U.S. patent application number 13/264983 was filed with the patent office on 2012-05-31 for anti-tnf-a antibodies and their uses.
Invention is credited to Yoshiko Akamatsu, Robert B. Dubridge, Fiona A. Harding, David B. Powers.
Application Number | 20120135005 13/264983 |
Document ID | / |
Family ID | 42470707 |
Filed Date | 2012-05-31 |
United States Patent
Application |
20120135005 |
Kind Code |
A1 |
Harding; Fiona A. ; et
al. |
May 31, 2012 |
ANTI-TNF-a ANTIBODIES AND THEIR USES
Abstract
The present disclosure relates to antibodies directed to the
tumor necrosis factor alpha ("TNF-.alpha.") and uses of such
antibodies, for example, to treat diseases associated with the
activity and/or overproduction of TNF-.alpha..
Inventors: |
Harding; Fiona A.; (Santa
Clara, CA) ; Akamatsu; Yoshiko; (Palo Alto, CA)
; Dubridge; Robert B.; (Belmont, CA) ; Powers;
David B.; (Fairfax, CA) |
Family ID: |
42470707 |
Appl. No.: |
13/264983 |
Filed: |
April 16, 2010 |
PCT Filed: |
April 16, 2010 |
PCT NO: |
PCT/US2010/031406 |
371 Date: |
February 13, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61170053 |
Apr 16, 2009 |
|
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Current U.S.
Class: |
424/158.1 ;
435/243; 435/320.1; 435/335; 435/69.6; 530/389.2; 536/23.53 |
Current CPC
Class: |
A61P 1/06 20180101; A61P
37/00 20180101; A61P 19/02 20180101; A61P 37/06 20180101; A61P
43/00 20180101; A61P 19/04 20180101; A61P 37/02 20180101; C07K
2317/92 20130101; A61P 17/00 20180101; A61P 1/00 20180101; A61P
1/04 20180101; C07K 2317/56 20130101; C07K 2317/76 20130101; A61P
29/00 20180101; C07K 16/241 20130101; A61P 3/10 20180101 |
Class at
Publication: |
424/158.1 ;
530/389.2; 536/23.53; 435/320.1; 435/243; 435/335; 435/69.6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 15/13 20060101 C12N015/13; C12N 15/63 20060101
C12N015/63; A61P 1/00 20060101 A61P001/00; C12N 5/10 20060101
C12N005/10; C12P 21/02 20060101 C12P021/02; A61P 19/02 20060101
A61P019/02; A61P 29/00 20060101 A61P029/00; C07K 16/22 20060101
C07K016/22; C12N 1/00 20060101 C12N001/00 |
Claims
1. An anti-TNF-.alpha. antibody or an anti-TNF-.alpha. binding
fragment of an antibody which comprises six complementarity
determining regions ("CDRs") having amino acid sequences
corresponding to SEQ ID NO:5 (CDR-H1), SEQ ID NO:6 (CDR-H2), SEQ ID
NO:7 (CDR-H3), SEQ ID NO:8 (CDR-L1), SEQ ID NO:9 (CDR-L2) and SEQ
ID NO:10 (CDR-H3), said CDR sequences being the CDR sequences of
antibody D2E7, wherein a. said CDRs include at least one
substitution selected from S3K in CDR-L2, S3R in CDR-L2, T4H in
CDR-L2, T4Q in CDR-L2, T4F in CDR-L2, T4W in CDR-L2, T4Y in CDR-L2,
L5R in CDR-L2, L5K in CDR-L2, Q6R in CDR-L2, Y2H in CDR-H1, A3G in
CDR-H1, and T3N in CDR-H2, and optionally one or more additional
mutations or combinations of mutations selected from one or more of
Tables 11 and 13 to 25, wherein the six CDR5 altogether have up to
17 amino acid substitutions as compared to CDR sequences of the
antibody D2E7. b. said CDRs include at least one substitution
selected from T4F in CDR-L2, T4W in CDR-L2, T4Y in CDR-L2, L5R in
CDR-L2, L5K in CDR-L2, Q6R in CDR-L2, Y2H in CDR-H1, A3G in CDR-H1,
and T3N in CDR-H2 and optionally one or more additional mutations
or combinations of mutations selected from one or more of Tables 11
and 13 to 25, wherein the six CDRs altogether have up to 17 amino
acid substitutions as compared to CDR sequences of the antibody
D2E7. c. said CDRs include at least one substitution selected from
T4F in CDR-L2, T4W in CDR-L2, T4Y in CDR-L2, L5R in CDR-L2, L5K in
CDR-L2, Q6R in CDR-L2, Y2H in CDR-H1, A3G in CDR-H1, and T3N in
CDR-H2, and optionally one or more additional mutations or
combinations of mutations selected from one or more of Tables 11
and 13 to 18, wherein the six CDRs altogether have up to 17 amino
acid substitutions as compared to CDR sequences of the antibody
D2E7. d. said CDRs include a substitution or combination of
substitutions selected from R7Q; A11S; R7Q+A11S; N8T; N8T+A11S;
I6T; A11G; I6T+AUG; Q4G; Q4G+A11S; Q4G+A11G; Q4H; Q4H+A11S; Q4R;
Q4R+A11S; G5S+A11S; N8S+A11S; I6T+A11S; N8T+A11G in CDR-L1, and
optionally one or more additional mutations or combinations of
mutations selected from one or more of Tables 12 to 25, wherein the
six CDRs altogether have up to 17 amino acid substitutions as
compared to CDR sequences of the antibody D2E7. e. said CDRs
includes a substitution or combination of substitutions selected
from R7Q; A11S; R7Q+A11S; N8T; N8T+A11S; I6T; A11G; I6T+A11G; Q4G;
Q4G+A11S; Q4G+A11G; Q4H; Q4H+A11S; Q4R; Q4R+A11S; G5S+A11S;
N8S+A11S; I6T+A11S; N8T+A11G in CDR-L1, and optionally one or more
additional mutations or combinations of mutations selected from one
or more of Tables 12 to 18, wherein the six CDRs altogether have up
to 17 amino acid substitutions as compared to CDR sequences of the
antibody D2E7. f. said CDRs include the substitutions G5S+A11S in
CDR-L1, and optionally one or more additional mutations or
combinations of mutations selected from one or more of Tables 11 to
25, wherein the six CDRs altogether have up to 17 amino acid
substitutions as compared to CDR sequences of the antibody D2E7. g.
wherein said CDRs include the substitutions G5S+A11G in CDR-L1, and
optionally one or more additional mutations or combinations of
mutations selected from one or more of Tables 11 to 25, wherein the
six CDRs altogether have up to 17 amino acid substitutions as
compared to CDR sequences of the antibody D2E7. h. said CDRs
include at least one substitution selected from S3N in CDR-L2, T4V
in CDR-L2, Q6K in CDR-L2, and D1G in CDR-H1 and at least one
substitution or combination of substitutions selected from Tables
11, 12 and 25, and optionally one or more additional mutations or
combinations of mutations selected from one or more of Tables 11 to
25, wherein the six CDRs altogether have up to 17 amino acid
substitutions as compared to CDR sequences of the antibody D2E7. i.
said CDR-L2 CDR includes at least one combination of substitutions
selected from: (i) S3K, T4H, L5R and Q6R; (ii) S3K, T4Q, L5R and
Q6K; (iii) S3K, T4Y and L5K; (iv) S3K and T4Y; (v) S3N, T4V, L5R
and Q6K; (vi) S3N, T4W, L5R and Q6R, (vii) S3R, T4F and L5R; (viii)
S3R, T4F, L5R and 06R; (ix) S3R, T4H and Q6K; (x) S3R, T4W, L5K and
06R; (xi) T4H, L5K and Q6K; (xii) T4H, L5K and Q6R; (xiii) T4W, L5R
and Q6R; and (xiv) T4Y and L5R, optionally wherein said
anti-TNF-.alpha. antibody or anti-TNF-.alpha. binding fragment
comprises one or more additional mutations or combinations of
mutations selected from one or more of Tables 11 to 24, wherein the
six CDRs altogether have up to 17 amino acid substitutions as
compared to CDR sequences of the antibody D2E7.
2-62. (canceled)
63. The anti-TNF-.alpha. antibody or anti-TNF-.alpha. binding
fragment of claim 1, wherein the six CDRs altogether have up to 16,
up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, up to
9, up to 8, up to 7, or up to 6 amino acid substitutions as
compared to CDR sequences of the antibody D2E7.
64. The anti-TNF-.alpha. antibody or anti-TNF-.alpha. binding
fragment of claim 1, wherein any individual CDR has no more than
three amino acid substitutions as compared to the corresponding CDR
sequence of the antibody D2E7.
