>
SEQ ID NO 438 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 438 His Gly Phe His Asp Asn 1 5 <210>
SEQ ID NO 439 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 439 His
Gly Ser His Asp Asn 1 5 <210> SEQ ID NO 440 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 440 His Gly Ser Arg Asp Asn 1 5 <210>
SEQ ID NO 441 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 441 His
Gly Ser Thr Asp Asn 1 5 <210> SEQ ID NO 442 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 442 His Gly Ser Ala Asp Asn 1 5 <210>
SEQ ID NO 443 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 443 His
Gly Ser Val Asp Asn 1 5 <210> SEQ ID NO 444 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 444 His Gly Ser Leu Asp Asn 1 5 <210>
SEQ ID NO 445 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 445 His
Gly Ser Ile Asp Asn 1 5 <210> SEQ ID NO 446 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 446 His Gly Ser Phe Asp Asn 1 5 <210>
SEQ ID NO 447 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 447 His
Gly Ser His Asp Asn 1 5 <210> SEQ ID NO 448 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 448 His Gly Ser His Ser Asn 1 5 <210>
SEQ ID NO 449 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 449 His
Gly Ser His Tyr Asn 1 5 <210> SEQ ID NO 450 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 450 His Gly Ser His His Asn 1 5 <210>
SEQ ID NO 451 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 451 His
Gly Ser His Arg Asn 1 5 <210> SEQ ID NO 452 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 452 His Gly Ser His Asn Asn 1 5 <210>
SEQ ID NO 453 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 453 His
Gly Ser His Gly Asn 1 5 <210> SEQ ID NO 454 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 454 His Gly Ser His Ala Asn 1 5 <210>
SEQ ID NO 455 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 455 His
Gly Ser His Val Asn 1 5 <210> SEQ ID NO 456 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 456 His Gly Ser His Ile Asn 1 5 <210>
SEQ ID NO 457 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 457 His
Gly Ser His Asp Ser 1 5 <210> SEQ ID NO 458 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 458 His Gly Ser His Asp His 1 5 <210>
SEQ ID NO 459 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 459 His
Gly Ser His Asp Lys 1 5 <210> SEQ ID NO 460 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 460 His Gly Ser His Asp Arg 1 5
<210> SEQ ID NO 461 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 461
His Gly Ser His Asp Asn 1 5 <210> SEQ ID NO 462 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 462 His Gly Ser His Asp Thr 1 5 <210>
SEQ ID NO 463 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 463 His
Gly Ser His Asp Gly 1 5 <210> SEQ ID NO 464 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 464 His Gly Ser His Asp Ala 1 5 <210>
SEQ ID NO 465 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 465 His
Gly Ser His Asp Leu 1 5 <210> SEQ ID NO 466 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 466 His Gly Ser His Asp Ile 1 5 <210>
SEQ ID NO 467 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 467 His
Gly Ser His Asp Pro 1 5 <210> SEQ ID NO 468 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 468 His Gly Ser His Asp Trp 1 5 <210>
SEQ ID NO 469 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 469 His
Gly Ser His Asp Phe 1 5 <210> SEQ ID NO 470 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 470 Ser Gly Gly Arg Ser Asn Ile Gly Asp Asn
Thr Val Lys 1 5 10 <210> SEQ ID NO 471 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 471 Ser Gly Gly Arg Ser Asn Ile Gly Cys Asn
Thr Val Lys 1 5 10 <210> SEQ ID NO 472 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 472 Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn
Thr Val Lys 1 5 10 <210> SEQ ID NO 473 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 473 Ser Gly Gly Arg Ser Asn Ile Gly Tyr Asn
Thr Val Lys 1 5 10 <210> SEQ ID NO 474 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 474 Ser Gly Gly Arg Ser Asn Ile Gly Lys Asn
Thr Val Lys 1 5 10 <210> SEQ ID NO 475 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 475 Ser Gly Gly Arg Ser Asn Ile Gly Arg Asn
Thr Val Lys 1 5 10 <210> SEQ ID NO 476 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 476 Ser Gly Gly Arg Ser Asn Ile Gly Asn Asn
Thr Val Lys 1 5 10 <210> SEQ ID NO 477 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 477 Ser Gly Gly Arg Ser Asn Ile Gly Thr Asn
Thr Val Lys 1 5 10 <210> SEQ ID NO 478 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 478 Ser Gly Gly Arg Ser Asn Ile Gly Pro Asn
Thr Val Lys 1 5 10 <210> SEQ ID NO 479 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 479 Ser Gly Gly Arg Ser Asn Ile Gly Ser Asp
Thr Val Lys 1 5 10 <210> SEQ ID NO 480 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 480 Ser Gly Gly Arg Ser Asn Ile Gly Ser Glu
Thr Val Lys 1 5 10 <210> SEQ ID NO 481 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 481 Ser Gly Gly Arg Ser Asn Ile Gly Ser Ser
Thr Val Lys 1 5 10 <210> SEQ ID NO 482 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 482 Ser Gly Gly Arg Ser Asn Ile Gly Ser Tyr
Thr Val Lys 1 5 10 <210> SEQ ID NO 483 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 483 Ser Gly Gly Arg Ser Asn Ile Gly Ser His
Thr Val Lys 1 5 10
<210> SEQ ID NO 484 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 484
Ser Gly Gly Arg Ser Asn Ile Gly Ser Lys Thr Val Lys 1 5 10
<210> SEQ ID NO 485 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 485
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Thr Val Lys 1 5 10
<210> SEQ ID NO 486 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 486
Ser Gly Gly Arg Ser Asn Ile Gly Ser Gln Thr Val Lys 1 5 10
<210> SEQ ID NO 487 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 487
Ser Gly Gly Arg Ser Asn Ile Gly Ser Thr Thr Val Lys 1 5 10
<210> SEQ ID NO 488 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 488
Ser Gly Gly Arg Ser Asn Ile Gly Ser Gly Thr Val Lys 1 5 10
<210> SEQ ID NO 489 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 489
Ser Gly Gly Arg Ser Asn Ile Gly Ser Met Thr Val Lys 1 5 10
<210> SEQ ID NO 490 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 490
Ser Gly Gly Arg Ser Asn Ile Gly Ser Ile Thr Val Lys 1 5 10
<210> SEQ ID NO 491 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 491
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Asp Val Lys 1 5 10
<210> SEQ ID NO 492 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 492
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Cys Val Lys 1 5 10
<210> SEQ ID NO 493 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 493
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Ser Val Lys 1 5 10
<210> SEQ ID NO 494 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 494
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Tyr Val Lys 1 5 10
<210> SEQ ID NO 495 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 495
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn His Val Lys 1 5 10
<210> SEQ ID NO 496 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 496
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Lys Val Lys 1 5 10
<210> SEQ ID NO 497 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 497
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Arg Val Lys 1 5 10
<210> SEQ ID NO 498 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 498
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Asn Val Lys 1 5 10
<210> SEQ ID NO 499 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 499
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Gln Val Lys 1 5 10
<210> SEQ ID NO 500 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 500
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Thr Val Lys 1 5 10
<210> SEQ ID NO 501 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 501
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Ala Val Lys 1 5 10
<210> SEQ ID NO 502 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 502
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Val Val Lys 1 5 10
<210> SEQ ID NO 503 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 503
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Leu Val Lys 1 5 10
<210> SEQ ID NO 504 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 504
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Ile Val Lys 1 5 10
<210> SEQ ID NO 505 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 505
Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Pro Val Lys 1 5 10
<210> SEQ ID NO 506 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
506
Asp Asn Asp Gln Arg Pro Ser 1 5 <210> SEQ ID NO 507
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 507 Glu Asn Asp Gln Arg Pro Ser
1 5 <210> SEQ ID NO 508 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
508 Cys Asn Asp Gln Arg Pro Ser 1 5 <210> SEQ ID NO 509
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 509 Ser Asn Asp Gln Arg Pro Ser
1 5 <210> SEQ ID NO 510 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
510 Tyr Asn Asp Gln Arg Pro Ser 1 5 <210> SEQ ID NO 511
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 511 His Asn Asp Gln Arg Pro Ser
1 5 <210> SEQ ID NO 512 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
512 Lys Asn Asp Gln Arg Pro Ser 1 5 <210> SEQ ID NO 513
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 513 Arg Asn Asp Gln Arg Pro Ser
1 5 <210> SEQ ID NO 514 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
514 Asn Asn Asp Gln Arg Pro Ser 1 5 <210> SEQ ID NO 515
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 515 Gln Asn Asp Gln Arg Pro Ser
1 5 <210> SEQ ID NO 516 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
516 Thr Asn Asp Gln Arg Pro Ser 1 5 <210> SEQ ID NO 517
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 517 Gly Asn Asp Gln Arg Pro Ser
1 5 <210> SEQ ID NO 518 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
518 Ala Asn Asp Gln Arg Pro Ser 1 5 <210> SEQ ID NO 519
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 519 Val Asn Asp Gln Arg Pro Ser
1 5 <210> SEQ ID NO 520 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
520 Met Asn Asp Gln Arg Pro Ser 1 5 <210> SEQ ID NO 521
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 521 Leu Asn Asp Gln Arg Pro Ser
1 5 <210> SEQ ID NO 522 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
522 Ile Asn Asp Gln Arg Pro Ser 1 5 <210> SEQ ID NO 523
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 523 Pro Asn Asp Gln Arg Pro Ser
1 5 <210> SEQ ID NO 524 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
524 Trp Asn Asp Gln Arg Pro Ser 1 5 <210> SEQ ID NO 525
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 525 Phe Asn Asp Gln Arg Pro Ser
1 5 <210> SEQ ID NO 526 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
526 Gly Asn Asp Ser Arg Pro Ser 1 5 <210> SEQ ID NO 527
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 527 Gly Asn Asp Tyr Arg Pro Ser
1 5 <210> SEQ ID NO 528 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
528 Gly Asn Asp Arg Arg Pro Ser 1 5 <210> SEQ ID NO 529
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens
<400> SEQUENCE: 529 Gly Asn Asp Gln Arg Pro Ser 1 5
<210> SEQ ID NO 530 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 530
Gly Asn Asp Thr Arg Pro Ser 1 5 <210> SEQ ID NO 531
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 531 Gly Asn Asp Ala Arg Pro Ser
1 5 <210> SEQ ID NO 532 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
532 Gly Asn Asp Ile Arg Pro Ser 1 5 <210> SEQ ID NO 533
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 533 Gly Asn Asp Pro Arg Pro Ser
1 5 <210> SEQ ID NO 534 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