65. The anti-TNF-.alpha. antibody or anti-TNF-.alpha. binding
fragment of claim 1, wherein any individual CDR has no more than
two amino acid substitutions as compared to the corresponding CDR
sequence of the antibody D2E7.
66. The anti-TNF-.alpha. antibody or an anti-TNF-.alpha. binding
fragment of claim 1, which is a human or humanized antibody, or
anti-TNF-.alpha. binding fragment of a humanized or humanized
antibody, respectively.
67. The anti-TNF-.alpha. antibody or an anti-TNF-.alpha. binding
fragment of claim 1, which has, other than said one or more
mutations, a V.sub.H sequence corresponding to SEQ ID NO:2 and a
V.sub.L sequence corresponding to SEQ ID NO:4.
68-70. (canceled)
71. A pharmaceutical composition comprising the anti-TNF-.alpha.
antibody or anti-TNF-.alpha. binding fragment of claim 1 and a
pharmaceutically acceptable carrier.
72. A nucleic acid comprising a nucleotide sequence encoding the
anti-TNF-.alpha. antibody or anti-TNF-.alpha. binding fragment of
claim 1.
73. A vector comprising the nucleic acid of claim 72.
74. A prokaryotic host cell transformed with a vector according to
claim 73.
75. A eukaryotic host cell transformed with a vector according to
claim 73.
76. A eukaryotic host cell engineered to express the nucleotide
sequence of claim 72.
77. The eukaryotic host cell of claim 76 which is a mammalian host
cell.
78. A method of producing anti-TNF-.alpha. antibody or
anti-TNF-.alpha. binding fragment, comprising: (a) culturing the
eukaryotic host cell of claim 76 and (b) recovering the
anti-TNF-.alpha. antibody or anti-TNF-.alpha., binding fragment
antibody.
79. A method of treating an immune disorder, comprising
administering to a human patient in need thereof a therapeutically
effective amount of the anti-TNF-.alpha. antibody or
anti-TNF-.alpha. binding fragment claim 1.
80. The method of claim 79, wherein the immune disorder is systemic
lupus erythematosus, rheumatoid arthritis, thyroidosis, graft
versus host disease, scleroderma, diabetes mellitus, Grave's
disease, sarcoidosis, chronic inflammatory bowel disease,
ulcerative colitis, or Crohn's disease.
Description
1. CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional
application No. 61/170,053, filed Apr. 16, 2009, the contents of
which are incorporated herein by reference in their entirety.
2. REFERENCE TO SEQUENCE LISTING
[0002] The Sequence Listing concurrently submitted herewith is
incorporated herein by reference.
3. FIELD OF THE INVENTION
[0003] The present invention relates to anti-TNF-.alpha.
antibodies, pharmaceutical compositions comprising anti-TNF-.alpha.
antibodies, and therapeutic uses of such antibodies.
4. BACKGROUND
[0004] Tumor necrosis factor alpha (TNF-.alpha.) is a
pro-inflammatory cytokine that is released by and interacts with
cells of the immune system. TNF-.alpha. has been shown to be
upregulated in a number of human diseases, including chronic
diseases such as rheumatoid arthritis, Crohn's disease, ulcerative
colitis and multiple sclerosis. For example, elevated levels of
TNF-.alpha. are found in the synovial fluid of rheumatoid arthritis
patients and play an important role in both the pathologic
inflammation and the joint destruction that are hallmarks of
rheumatoid arthritis.
[0005] Human TNF-.alpha. is a 17 kDa protein, and the active form
exists as a homotrimer (Pennica et al., 1984, Nature 312:724-729;
Davis et al., 1987, Biochemistry 26:1322-1326; Jones et al., 1989,
Nature 338:225-228). TNF-.alpha. exerts its biological effects
through interaction with two structurally related but functionally
distinct cell surface receptors, p55 and p75, that are co-expressed
on most cell types (Loetscher et al., 1990, Cell 61:351-9; Smith et
al., 1990, Science 248(4958):1019-23). p55 is also known as p55R;
p55TNFR; CD120a; TNFR I; TNFR 1 and TNFRSFIa. p75 is also known as
p75R; p75TNFR; CD120b; TNFR II; TNFR 2 and TNFRSFIb. Both receptors
are also proteolytically released as soluble molecules capable of
binding TNF-.alpha..
[0006] Inhibition of TNF-.alpha. activity as a method of treating
disease, in particular, rheumatoid arthritis, has been achieved by
a number of different means using inhibitors such as antibodies and
soluble receptors. Examples include etanercept, marketed by Immunex
Corporation as ENBREL.RTM. which is a recombinant fusion protein
comprising two p75 soluble TNF-receptor domains linked to the Fc
portion of a human immunoglobulin. Infliximab, marketed by Centocor
Corporation as REMICADE.RTM., is a chimeric antibody having murine
anti-TNF-.alpha. variable domains and human IgG.sub.1 constant
domains. Other inhibitors include engineered TNF-.alpha. molecules
which form trimers with native TNF-.alpha. and prevent receptor
binding (Steed et al., 2003, Science 301:1895-1898; WO 03/033720;
WO 01/64889). These current methods of inhibiting TNF-.alpha.
activity block binding of TNF-.alpha. to both the p55 and p75
receptors (See, for example, Mease, 2005, Expert Opin. Biol.
Therapy 5(11):1491-1504). Adalimumab, marketed by Abbott
Laboratories as HUMIRA.RTM., is a recombinant, fully human
anti-TNF-.alpha. antibody (Tussirot and Wendling, 2004, Expert
Opin. Pharmacother. 5:581-594). Adalimumab binds specifically to
TNF-.alpha. and blocks its interaction with the p55 and p75 cell
surface TNF-.alpha. receptors. Adalimumab also lyses surface
TNF-.alpha. expressing cells in vitro via complement-dependent
cytotoxicity ("CDC") and antibody-dependent cell-mediated
cytotoxicity ("ADCC"). Adalimumab does not bind or inactivate
lymphotoxin (TNF-.beta.). Adalimumab also modulates biological
responses that are induced or regulated by TNF, including changes
in the levels of adhesion molecules responsible for leukocyte
migration (ELAM-1, VCAM-1, and ICAM-1 with an IC.sub.50 of
1-2.times.10.sup.-10 M).
[0007] Despite being a human antibody, Adalimumab can elicit an
immune response when administered to humans. Such an immune
response can result in an immune complex-mediated clearance of the
antibodies or fragments from the circulation and make repeated
administration unsuitable for therapy, thereby reducing the
therapeutic benefit to the patient and limiting the
readministration of the antibody.
[0008] Accordingly, there is a need to provide improved
anti-TNF-.alpha. antibodies or fragments that overcome one more of
these problems, for example, by generating variants with higher
affinity than Adalimumab that can be administered at reduced
dosages or variants with reduced immunogenicity as compared to
Adalimumab.
[0009] Citation or identification of any reference in Section 4 or
in any other section of this application shall not be construed as
an admission that such reference is available as prior art to the
present disclosure.
5. SUMMARY
[0010] The present disclosure relates to variants of the
anti-TNF-.alpha. antibody D2E7 with improved binding to TNF-.alpha.
and/or reduced immunogenicity as compared to D2E7. D2E7 has three
heavy chain CDRs, referred to herein (in amino- to carboxy-terminal
order) as CDR-H1 (SEQ ID NO:5), CDR-H2 (SEQ ID NO:6), and CDR-H3
(SEQ ID NO:7), and three light chain CDRs, referred to herein (in
amino- to carboxy-terminal order) as CDR-L1 (SEQ ID NO:8), CDR-L2
(SEQ ID NO:9), and CDR-L3 (SEQ ID NO:10). The anti-TNF-.alpha.
antibodies and anti-TNF-.alpha. binding fragments of the disclosure
generally have at least one amino acid substitution in at least one
CDR as compared to D2E7.
[0011] In certain aspects, at least one amino acid substitution or
combination of substitutions is selected from Table 11, Table 12
and/or Table 25. Further mutations (including substitutions,
deletions or insertions) can be selected from one or more of Tables
13-25.
[0012] In certain aspects, the present disclosure relates to
variants of the anti-TNF-.alpha. antibody D2E7 with improved
binding properties, e.g., improved affinity, to TNF-.alpha. as
compared to D2E7. In specific embodiments, the antibodies of the
disclosure have a greater affinity than D2E7 towards TNF-.alpha.,
for example improved K.sub.D as measured by BIAcore and/or improved
affinity as measured by competition ELISA.