534 Gln Ser Tyr Asp Arg Gly Thr His Pro Ala Leu Leu 1 5 10
<210> SEQ ID NO 535 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 535
Gln Ser Tyr Cys Arg Gly Thr His Pro Ala Leu Leu 1 5 10 <210>
SEQ ID NO 536 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 536 Gln
Ser Tyr Ser Arg Gly Thr His Pro Ala Leu Leu 1 5 10 <210> SEQ
ID NO 537 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 537 Gln Ser Tyr Tyr
Arg Gly Thr His Pro Ala Leu Leu 1 5 10 <210> SEQ ID NO 538
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 538 Gln Ser Tyr Asn Arg Gly Thr
His Pro Ala Leu Leu 1 5 10 <210> SEQ ID NO 539 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 539 Gln Ser Tyr Gln Arg Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 540 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 540 Gln Ser Tyr Thr Arg Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 541 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 541 Gln Ser Tyr Gly Arg Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 542 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 542 Gln Ser Tyr Ala Arg Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 543 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 543 Gln Ser Tyr Leu Arg Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 544 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 544 Gln Ser Tyr Ile Arg Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 545 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 545 Gln Ser Tyr Trp Arg Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 546 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 546 Gln Ser Tyr Phe Arg Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 547 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 547 Gln Ser Tyr Asp Asp Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 548 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 548 Gln Ser Tyr Asp Cys Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 549 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 549 Gln Ser Tyr Asp Ser Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 550 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 550 Gln Ser Tyr Asp Tyr Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 551 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 551 Gln Ser Tyr Asp Arg Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 552 <211> LENGTH: 12
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 552 Gln
Ser Tyr Asp Asn Gly Thr His Pro Ala Leu Leu 1 5 10 <210> SEQ
ID NO 553 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 553 Gln Ser Tyr Asp
Gln Gly Thr His Pro Ala Leu Leu 1 5 10 <210> SEQ ID NO 554
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 554 Gln Ser Tyr Asp Thr Gly Thr
His Pro Ala Leu Leu 1 5 10 <210> SEQ ID NO 555 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 555 Gln Ser Tyr Asp Gly Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 556 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 556 Gln Ser Tyr Asp Ala Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 557 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 557 Gln Ser Tyr Asp Val Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 558 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 558 Gln Ser Tyr Asp Met Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 559 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 559 Gln Ser Tyr Asp Leu Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 560 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 560 Gln Ser Tyr Asp Ile Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 561 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 561 Gln Ser Tyr Asp Pro Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 562 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 562 Gln Ser Tyr Asp Trp Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 563 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 563 Gln Ser Tyr Asp Arg Asp Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 564 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 564 Gln Ser Tyr Asp Arg Cys Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 565 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 565 Gln Ser Tyr Asp Arg Ser Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 566 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 566 Gln Ser Tyr Asp Arg Tyr Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 567 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 567 Gln Ser Tyr Asp Arg His Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 568 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 568 Gln Ser Tyr Asp Arg Arg Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 569 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 569 Gln Ser Tyr Asp Arg Asn Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 570 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 570 Gln Ser Tyr Asp Arg Gln Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 571 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 571 Gln Ser Tyr Asp Arg Thr Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 572 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 572 Gln Ser Tyr Asp Arg Gly Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 573 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 573 Gln Ser Tyr Asp Arg Ala Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 574 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 574 Gln Ser Tyr Asp Arg Val Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 575
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 575 Gln Ser Tyr Asp Arg Leu Thr