[0013] In certain aspects, the anti-TNF-.alpha. antibodies and
anti-TNF-.alpha. binding fragments include at least one
substitution selected from S3K in CDR-L2 (SEQ ID NO:9), S3R in
CDR-L2 (SEQ ID NO:9), S3N in CDR-L2 (SEQ ID NO:9), T4H in CDR-L2
(SEQ ID NO:9), T4Q in CDR-L2 (SEQ ID NO:9), T4V in CDR-L2 (SEQ ID
NO:9), T4F in CDR-L2 (SEQ ID NO:9), T4W in CDR-L2 (SEQ ID NO:9),
T4Y in CDR-L2 (SEQ ID NO:9); L5R in CDR-L2 (SEQ ID NO:9), L5K in
CDR-L2 (SEQ ID NO:9), Q6K in CDR-L2 (SEQ ID NO:9), Q6R in CDR-L2
(SEQ ID NO:9), D1G in CDR-H1 (SEQ ID NO:5), Y2H in CDR-H1 (SEQ ID
NO:5); A3G in CDR-H1 (SEQ ID NO:5), and T3N in CDR-H2 (SEQ ID
NO:6). Additional mutations that can be incorporated into the
improved affinity variant anti-TNF-.alpha. antibodies and
anti-TNF-.alpha. binding fragments can be deimmunizing
substitutions, such as those described in Table 11, as well as
other mutations, e.g., substitutions, that do not destroy the
ability of the anti-TNF-.alpha. antibodies and anti-TNF-.alpha.
binding fragments to bind TNF-.alpha., including but not limited to
the known mutations described in Tables 13 to 24 or the mutations
described in Table 25.
[0014] In certain aspects, the anti-TNF-.alpha. antibodies and
anti-TNF-.alpha. binding fragments include at least one
substitution selected from T4F in CDR-L2, T4W in CDR-L2, T4Y in
CDR-L2, L5R in CDR-L2, L5K in CDR-L2, Q6R in CDR-L2, Y2H in CDR-H1,
A3G in CDR-H1, and T3N in CDR-H2. Additional mutations or
combinations of mutations that can be incorporated into such
anti-TNF-.alpha. antibodies and anti-TNF-.alpha. binding fragments
can be selected from one or more of Tables 11 and 13 to 25.
[0015] In certain other aspects, the anti-TNF-.alpha. antibodies
and anti-TNF-.alpha. binding fragments include at least one
substitution selected from T4F in CDR-L2, T4W in CDR-L2, T4Y in
CDR-L2, L5R in CDR-L2, L5K in CDR-L2, Q6R in CDR-L2, Y2H in CDR-H1,
A3G in CDR-H1, and T3N in CDR-H2. Additional mutations or
combinations of mutations that can be incorporated into such
anti-TNF-.alpha. antibodies and anti-TNF-.alpha. binding fragments
can be selected from one or more of Tables 11 and 13 to 18.
[0016] In yet other aspects, the anti-TNF-.alpha. antibodies and
anti-TNF-.alpha. binding fragments include the substitutions
G5S+A11S or G5S+A11G in CDR-L1. Additional mutations or
combinations of mutations that can be incorporated into such
anti-TNF-.alpha. antibodies and anti-TNF-.alpha. binding fragments
can be selected from one or more of Tables 11 to 25.
[0017] In certain aspects, the anti-TNF-.alpha. antibodies and
anti-TNF-.alpha. binding fragments include the substitutions
selected from S3N in CDR-L2, T4V in CDR-L2, Q6K in CDR-L2, and D1G
in CDR-H1 in combination with at least one substitution selected
from Tables 11, 12, and 25. Additional mutations or combinations of
mutations that can be incorporated into such anti-TNF-.alpha.
antibodies and anti-TNF-.alpha. binding fragments can be selected
from one or more of Tables 11 to 24.
[0018] In certain aspects, the anti-TNF-.alpha. antibodies and
anti-TNF-.alpha. binding fragments include the substitutions
selected from S3N in CDR-L2, T4V in CDR-L2, Q6K in CDR-L2, and D1G
in CDR-H1 in combination with at least one substitution selected
from S3K in CDR-L2, S3R in CDR-L2, T4H in CDR-L2, T4Q in CDR-L2,
T4F in CDR-L2, T4W in CDR-L2, T4Y in CDR-L2, L5R in CDR-L2, L5K in
CDR-L2, Q6R in CDR-L2, Y2H in CDR-H1, A3G in CDR-H1, and T3N in
CDR-H2.
[0019] In certain aspects, the anti-TNF-.alpha. antibodies and
anti-TNF-.alpha. binding fragments include the combination of
substitutions selected from at least one of S3K, T4H, L5R and Q6R;
S3K, T4Q, L5R and Q6K; S3K, T4Y and L5K; S3K and T4Y; S3N, T4V, L5R
and Q6K; S3N, T4W, L5R and Q6R; S3R, T4F and L5R; S3R, T4F, L5R and
Q6R; S3R, T4H and Q6K; S3R, T4W, L5K and Q6R; T4H, L5K and Q6K;
T4H, L5K and Q6R; T4W, L5R and Q6R; and T4Y and L5R in CDR-L2,
wherein the six CDRs altogether have up to 17 amino acid
substitutions as compared to CDR sequences of the antibody D2E7.
The anti-TNF-.alpha. antibodies or anti-TNF-.alpha. binding
fragments optionally include one or more additional mutations or
combinations of mutations which can be selected from one or more of
Tables 11 to 24.
[0020] In certain aspects, the anti-TNF-.alpha. antibodies and
anti-TNF-.alpha. binding fragments include one or more
substitutions or combinations of substitutions selected from S3K,
S3R, S3N, T4F, T4W, T4Y, T4H, T4Q, T4V, L5R, L5K, Q6R, and Q6K in
CDR-L2. Additional mutations or combinations of mutations that can
be incorporated into such anti-TNF-.alpha. antibodies and
anti-TNF-.alpha. binding fragments can be selected from one or more
of Tables 11 to 24.
[0021] In other aspects, the present disclosure relates to variants
of the anti-TNF-.alpha. antibody D2E7 with reduced immunogenicity
as compared to D2E7. In certain aspects, the anti-TNF-.alpha.
antibodies and anti-TNF-.alpha. binding fragments include at least
one substitution or combination of substitution(s) in CDR-L1 (SEQ
ID NO:8) selected from R7Q; A11S; R7Q+A11S; N8T; N8T+A11S; I6T;
A11G; I6T+A11G; Q4G; Q4G+A11S; Q4G+A11G; Q4H; Q4H+A11S; Q4R;
Q4R+A11S; G5S; G5S+A11S; N8S+A11S; I6T+A11S; and N8T+A11G.
Additional mutations that can be incorporated into the
anti-TNF-.alpha. antibodies and anti-TNF-.alpha. binding fragments
with reduced antigenicity include substitutions that improve
binding properties to TNF-.alpha., such as those described in Table
12 and/or Table 25, as well as other mutations, e.g., substitutions
that do not destroy the ability of the anti-TNF-.alpha. antibodies
and anti-TNF-.alpha. binding fragments to bind TNF-.alpha.,
including but not limited to the known mutations described in
Tables 13 to 25.
[0022] In certain aspects, the anti-TNF-.alpha. antibodies and
anti-TNF-.alpha. binding fragments of the disclosure have VH and VL
sequences having 80% to 99% sequence identity to the VH and VL
sequences of D2E7, and include at least one amino acid substitution
in at least one CDR as compared to D2E7. In specific embodiments,
the percentage sequence identity for the heavy chain and the light
chain compared to the VH and VL sequences of D2E7 is each
independently selected from at least 80%, at least 85%, at least
90%, or at least 95% sequence identity.
[0023] In certain aspects, the anti-TNF-.alpha. antibodies and
anti-TNF-.alpha. binding fragments of the disclosure have up to 17
amino acid substitutions in their CDRs as compared to the CDRs of
D2E7. Variant antibodies with 17 amino acid substitutions that
maintain their target binding capability have been generated by
Bostrom et al., 2009, Science 323:1610-14. The anti-TNF-.alpha.
antibodies and anti-TNF-.alpha. binding fragments of the disclosure
can also have up to 16, up to 15, up to 14, up to 13, up to 12, up
to 11, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, or up
to 4 amino acid substitutions in their CDRs as compared to CDR
sequences of the antibody D2E7.