His Pro Ala Leu Leu 1 5 10 <210> SEQ ID NO 576 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 576 Gln Ser Tyr Asp Arg Ile Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 577 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 577 Gln Ser Tyr Asp Arg Pro Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 578 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 578 Gln Ser Tyr Asp Arg Trp Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 579 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 579 Gln Ser Tyr Asp Arg Phe Thr His Pro Ala
Leu Leu 1 5 10 <210> SEQ ID NO 580 <211> LENGTH: 48
<212> TYPE: DNA <213> ORGANISM: synthetic construct
<220> FEATURE: <223> OTHER INFORMATION: nucleotides at
positions 16 to 34 can be substituted with any nucleotide such that
the randomized nucleotides represent 12% of the sequence
<400> SEQUENCE: 580 tgtcccttgg ccccagtagt catagctccc
actggtcgta cagtaata 48 <210> SEQ ID NO 581 <211>
LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: synthetic
construct <400> SEQUENCE: 581 gacacctcga tcagcggata
acaatttcac acagg 35 <210> SEQ ID NO 582 <211> LENGTH:
15 <212> TYPE: DNA <213> ORGANISM: synthetic construct
<400> SEQUENCE: 582 tggggccaag ggaca 15 <210> SEQ ID NO
583 <211> LENGTH: 45 <212> TYPE: DNA <213>
ORGANISM: synthetic construct <400> SEQUENCE: 583 attcgtccta
taccgttcta ctttgtcgtc tttccagacg ttagt 45 <210> SEQ ID NO 584
<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:
synthetic construct <400> SEQUENCE: 584 attcgtccta taccgttc
18 <210> SEQ ID NO 585 <211> LENGTH: 66 <212>
TYPE: DNA <213> ORGANISM: synthetic construct <220>
FEATURE: <223> OTHER INFORMATION: nucleotides from position
28 to 42 can be substituted with any nucleotide such that the
randomized nucleotides represent 12% of the sequence <400>
SEQUENCE: 585 ggtcccagtt ccgaagaccc tcgaacccct caggctgctg
tcatatgact ggcagtaata 60 gtcagc 66 <210> SEQ ID NO 586
<211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM:
synthetic construct <400> SEQUENCE: 586 tggggccaag ggaca 15
<210> SEQ ID NO 587 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: synthetic construct <400> SEQUENCE:
587 tgaagagacg gtgaccattg tccc 24 <210> SEQ ID NO 588
<211> LENGTH: 16 <212> TYPE: DNA <213> ORGANISM:
synthetic construct <400> SEQUENCE: 588 gacacctcga tcagcg 16
<210> SEQ ID NO 589 <211> LENGTH: 48 <212> TYPE:
DNA <213> ORGANISM: synthetic construct <400> SEQUENCE:
589 gagtcattct cgacttgcgg ccgcacctag gacggtcagc ttggtccc 48
<210> SEQ ID NO 590 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 590
Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser Met Val 1 5 10 <210>
SEQ ID NO 591 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <220> FEATURE: <223>
OTHER INFORMATION: Xaa is encoded by a randomized codon of sequence
NNS with N being any nucleotide and S being either deoxycytosine or
deoxyguanidine <400> SEQUENCE: 591 Xaa Xaa Xaa Xaa Xaa Xaa
Phe Thr Gly Ser Met Val 1 5 10 <210> SEQ ID NO 592
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:
Xaa is encoded by a randomized codon of sequence NNS with N being
any nucleotide and S being either deoxycytosine or deoxyguanidine
<400> SEQUENCE: 592 Gln Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Ser
Met Val 1 5 10 <210> SEQ ID NO 593 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <223> OTHER INFORMATION: Xaa is encoded
by a randomized codon of sequence NNS with N being any nucleotide
and S being either deoxycytosine or deoxyguanidine <400>
SEQUENCE: 593 Gln Ser Tyr Asp Arg Gly Xaa Xaa Xaa Xaa Xaa X aa 1 5
10 <210> SEQ ID NO 594 <211> LENGTH: 100 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
594 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asp His 20 25 30 Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Gly Arg Thr Arg Asn Lys Ala Asn Ser Tyr
Thr Thr Glu Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn
Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg
100
<210> SEQ ID NO 595 <211> LENGTH: 100 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 595
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp
His 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Gln Gly Lys Gly Leu
Glu Leu Val 35 40 45 Gly Leu Ile Arg Asn Lys Ala Asn Ser Tyr Thr
Thr Glu Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Leu Thr Ile Ser
Arg Glu Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser
Leu Lys Thr Glu Asp Leu Ala Val Tyr 85 90 95 Tyr Cys Ala Arg 100
<210> SEQ ID NO 596 <211> LENGTH: 100 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 596
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp
His 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Gln Gly Lys Gly Leu
Glu Leu Val 35 40 45 Gly Leu Ile Arg Asn Lys Ala Asn Ser Tyr Thr
Thr Glu Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Leu Thr Ile Ser
Arg Glu Asp Ser Lys Asn Thr 65 70 75 80 Met Tyr Leu Gln Met Ser Asn
Leu Lys Thr Glu Asp Leu Ala Val Tyr 85 90 95 Tyr Cys Ala Arg 100
<210> SEQ ID NO 597 <211> LENGTH: 100 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 597
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp
His 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Gln Gly Lys Gly Leu
Glu Leu Val 35 40 45 Gly Leu Ile Arg Asn Lys Ala Asn Ser Tyr Thr
Thr Glu Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Leu Thr Ile Ser
Arg Glu Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser
Leu Lys Thr Glu Asp Leu Ala Val Tyr 85 90 95 Tyr Cys Ala Arg 100
<210> SEQ ID NO 598 <211> LENGTH: 98 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 598
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp
Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys <210>
SEQ ID NO 599 <211> LENGTH: 98 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 599 Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr
20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Leu Tyr His Cys 85 90 95 Ala Arg <210> SEQ ID
NO 600 <211> LENGTH: 98 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 600 Glu Val Gln Leu
Val Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30
Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr
Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys <210> SEQ ID NO 601
<211> LENGTH: 98 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 601 Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser
Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg <210> SEQ ID NO 602 <211>
LENGTH: 98 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 602 Gln Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser
Ser Ser Ser Ser Tyr Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 603 <211> LENGTH: 100
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 603 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Gly Ser 20 25 30 Ala Met His Trp Val Arg
Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg
Ser Lys Ala Asn Ser Tyr Ala Thr Ala Tyr Ala Ala 50 55 60 Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85
90 95
Tyr Cys Thr Arg 100 <210> SEQ ID NO 604 <211> LENGTH:
100 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 604 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys
Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85
90 95 Tyr Cys Thr Thr 100 <210> SEQ ID NO 605 <211>
LENGTH: 100 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 605 Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg
Ile Glu Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60
Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65
70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala
Val Tyr 85 90 95 Tyr Cys Thr Thr 100 <210> SEQ ID NO 606
<211> LENGTH: 100 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 606 Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly
Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala
Val Tyr 85 90 95 Tyr Cys Thr Thr 100 <210> SEQ ID NO 607
<211> LENGTH: 100 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 607 Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly
Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asn Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala
Val Tyr 85 90 95 Tyr Cys Thr Thr 100 <210> SEQ ID NO 608
<211> LENGTH: 100 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 608 Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly
Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala
Val Tyr 85 90 95 Tyr Cys Thr Thr 100 <210> SEQ ID NO 609
<211> LENGTH: 100 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 609 Glu Val Gln Leu Val Glu Ser
Gly Gly Ala Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly
Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55
60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala
Val Tyr 85 90 95 Tyr Cys Thr Thr 100 <210> SEQ ID NO 610
<211> LENGTH: 98 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 610 Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Pro Ala Ser Gly Phe Thr Phe Ser Asn His 20 25 30 Tyr Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Tyr Ile Ser Gly Asp Ser Gly Tyr Thr Asn Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Ser Pro Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Val Lys <210> SEQ ID NO 611 <211>
LENGTH: 98 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 611 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn His 20 25 30 Tyr Thr Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ser Ser
Gly Asn Ser Gly Tyr Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Val Lys <210> SEQ ID NO 612 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 612 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Ser 20 25 30 Asp Met Asn Trp Val His
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Gly Val Ser Trp Asn Gly Ser Arg Thr His Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Thr Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Val Arg <210> SEQ ID NO 613 <211>
LENGTH: 98 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 613 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Ser 20 25 30 Asp Met Asn Trp Ala Arg
Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Val Ser
Trp Asn Gly Ser Arg Thr His Tyr Val Asp Ser Val 50 55 60 Lys Arg
Arg Phe Ile Ile Ser Arg Asp Asn Ser Arg Asn Ser Leu Tyr 65 70 75 80
Leu Gln Lys Asn Arg Arg Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys 85
90 95 Val Arg <210> SEQ ID NO 614 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 614 Thr Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Glu Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Ser 20 25 30 Asp Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Val Ser
Trp Asn Gly Ser Arg Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Ile Ile Ser Arg Asp Asn Ser Arg Asn Phe Leu Tyr 65 70 75 80
Gln Gln Met Asn Ser Leu Arg Pro Glu Asp Met Ala Val Tyr Tyr Cys 85
90 95 Val Arg <210> SEQ ID NO 615 <211> LENGTH: 97
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 615 Glu Val His Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ala Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Asp Met His Trp Val Arg
Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Asn Gly
Thr Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys 50 55 60 Gly Arg
Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95 Arg <210> SEQ ID NO 616 <211> LENGTH: 97
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 616 Glu Val Gln Leu Val Glu Thr Gly Gly Gly
Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr
Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95 Arg <210> SEQ ID NO 617 <211> LENGTH: 97
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 617 Glu Val Gln Leu Val Gln Ser Gly Gly Gly
Leu Val His Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly
Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala 85
90 95 Arg <210> SEQ ID NO 618 <211> LENGTH: 97
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 618 Glu Val Gln Leu Val Gln Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly
Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala 85