[0024] In specific embodiments, an anti-TNF-.alpha. antibody or
anti-TNF-.alpha. binding fragment of the disclosure has,
independently: [0025] up to one, or up to two, or up to three
CDR-H1 substitutions as compared to the corresponding CDR of D2E7;
[0026] up to one, up to two, up to three, up to four, up to five or
up to six CDR-H2 substitutions as compared to the corresponding CDR
of D2E7; [0027] up to one, up to two, up to three, up to four, or
up to five CDR-H3 substitutions as compared to the corresponding
CDR of D2E7; [0028] up to one, up to two, up to three, or up to
four CDR-L1 substitutions as compared to the corresponding CDR of
D2E7; [0029] up to one, up to two, up to three, or up to four
CDR-L2 substitutions as compared to the corresponding CDR of D2E7;
and [0030] up to one, up to two, up to three, or up to four CDR-L3
substitutions as compared to the corresponding CDR of D2E7.
[0031] The present disclosure further provides pharmaceutical
compositions comprising modified anti-TNF-.alpha. antibodies and
anti-TNF-.alpha. binding fragments having increased affinity to
TNF-.alpha. and/or reduced immunogenicity as compared to D2E7.
[0032] In certain aspects, an anti-TNF-.alpha. antibody or
anti-TNF-.alpha. binding fragment of the disclosure can be a
bispecific antibody or a TNF-.alpha. binding fragment of a
bispecific antibody. The bispecific antibody can be specific to
TNF-.alpha. and another pro-inflammatory cytokine (such as, for
example, lymphotoxin, interferon-.gamma., or interleukin-1).
[0033] Nucleic acids comprising nucleotide sequences encoding the
anti-TNF-.alpha. antibodies and anti-TNF-.alpha. binding fragments
of the disclosure are provided herein, as are vectors comprising
nucleic acids. Additionally, prokaryotic and eukaryotic host cells
transformed with a vector comprising a nucleotide sequence encoding
an anti-TNF-.alpha. antibody or anti-TNF-.alpha. binding fragment
are provided herein, as well as eukaryotic (such as mammalian) host
cells engineered to express the nucleotide sequences. Methods of
producing anti-TNF-.alpha. antibodies and anti-TNF-.alpha. binding
fragments by culturing host cells are also provided.
[0034] The anti-TNF-.alpha. antibodies and anti-TNF-.alpha. binding
fragments of the disclosure are useful in the treatment of immune
disorders, e.g., systemic lupus erythematosus, rheumatoid
arthritis, thyroidosis, graft versus host disease, scleroderma,
diabetes mellitus, Grave's disease, sarcoidosis, chronic
inflammatory bowel disease, ulcerative colitis, or Crohn's
disease.
[0035] It should be noted that the indefinite articles "a" and "an"
and the definite article "the" are used in the present application,
as is common in patent applications, to mean one or more unless the
context clearly dictates otherwise. Further, the term "or" is used
in the present application, as is common in patent applications, to
mean the disjunctive "or" or the conjunctive "and."
[0036] All publications mentioned in this specification are herein
incorporated by reference. Any discussion of documents, acts,
materials, devices, articles or the like that has been included in
this specification is solely for the purpose of providing a context
for the present disclosure. It is not to be taken as an admission
that any or all of these matters form part of the prior art base or
were common general knowledge in the field relevant to the present
disclosure as it existed anywhere before the priority date of this
application.
[0037] The features and advantages of the disclosure will become
further apparent from the following detailed description of
embodiments thereof.
6. BRIEF DESCRIPTION OF THE TABLES AND FIGURES
[0038] Table 1 shows D2E7 VH peptides and D2E7 VL peptides,
respectively, that were tested for immunogenicity.
[0039] Table 2 shows identified CD4+ T cell epitope regions in
D2E7. CDR regions are underlined.
[0040] Table 3 shows HLA class II associations and relative risk of
response to the D2E7 VL region peptide epitopes.
[0041] Table 4 shows sequences of D2E7 VL CDR1 epitope variants. A
total of 99 donors were tested. The number of responders, the
percent of responders, and the average stimulation index is
indicated for each peptide tested.
[0042] Table 5 shows candidate mutations in CDR-L1 for lowering
immunogenicity of D2E7. The numbering of the amino acids in Table 5
corresponds to the positions in the context of the D2E7 light
chain.
[0043] Table 6 shows BIAcore and ELISA results for substitutions in
CDR-L1 that do not result in significantly decreased binding as
compared to D2E7. The numbering of the amino acids in Table 6
corresponds to the positions in the context of the D2E7 light
chain. Improvement in K.sub.D (as measured by BIAcore) and
IC.sub.50 of binding (as measured by ELISA) are indicated by "WTx".
CV % refers to the standard deviation as a percentage of the total
value measure.
[0044] Table 7 shows T-cell assay results for all single and double
mutations to the D2E7 epitope. Peptide 1 is the parent peptide.
Modifications to the parent peptide are in bold-faced type.
[0045] Table 8 shows the preferred epitope peptide variants based
solely on T cell assay results. The numbering of the amino acids in
Table 8 corresponds to the positions in the context of the D2E7
light chain.
[0046] Table 9 shows anti-proliferation bioactivity of antibodies
constructed to contain the preferred variant epitope peptides. The
parent is unmodified D2E7 antibody. The numbering of the amino
acids in Table 9 corresponds to the positions in the context of the
D2E7 light chain.
[0047] Table 10 shows binding kinetics of D2E7 and the D2E7
variants against TNF-.alpha. as analyzed by BIAcore. The numbering
of the amino acids in Table 10 corresponds to the positions in the
context of the D2E7 light chain.
[0048] Table 11 shows CDR-L1 substitutions or combinations of
substitutions that can be incorporated into D2E7-related antibodies
to reduce their immunogenicity.
[0049] Table 12 shows CDR amino acid substitutions outside CDR-L1
resulting in improved K.sub.D (as analyzed by BIAcore), affinity
(as measured by ELISA), or both as compared to D2E7. The numbering
of the amino acids in Table 12 corresponds to the positions in the
context of the D2E7 light and heavy chains. Improvement in K.sub.D
(as measured by BIAcore) and IC.sub.50 of binding (as measured by
ELISA) are indicated by "WTx". CV % refers to the standard
deviation as a percentage of the total value measure and "ND" means
"not done".
[0050] Table 13 shows known mutations in CDR-H1 that can be
incorporated into the antibodies of the disclosure.
[0051] Table 14 shows known mutations in CDR-H2 that can be
incorporated into the antibodies of the disclosure. The inclusion
of 2 amino acids into a single cell indicates a CDR variant that
incorporates an addition to or insertion into the CDR. Shading of a
cell indicates a CDR variant that lacks the shaded amino acid
residues.
[0052] Table 15 shows known mutations in CDR-H3 that can be
incorporated into the antibodies of the disclosure.
[0053] Table 16 shows known mutations in CDR-L1 that can be
incorporated into the antibodies of the disclosure. The inclusion
of 2 amino acids into a single cell indicates a CDR variant that
incorporates an addition to or insertion into the CDR.
[0054] Table 17 shows known mutations in CDR-L2 that can be
incorporated into the antibodies of the disclosure. The inclusion
of 2 amino acids into a single cell indicates a CDR variant that
incorporates the indicated additional N-terminal amino acid into
the CDR.
[0055] Table 18 shows known mutations in CDR-L3 that can be
incorporated into the antibodies of the disclosure. The inclusion
of 2 amino acids into a single cell indicates a CDR variant that
incorporates the indicated additional N-terminal amino acid into
the CDR.
[0056] Table 19 shows further known mutations in CDR-H1 that can be
incorporated into the antibodies of the disclosure.
[0057] Table 20 shows further known mutations in CDR-H2 that can be
incorporated into the antibodies of the disclosure.
[0058] Table 21 shows further known mutations in CDR-H3 that can be
incorporated into the antibodies of the disclosure.
[0059] Table 22 shows further known mutations in CDR-L1 that can be
incorporated into the antibodies of the disclosure.
[0060] Table 23 shows further known mutations in CDR-L2 that can be
incorporated into the antibodies of the disclosure.
[0061] Table 24 shows further known mutations in CDR-L3 that can be
incorporated into the antibodies of the disclosure.
[0062] Table 25 shows combinations of point mutations in CDR-L2
resulting in improved K.sub.D (as analyzed by BIAcore), affinity
(as measured by ELISA), or both as compared to D2E7. The point
mutations can be incorporated singly or in combination into the
antibodies of the disclosure.
[0063] FIGS. 1A-1E. FIG. 1A shows the amino acid sequences of the
D2E7 heavy and light chains, with CDR regions in bold, underlined
text. FIG. 1B shows the CDR sequences and corresponding sequence
identifiers of D2E7. FIG. 1C shows a correspondence chart between
the heavy chain CDR numbering and the heavy chain Kabat numbering.
FIG. 1D shows a correspondence chart between the light chain CDR
numbering and the light chain Kabat numbering. FIG. 1E shows the
nucleotide sequences of the heavy and light chain variable regions
of D2E7 (SEQ ID NO:1 and SEQ ID NO:3, respectively) as published in
U.S. Pat. No. 6,090,382.
[0064] FIG. 2 shows percent responses (bottom) and average
stimulation indexes (top) to the D2E7 VL peptides.
[0065] FIG. 3 shows average stimulation indexes (top) and percent
responses (bottom) to the D2E7 VH peptides. Peptide #27 had an
anomalous stimulation index in one donor, and is indicated in
darker shading.
[0066] FIG. 4 shows D2E7 VL CDR1 epitope peptide variants. Open
symbols indicate multiple retests of the unmodified parent peptide
within the dataset. Filled symbols represent unique peptide alanine
scan variants. The sequence of the most reduced response-inducing
variants is indicated.
[0067] FIG. 5 shows D2E7 VL CDR1 epitope peptide variants. Open
symbols indicate multiple retests of the unmodified parent peptide
within the dataset. Filled symbols represent unique peptide
variants. The most reduced response-inducing variants are indicated
by a circle. This figure graphically represents data from Table
7.
[0068] FIG. 6 shows the results of competition ELISA of D2E7
variant antibodies. ELISA plates were coated with TNF-.alpha..
Biotinylated D2E7 was included in all wells at a single
concentration, and the variant antibody was titrated in. The
IC.sub.50 values were calculated for each antibody. The experiment
was performed three times. The Y axis shows average results as a
percent of the parent antibody binding.
7. DETAILED DESCRIPTION
7.1 Anti-TNF-.alpha. Antibodies
[0069] The present disclosure provides anti-TNF-.alpha. antibodies.
Unless indicated otherwise, the term "antibody" (Ab) refers to an
immunoglobulin molecule that specifically binds to, or is
immunologically reactive with, a particular antigen, and includes
polyclonal, monoclonal, genetically engineered and otherwise
modified forms of antibodies, including but not limited to chimeric
antibodies, humanized antibodies, heteroconjugate antibodies (e.g.,
bispecific antibodies, diabodies, triabodies, and tetrabodies), and
antigen binding fragments of antibodies, including, e.g., Fab',
F(ab').sub.2, Fab, Fv, rIgG, and scFv fragments. Moreover, unless
otherwise indicated, the term "monoclonal antibody" (mAb) is meant
to include both intact molecules, as well as, antibody fragments
(such as, for example, Fab and F(ab').sub.2 fragments) which are
capable of specifically binding to a protein. Fab and F(ab').sub.2
fragments lack the Fc fragment of intact antibody, clear more
rapidly from the circulation of the animal or plant, and may have
less non-specific tissue binding than an intact antibody (Wahl et
al., 1983, J. Nucl. Med. 24:316).
[0070] The term "scFv" refers to a single chain Fv antibody in
which the variable domains of the heavy chain and the light chain
from a traditional antibody have been joined to form one chain.
[0071] References to "VH" refer to the variable region of an
immunoglobulin heavy chain of an antibody, including the heavy
chain of an Fv, scFv, or Fab. References to "VL" refer to the
variable region of an immunoglobulin light chain, including the
light chain of an Fv, scFv, dsFv or Fab. Antibodies (Abs) and
immunoglobulins (Igs) are glycoproteins having the same structural
characteristics. While antibodies exhibit binding specificity to a
specific target, immunoglobulins include both antibodies and other
antibody-like molecules which lack target specificity. Native
antibodies and immunoglobulins are usually heterotetrameric
glycoproteins of about 150,000 Daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each heavy
chain has at the amino terminus a variable domain (VH) followed by
a number of constant domains. Each light chain has a variable
domain at the amino terminus (VL) and a constant domain at the
carboxy terminus.
[0072] The anti-TNF-.alpha. antibodies of the disclosure bind to
human TNF-.alpha. and inhibit TNF-.alpha. receptor activity in a
cell. Without being bound by any one theory, the inventors believe
that the antibodies reduce the binding of TNF-.alpha. to both the
low affinity TNF-.alpha. receptor (p75) and the high affinity
TNF-.alpha. receptor (p55).
[0073] The anti-TNF-.alpha. antibodies of the disclosure contain
complementarity determining regions (CDRs) that are related in
sequence to the CDRs of the antibody D2E7 (also known as Adalimumab
or HUMIRA.RTM.).
[0074] CDRs are also known as hypervariable regions both in the
light chain and the heavy chain variable domains. The more highly
conserved portions of variable domains are called the framework
(FR). As is known in the art, the amino acid position/boundary
delineating a hypervariable region of an antibody can vary,
depending on the context and the various definitions known in the
art. Some positions within a variable domain may be viewed as
hybrid hypervariable positions in that these positions can be
deemed to be within a hypervariable region under one set of
criteria while being deemed to be outside a hypervariable region
under a different set of criteria. One or more of these positions
can also be found in extended hypervariable regions. The disclosure
provides antibodies comprising modifications in these hybrid
hypervariable positions. The variable domains of native heavy and
light chains each comprise four FR regions, largely by adopting a
(.beta.-sheet configuration, connected by three CDRs, which form
loops connecting (and in some cases forming part of) the
.beta.-sheet structure. The CDRs in each chain are held together in
close proximity by the FR regions in the order
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and with the CDRs from the other
chain, contribute to the formation of the target binding site of
antibodies (See Kabat et al., Sequences of Proteins of
Immunological Interest (National Institute of Health, Bethesda, Md.
1987)). As used herein, numbering of immunoglobulin amino acid
residues is done according to the immunoglobulin amino acid residue
numbering system of Kabat et al. unless otherwise indicated.
[0075] The sequences of the heavy and light chain variable regions
of D2E7 are represented by SEQ ID NO:2 and SEQ ID NO:4,
respectively, and encoded by SEQ ID NO.:1 and SEQ ID NO.:3,
respectively. The sequences of the heavy and light chain variable
regions are also depicted in FIG. 1A. The sequences of the CDRs of
D2E7, and their corresponding identifiers, are presented in FIG.
1B. The sequences of the heavy and light chain variable regions of
D2E7 (as published in U.S. Pat. No. 6,090,382) are shown in FIG.
1C. Any nucleotide sequences encoding SEQ ID NO:2 or SEQ ID NO:4
can be used in the compositions and methods of the present
disclosure.
[0076] The present disclosure further provides anti-TNF-.alpha.
antibody fragments comprising CDR sequences that are related to the
CDR sequences of D2E7. The term "antibody fragment" refers to a
portion of a full-length antibody, generally the target binding or
variable region. Examples of antibody fragments include Fab, Fab',
F(ab')2 and Fv fragments. An "Fv" fragment is the minimum antibody
fragment which contains a complete target recognition and binding
site. This region consists of a dimer of one heavy and one light
chain variable domain in a tight, noncovalent association (VH-VL
dimer). It is in this configuration that the three CDRs of each
variable domain interact to define a target binding site on the
surface of the VH-VL dimer. Often, the six CDRs confer target
binding specificity to the antibody. However, in some instances
even a single variable domain (or half of an Fv comprising only
three CDRs specific for a target) can have the ability to recognize
and bind target. "Single chain Fv" or "scFv" antibody fragments
comprise the VH and VL domains of an antibody in a single
polypeptide chain. Generally, the Fv polypeptide further comprises
a polypeptide linker between the VH and VL domain that enables the
scFv to form the desired structure for target binding. "Single
domain antibodies" are composed of a single VH or VL domains which
exhibit sufficient affinity to the TNF-.alpha.. In a specific
embodiment, the single domain antibody is a camelid antibody (see,
e.g., Riechmann, 1999, Journal of Immunological Methods
231:25-38).
[0077] The Fab fragment contains the constant domain of the light
chain and the first constant domain (CH.sub.1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxyl terminus of the heavy chain CH.sub.1
domain including one or more cysteines from the antibody hinge
region. F(ab') fragments are produced by cleavage of the disulfide
bond at the hinge cysteines of the F(ab').sub.2 pepsin digestion
product. Additional chemical couplings of antibody fragments are
known to those of ordinary skill in the art.
[0078] In certain embodiments, the anti-TNF-.alpha. antibodies of
the disclosure are monoclonal antibodies. The term "monoclonal
antibody" as used herein is not limited to antibodies produced
through hybridoma technology. The term "monoclonal antibody" refers
to an antibody that is derived from a single clone, including any
eukaryotic, prokaryotic, or phage clone and not the method by which
it is produced. Monoclonal antibodies useful in connection with the
present disclosure can be prepared using a wide variety of
techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies or a combination
thereof. The anti-TNF-.alpha. antibodies of the disclosure include
chimeric, primatized, humanized, or human antibodies.