90 95 Arg <210> SEQ ID NO 619 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 619 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser
Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys <210> SEQ ID NO 620 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 620 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ala Ile Ser
Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Val Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Val Lys <210> SEQ ID NO 621 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 621 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45
Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Val Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Val Lys <210> SEQ ID NO 622 <211>
LENGTH: 98 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 622 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ala Ile Ser
Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 623 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 623 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser
Gly Ser Gly Gly Ser Thr Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys <210> SEQ ID NO 624 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 624 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 625 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 625 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 626 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 626 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 627 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 627 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 628 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 628 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 629 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 629 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 630 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 630 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> SEQ ID NO 631
<211> LENGTH: 98 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 631 Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg <210> SEQ ID NO 632 <211>
LENGTH: 98 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 632 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 633 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 633 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ala Ile Ser
Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Val Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Val Lys <210> SEQ ID NO 634 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 634 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ala Ile Ser
Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 635 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 635 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Ala Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 636 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 636 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 637 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 637 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys <210> SEQ ID NO 638 <211> LENGTH: 97
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 638 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met His Trp Val Arg
Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly
Thr Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys 50 55 60 Gly Arg
Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95 Arg <210> SEQ ID NO 639 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 639 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> SEQ ID NO 640
<211> LENGTH: 98 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 640 Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Leu Arg Ala Arg Leu Cys Ile
Thr Val 85 90 95 Arg Glu <210> SEQ ID NO 641 <211>
LENGTH: 98 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 641 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 642 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 642 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 643 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 643 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 644 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 644 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 645 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 645 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Arg Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 646 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 646 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 647 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 647 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 648 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 648 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Gly Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> SEQ ID NO 649
<211> LENGTH: 98 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 649 Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys <210> SEQ ID NO 650 <211>
LENGTH: 98 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 650 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys <210> SEQ ID NO 651 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 651 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 652 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 652 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys <210> SEQ ID NO 653 <211> LENGTH: 95
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 653 Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Arg Lys 85 90
95 <210> SEQ ID NO 654 <211> LENGTH: 98 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
654 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys
Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
<210> SEQ ID NO 655 <211> LENGTH: 98 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 655