[0079] The anti-TNF-.alpha. antibodies of the disclosure can be
chimeric antibodies. The term "chimeric" antibody as used herein
refers to an antibody having variable sequences derived from a
non-human immunoglobulin, such as rat or mouse antibody, and human
immunoglobulin constant regions, typically chosen from a human
immunoglobulin template. Methods for producing chimeric antibodies
are known in the art. See, e.g., Morrison, 1985, Science
229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies
et al., 1985, J. Immunol. Methods 125:191-202; U.S. Pat. Nos.
5,807,715; 4,816,567; and 4,816,397, which are incorporated herein
by reference in their entireties.
[0080] The anti-TNF-.alpha. antibodies of the disclosure can be
humanized. "Humanized" forms of non-human (e.g., murine) antibodies
are chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
target-binding subdomains of antibodies) which contain minimal
sequences derived from non-human immunoglobulin. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin sequence. The humanized antibody
can also comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin consensus
sequence. Methods of antibody humanization are known in the art.
See, e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos.
5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 to Queen
et al.; EP239400; PCT publication WO 91/09967; U.S. Pat. No.
5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol.,
28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska
et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Pat. No.
5,565,332, all of which are hereby incorporated by reference in
their entireties.
[0081] The anti-TNF-.alpha. antibodies of the disclosure can be
human antibodies. Completely "human" anti-TNF-.alpha. antibodies
can be desirable for therapeutic treatment of human patients. As
used herein, "human antibodies" include antibodies having the amino
acid sequence of a human immunoglobulin and include antibodies
isolated from human immunoglobulin libraries or from animals
transgenic for one or more human immunoglobulin and that do not
express endogenous immunoglobulins. Human antibodies can be made by
a variety of methods known in the art including phage display
methods using antibody libraries derived from human immunoglobulin
sequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT
publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO
96/34096; WO 96/33735; and WO 91/10741, each of which is
incorporated herein by reference in its entirety. Human antibodies
can also be produced using transgenic mice which are incapable of
expressing functional endogenous immunoglobulins but which can
express human immunoglobulin genes. See, e.g., PCT publications WO
98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S. Pat. Nos.
5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;
5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are
incorporated by reference herein in their entireties. In addition,
companies such as Medarex (Princeton, N.J.), Astellas Pharma
(Deerfield, Ill.), Amgen (Thousand Oaks, Calif.) and Regeneron
(Tarrytown, N.Y.) can be engaged to provide human antibodies
directed against a selected antigen using technology similar to
that described above. Completely human antibodies that recognize a
selected epitope can be generated using a technique referred to as
"guided selection." In this approach a selected non-human
monoclonal antibody, e.g., a mouse antibody, is used to guide the
selection of a completely human antibody recognizing the same
epitope (Jespers et al., 1988, Biotechnology 12:899-903).
[0082] The anti-TNF-.alpha. antibodies of the disclosure can be
primatized. The term "primatized antibody" refers to an antibody
comprising monkey variable regions and human constant regions.
Methods for producing primatized antibodies are known in the art.
See e.g., U.S. Pat. Nos. 5,658,570; 5,681,722; and 5,693,780, which
are incorporated herein by reference in their entireties.
[0083] The anti-TNF-.alpha. antibodies of the disclosure can be
bispecific antibodies. Bispecific antibodies are monoclonal, often
human or humanized, antibodies that have binding specificities for
at least two different antigens. In the present disclosure, one of
the binding specificities can be directed towards TNF-.alpha., the
other can be for any other antigen, e.g., for a cell-surface
protein, receptor, receptor subunit, tissue-specific antigen,
virally derived protein, virally encoded envelope protein,
bacterially derived protein, or bacterial surface protein, etc.
[0084] The anti-TNF-.alpha. antibodies of the disclosure include
derivatized antibodies. For example, but not by way of limitation,
derivatized antibodies are typically modified by glycosylation,
acetylation, pegylation, phosphorylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein (see Section 7.6 for a
discussion of antibody conjugates), etc. Any of numerous chemical
modifications can be carried out by known techniques, including,
but not limited to, specific chemical cleavage, acetylation,
formylation, metabolic synthesis of tunicamycin, etc. Additionally,
the derivative can contain one or more non-natural amino acids,
e.g., using ambrx technology (See, e.g., Wolfson, 2006, Chem. Biol.
13(10):1011-2).
[0085] In yet another embodiment of the disclosure, the
anti-TNF-.alpha. antibodies or fragments thereof can be antibodies
or antibody fragments whose sequence has been modified to alter at
least one constant region-mediated biological effector function
relative to the corresponding wild type sequence. For example, in
some embodiments, an anti-TNF-.alpha. antibody of the disclosure
can be modified to reduce at least one constant region-mediated
biological effector function relative to an unmodified antibody,
e.g., reduced binding to the Fc receptor (Fc.gamma.R). Fc.gamma.R
binding can be reduced by mutating the immunoglobulin constant
region segment of the antibody at particular regions necessary for
Fc.gamma.R interactions (See, e.g., Canfield and Morrison, 1991, J.
Exp. Med. 173:1483-1491; and Lund et al., 1991, J. Immunol.
147:2657-2662). Reduction in Fc.gamma.R binding ability of the
antibody can also reduce other effector functions which rely on
Fc.gamma.R interactions, such as opsonization, phagocytosis and
antigen-dependent cellular cytotoxicity ("ADCC").
[0086] In other embodiments of the disclosure, an anti-TNF-.alpha.
antibody or fragment thereof can be modified to acquire or improve
at least one constant region-mediated biological effector function
relative to an unmodified antibody, e.g., to enhance Fc.gamma.R
interactions (See, e.g., US 2006/0134709). For example, an
anti-TNF-.alpha. antibody of the disclosure can have a constant
region that binds Fc.gamma.RIIA, Fc.gamma.RIIB and/or
Fc.gamma.RIIIA with greater affinity than the corresponding wild
type constant region.
[0087] Thus, antibodies of the disclosure can have alterations in
biological activity that result in increased or decreased
opsonization, phagocytosis, or ADCC. Such alterations are known in
the art. For example, modifications in antibodies that reduce ADCC
activity are described in U.S. Pat. No. 5,834,597. An exemplary
ADCC lowering variant corresponds to "mutant 3" (shown in FIG. 4 of
U.S. Pat. No. 5,834,597) in which residue 236 is deleted and
residues 234, 235 and 237 (using EU numbering) are substituted with
alanines.
[0088] In some embodiments, the anti-TNF-.alpha. antibodies of the
disclosure have low levels of or lack fucose. Antibodies lacking
fucose have been correlated with enhanced ADCC activity, especially
at low doses of antibody. See Shields et al., 2002, J. Biol. Chem.
277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-73.
Methods of preparing fucose-less antibodies include growth in rat
myeloma YB2/0 cells (ATCC CRL 1662). YB2/0 cells express low levels
of FUT8 mRNA, which encodes .alpha.-1,6-fucosyltransferase, an
enzyme necessary for fucosylation of polypeptides.
[0089] In yet another aspect, the anti-TNF-.alpha. antibodies or
fragments thereof can be antibodies or antibody fragments that have
been modified to increase or reduce their binding affinities to the
fetal Fc receptor, FcRn, for example, by mutating the
immunoglobulin constant region segment at particular regions
involved in FcRn interactions (See, e.g., WO 2005/123780). In
particular embodiments, an anti-TNF-.alpha. antibody of the IgG
class is mutated such that at least one of amino acid residues 250,
314, and 428 of the heavy chain constant region is substituted
alone, or in any combinations thereof, such as at positions 250 and
428, or at positions 250 and 314, or at positions 314 and 428, or
at positions 250, 314, and 428, with positions 250 and 428 a
specific combination. For position 250, the substituting amino acid
residue can be any amino acid residue other than threonine,
including, but not limited to, alanine, cysteine, aspartic acid,
glutamic acid, phenylalanine, glycine, histidine, isoleucine,
lysine, leucine, methionine, asparagine, proline, glutamine,
arginine, serine, valine, tryptophan, or tyrosine. For position
314, the substituting amino acid residue can be any amino acid
residue other than leucine, including, but not limited to, alanine,
cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,
histidine, isoleucine, lysine, methionine, asparagine, proline,
glutamine, arginine, serine, threonine, valine, tryptophan, or
tyrosine.