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Thr Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210>
SEQ ID NO 656 <211> LENGTH: 98 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 656 Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> SEQ ID
NO 657 <211> LENGTH: 98 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 657 Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> SEQ ID NO 658
<211> LENGTH: 97 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 658 Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 50 55
60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu
65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg <210> SEQ ID NO 659 <211> LENGTH:
98 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 659 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser
Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 660 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 660 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser
Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 661 <211> LENGTH: 97
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 661 Glu Asp Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Pro Ser Cys Ala Ala
Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Val Leu His Trp Val Arg
Arg Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ala Ile Gly
Thr Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val Met 50 55 60 Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Ile Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala 85
90 95 Arg <210> SEQ ID NO 662 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 662 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Ser Arg Ile Asn
Ser Asp Gly Ser Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 663 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 663 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Ser Arg Ile Asn
Ser Asp Gly Ser Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 664 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 664 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys
Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg <210> SEQ ID NO 665 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 665 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Ser Arg Ile Asn
Ser Asp Gly Ser Ser Thr Ser Tyr Ala Asp Ser Met 50 55 60 Lys Gly
Gln Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys 85
90 95 Thr Arg <210> SEQ ID NO 666 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 666 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr
Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> SEQ ID
NO 667 <211> LENGTH: 98 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 667 Gln Val Gln Leu
Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Thr Thr <210> SEQ ID NO 668
<211> LENGTH: 98 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 668 Gln Ser Val Leu Thr Gln Pro
Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser
Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile
Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55
60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln
65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser
Ser Leu 85 90 95 Ser Ala <210> SEQ ID NO 669 <211>
LENGTH: 98 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 669 Gln Ser Val Leu Thr Gln Pro Pro Ser Val
Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asp Met Gly Asn Tyr 20 25 30 Ala Val Ser Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Glu Asn
Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Trp 65 70 75 80
Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Ala Trp Asp Thr Ser Pro 85
90 95 Arg Ala <210> SEQ ID NO 670 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 670 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn
Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85
90 95 Asn Gly <210> SEQ ID NO 671 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 671 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn
Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85
90 95 Ser Gly <210> SEQ ID NO 672 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 672 Gln Ser Val Leu Thr Gln Pro Pro Ser Val
Ser Glu Ala Pro Arg Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Ala Val Asn Trp Tyr Gln
Gln Leu Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asp
Asp Leu Leu Pro Ser Gly Val Ser Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85
90 95 Asn Gly <210> SEQ ID NO 673 <211> LENGTH: 99
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 673 Gln Ser Val Leu Thr Gln Pro Pro Ser Val
Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly
Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val Val His Trp Tyr
Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly
Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Gln Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80
Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Lys Ala Trp Asp Asn Ser 85
90 95 Leu Asn Ala <210> SEQ ID NO 674 <211> LENGTH: 99
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 674 Gln Ser Val Val Thr Gln Pro Pro Ser Val
Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly
Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr
Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly
Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85
90 95 Leu Ser Gly <210> SEQ ID NO 675 <211> LENGTH: 98
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 675 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val
Ser Gly Thr Pro Gly Gln 1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20
25 30 Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45 Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Thr Gly Val Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Gln Ser Tyr Asp Ser Ser Leu 85 90 95 Arg Gly
* * * * *