[0090] For position 428, the substituting amino acid residues can
be any amino acid residue other than methionine, including, but not
limited to, alanine, cysteine, aspartic acid, glutamic acid,
phenylalanine, glycine, histidine, isoleucine, lysine, leucine,
asparagine, proline, glutamine, arginine, serine, threonine,
valine, tryptophan, or tyrosine. Specific combinations of suitable
amino acid substitutions are identified in Table 1 of U.S. Pat. No.
7,217,797, which table is incorporated by reference herein in its
entirety. Such mutations increase the antibody's binding to FcRn,
which protects the antibody from degradation and increases its
half-life.
[0091] In yet other aspects, an anti-TNF-.alpha. antibody has one
or more amino acids inserted into one or more of its hypervariable
regions, for example as described in Jung and Pliickthun, 1997,
Protein Engineering 10:9, 959-966; Yazaki et al., 2004, Protein
Eng. Des Sel. 17(5):481-9. Epub 2004 Aug. 17; and U.S. Pat. App.
No. 2007/0280931.
7.2 Nucleic Acids and Expression Systems
[0092] The present disclosure encompasses nucleic acid molecules
and host cells encoding the anti-TNF-.alpha. antibodies of the
disclosure.
[0093] An anti-TNF-.alpha. antibody of the disclosure 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,
optionally, 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 Molecular Cloning; A Laboratory
Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold
Spring Harbor, N.Y., 1989), Current Protocols in Molecular Biology
(Ausubel, F. M. et al., eds., Greene Publishing Associates, 1989)
and in U.S. Pat. No. 4,816,397.
[0094] In one embodiment, the anti-TNF-.alpha. antibodies are
similar to D2E7 but for changes in one or more CDRs (referred to
hereinbelow as having "D2E7-related" sequences). In another
embodiment, the anti-TNF-.alpha. antibodies are similar to D2E7 but
for changes in one or more framework regions. In yet another
embodiment, the anti-TNF-.alpha. antibodies are similar to D2E7 but
for changes in one or more CDRs and in one or more framework
regions. To generate nucleic acids encoding such anti-TNF-.alpha.
antibodies, DNA fragments encoding the light and heavy chain
variable regions are first obtained. These DNAs can be obtained by
amplification and modification of germline DNA or cDNA encoding
light and heavy chain variable sequences, for example using the
polymerase chain reaction (PCR). Germline DNA sequences for human
heavy and light chain variable region genes are known in the art
(See, e.g., the "VBASE" human germline sequence database; see also
Kabat, E. A. et al., 1991, Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242; Tomlinson et al., 1992, J.
Mol. Biol. 22T:116-198; and Cox et al., 1994, Eur. J. Immunol.
24:827-836; the contents of each of which are incorporated herein
by reference). A DNA fragment encoding the heavy or light chain
variable region of D2E7, the sequences of which are shown in FIG.
1C, can be synthesized and used as a template for mutagenesis to
generate a variant as described herein using routine mutagenesis
techniques; alternatively, a DNA fragment encoding the variant can
be directly synthesized.
[0095] Once DNA fragments encoding D2E7 or D2E7-related VH and VL
segments are obtained, these DNA fragments can be 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.
[0096] 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 (CH.sub.I, CH.sub.2, CH.sub.3 and optionally
CH.sub.4). 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 IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA,
IgE, IgM or IgD constant region, but in certain embodiments is an
IgG.sub.1 or IgG.sub.4 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 CH.sub.1
constant region.
[0097] 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 in certain embodiments is a kappa
constant region. 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.about.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., 1990, Nature
348:552-554).
[0098] To express the anti-TNF-.alpha. antibodies of the
disclosure, 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 vectors or, more typically, both
genes are inserted into the same expression vector.
[0099] 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 D2E7 or D2E7-related light or heavy chain sequences, the
expression vector can already carry antibody constant region
sequences. For example, one approach to converting the D2E7 or
D2E7-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).
[0100] In addition to the antibody chain genes, the recombinant
expression vectors of the disclosure 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. Suitable 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.
[0101] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the disclosure can
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,
puromycin, blasticidin, hygromycin or methotrexate, on a host cell
into which the vector has been introduced. Suitable 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). 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,
lipofection, calcium-phosphate precipitation, DEAE-dextran
transfection and the like.
[0102] It is possible to express the antibodies of the disclosure
in either prokaryotic or eukaryotic host cells. In certain
embodiments, expression of antibodies is performed in eukaryotic
cells, e.g., mammalian host cells, for optimal secretion of a
properly folded and immunologically active antibody. Exemplary
mammalian host cells for expressing the recombinant antibodies of
the disclosure include Chinese Hamster Ovary (CHO cells) (including
DHFR.sup.- 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 Kaufman and Sharp, 1982, Mol. Biol.
159:601-621), NS0 myeloma cells, COS cells, 293 cells and SP2/0
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 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. Host
cells can also be used to produce portions of intact antibodies,
such as Fab fragments or scFv molecules. It is understood that
variations on the above procedure are within the scope of the
present disclosure. For example, it can be desirable to transfect a
host cell with DNA encoding either the light chain or the heavy
chain (but not both) of an anti-TNF-.alpha. antibody of this
disclosure.
[0103] Recombinant DNA technology can 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 TNF-.alpha.. The
molecules expressed from such truncated DNA molecules are also
encompassed by the antibodies of the disclosure.
[0104] In addition, bifunctional antibodies can be produced in
which one heavy and one light chain are an antibody of the
disclosure and the other heavy and light chain are specific for an
antigen other than TNF-.alpha. by crosslinking an antibody of the
disclosure to a second antibody by standard chemical crosslinking
methods. Bifunctional antibodies can also be made by expressing a
nucleic acid engineered to encode a bifunctional antibody.
[0105] In certain embodiments, dual specific antibodies, i.e.
antibodies that bind TNF-.alpha. and an unrelated antigen using the
same binding site, can be produced by mutating amino acid residues
in the light chain and/or heavy chain CDRs. In various embodiments,
dual specific antibodies that bind TNF-.alpha. and another antigen,
for example, another proinflammatory cytokine (such as, for
example, lymphotoxin, interferon-.gamma., or interleukin-1) can be
produced by mutating amino acid residues in the periphery of the
antigen binding site (See, e.g., Bostrom et al., 2009, Science
323:1610-1614). Dual functional antibodies can be made by
expressing a nucleic acid engineered to encode a dual specific
antibody.
[0106] For recombinant expression of an anti-TNF-.alpha. antibody
of the disclosure, the host cell can be co-transfected with two
expression vectors of the disclosure, the first vector encoding a
heavy chain derived polypeptide and the second vector encoding a
light chain derived polypeptide. Typically, the two vectors each
contain a separate selectable marker. Alternatively, a single
vector can be used which encodes both heavy and light chain
polypeptides.
[0107] Once a nucleic acid encoding one or more portions of D2E7 or
of an anti-TNF-.alpha. antibody with CDR sequences related to the
CDR sequences of D2E7 is generated, further alterations or
mutations can be introduced into the coding sequence, for example
to generate nucleic acids encoding antibodies with different CDR
sequences, antibodies with reduced affinity to the Fc receptor, or
antibodies of different subclasses.
[0108] The anti-TNF-.alpha. antibodies of the disclosure can also
be produced by chemical synthesis (e.g., by the methods described
in Solid Phase Peptide Synthesis, 2.sup.nd ed., 1984 The Pierce
Chemical Co., Rockford, Ill.). Variant antibodies can also be
generated using a cell-free platform (see, e.g., Chu et al.,
Biochemia No. 2, 2001 (Roche Molecular Biologicals)).
[0109] Once an anti-TNF-.alpha. antibody of the disclosure has been
produced by recombinant expression, it can be purified by any
method known in the art for purification of an immunoglobulin
molecule, for example, by chromatography (e.g., ion exchange,
affinity, particularly by affinity for TNF-.alpha. after Protein A
or Protein G selection, and sizing column chromatography),
centrifugation, differential solubility, or by any other standard
technique for the purification of proteins. Further, the
anti-TNF-.alpha. antibodies of the present disclosure or fragments
thereof can be fused to heterologous polypeptide sequences
described herein or otherwise known in the art to facilitate
purification.
[0110] Once isolated, an anti-TNF-.alpha. antibody can, if desired,
be further purified, e.g., by high performance liquid
chromatography (See, e.g., Fisher, Laboratory Techniques In
Biochemistry And Molecular Biology (Work and Burdon, eds.,
Elsevier, 1980)), or by gel filtration chromatography on a
Superdex.TM. 75 column (Pharmacia Biotech AB, Uppsala, Sweden).
[0111] 7.3 Biological Activities of Anti-TNF-.alpha. Antibodies
[0112] In certain embodiments, the anti-TNF-.alpha. antibodies of
the disclosure have certain biological activities, such as
competing with D2E7 for binding to TNF-.alpha. or neutralizing
TNF-.alpha. activity.
[0113] Accordingly, in certain embodiments, anti-TNF-.alpha.
antibodies of the disclosure compete with D2E7 for binding to
TNF-.alpha.. The ability to compete for binding to TNF-.alpha. can
be tested using a competition assay. In one example of a
competition assay, TNF-.alpha. is adhered onto a solid surface,
e.g., a microwell plate, by contacting the plate with a solution of
TNF-.alpha. (e.g., at a concentration of 1 .mu.g/mL in PBS over
night at 4.degree. C.). The plate is washed (e.g., 0.1% Tween 20 in
PBS) and blocked (e.g., in Superblock, Thermo Scientific, Rockford,
Ill.). A mixture of sub-saturating amount of biotinylated D2E7 (80
ng/mL) and unlabeled D2E7 (the "reference" antibody) or competing
anti-TNF-.alpha. antibody (the "test" antibody) antibody in serial
dilution (e.g., at a concentration of 2.8 .mu.g/mL, 8.3 .mu.g/mL,
or 25 .mu.g/mL) in ELISA buffer (e.g., 1% BSA and 0.1% Tween 20 in
PBS) is added to wells and plates are incubated for 1 hour with
gentle shaking The plate is washed, 1 .mu.g/mL HRP-conjugated
Streptavidin diluted in ELISA buffer was added to each well and the
plates incubated for 1 hour. Plates are washed and bound antibodies
were detected by addition of substrate (e.g., TMB, Biofx
Laboratories Inc., Owings Mills, Md.). The reaction is terminated
by addition of stop buffer (e.g., Bio FX Stop Reagents, Biofx
Laboratories Inc., Owings Mills, Md.) and the absorbance was
measured at 650 nm using microplate reader (e.g., VERSAmax,
Molecular Devices, Sunnyvale, Calif.). Variations on this
competition assay can also be used to test competition between an
anti-TNF-.alpha. antibody of the disclosure and D2E7. For example,
in certain aspects, the anti-TNF-.alpha. antibody is used as a
reference antibody and D2E7 is used as a test antibody.
Additionally, instead of soluble TNF-.alpha., membrane-bound
TNF-.alpha. expressed on cell surface (for example mammalian cells
such as 293S) in culture can be used. Alternatively, instead of
soluble D2E7 and test antibodies, those expressed on cell surface
(for example mammalian cells such as 293c18) in culture can be used
too. Generally, about 10.sup.4 to 10.sup.6 transfectants, e.g.,
about 10.sup.5 transfectants, are used. Other formats for
competition assays are known in the art and can be employed.
[0114] In various embodiments, an anti-TNF-.alpha. antibody of the
disclosure reduces the binding of labeled D2E7 by at least 40%, by
at least 50%, by at least 60%, by at least 70%, by at least 80%, by
at least 90%, or by a percentage ranging between any of the
foregoing values (e.g., an anti-TNF-.alpha. antibody of the
disclosure reduces the binding of labeled D2E7 by 50% to 70%) when
the anti-TNF-.alpha. antibody is used at a concentration of 0.08
.mu.g/mL, 0.4 .mu.g/mL, 2 .mu.g/mL, 10 .mu.g/mL, 50 .mu.g/mL, 100
.mu.g/mL or at a concentration ranging between any of the foregoing
values (e.g., at a concentration ranging from 2 .mu.g/mL to 10
.mu.g/mL).
[0115] In other embodiments, D2E7 reduces the binding of a labeled
anti-TNF-.alpha. antibody of the disclosure by at least 40%, by at
least 50%, by at least 60%, by at least 70%, by at least 80%, by at
least 90%, or by a percentage ranging between any of the foregoing
values (e.g., D2E7 reduces the binding of a labeled an
anti-TNF-.alpha. antibody of the disclosure by 50% to 70%) when
D2E7 is used at a concentration of 0.4 .mu.g/mL, 2 .mu.g/mL, 10
.mu.g/mL, 50 .mu.g/mL, 250 .mu.g/mL or at a concentration ranging
between any of the foregoing values (e.g., at a concentration
ranging from 2 .mu.g/mL to 10 .mu.g/mL).
[0116] In other aspects, an anti-TNF-.alpha. antibody of the
disclosure inhibits TNF-.alpha. activity in a range of in vitro
assays, such as cell cytotoxicity, mitogenesis, cytokine induction,
and induction of adhesion molecules. Alternatively, activity of an
anti-TNF-.alpha. antibody of the disclosure can be measured by in
vitro assays using membrane bound TNF-.alpha. naturally or
recombinantly expressed on cells, such as ability to induce reverse
signaling, cytokine induction, induction of adhesion molecules, CDC
and ADCC. An exemplary TNF-.alpha. neutralization assay that
measures inhibition of soluble TNF-.alpha. cytotoxicity using cells
sensitive to TNF-.alpha. (e.g., L929) is described below. Other
TNF-.alpha. cytotoxicity assays can also be used to assess the
activity of the anti-TNF-.alpha. antibodies of the disclosure.
[0117] Thus, in an exemplary embodiment, an anti-TNF-.alpha.
cytotoxicity assays entails plating 3.times.10.sup.4 murine L929
cells into individual wells of a flat bottomed 96-well microtiter
plate. The cells are incubated overnight at 37.degree. C. in a
humidified 5% CO.sub.2 incubator. The next day, serial dilutions of
the anti-TNF-.alpha. antibody (e.g., 0.712 .mu.g/mL, 0.949
.mu.g/mL, 1.27 .mu.g/mL, 1.69 .mu.g/mL, 2.25 .mu.g/mL or 3
.mu.g/mL) are prepared in 25 .mu.L of serum-free medium and added
to cells (e.g. final concentration in 150 .mu.L culture is 119
ng/mL, 158 ng/mL, 211 ng/mL, 282 ng/mL, 375 ng/mL or 500 ng/mL).
After a 2-hour incubation at 37.degree. C., 5% CO.sub.2,
TNF-.alpha. is added at final concentration of 40 ng/mL (e.g., 254,
of 240 ng/mL) and the cells were further incubated for 48 hours at
37.degree. C., 5% CO.sub.2. The wells are scored for cytotoxicity
as compared to control plates (which in certain embodiments were
treated with TNF-.alpha. that were not incubated with an
anti-TNF-.alpha. antibody, e.g., were incubated with an isotype
control antibody and in other embodiments were treated with D2E7)
using a viability assay (e.g., CellTiter-Blue, Promega, Madison,
Wis.). Other formats for TNF-.alpha. neutralization assays are
known in the art and can be employed.
[0118] In various embodiments, an anti-TNF-.alpha. antibody of the
disclosure neutralizes TNF-.alpha. by at least 30%, by at least
40%, by at least 50%, by at least 60%, by at least 70%, by at least
80%, by at least 90%, or by a percentage ranging between any of the
foregoing values (e.g., an anti-TNF-.alpha. antibody of the
disclosure neutralizes TNF-.alpha. activity by 50% to 70%) when the
anti-TNF-.alpha. antibody is used at a concentration of 2 ng/mL, 5
ng/mL, 10 ng/mL, 20 ng/mL, 0.1 .mu.g/mL, 0.2 .mu.g/mL, 1 .mu.g/mL,
2 .mu.g/mL, 5 .mu.g/mL, 10 .mu.g/mL, 20 .mu.g/mL, or at a
concentration ranging between any of the foregoing values (e.g., at
a concentration ranging from 1 .mu.g/mL to 5 .mu.g/mL). In some
embodiments, an anti-TNF-.alpha. antibody of the disclosure is at
least 80% as effective, at least 90% as effective, at least 100% as
effective, at least 110% as effective, at least 125% as effective
or at least 150% as effective, and up to 110% as effective, up to
125% as effective, up to 150% as effective or up to 200% as
effective as D2E7 at neutralizing TNF-.alpha., or any range between
any pair of the foregoing values (e.g., 80% to 125% as effective as
D2E7 or 125% to 200% as effective as D2E7 in neutralizing
TNF-.alpha.).
[0119] In certain embodiments, the anti-TNF-.alpha. antibodies of
the disclosure have a high binding affinity for TNF-.alpha.. In
specific embodiments, the anti-TNF-.alpha. antibodies of the
present disclosure have specific association rate constants
(k.sub.on or k.sub.a values), dissociation rate constants
(k.sub.off or k.sub.d values), affinity constants (K.sub.A values),
dissociation constants (K.sub.D values) and/or IC.sub.50 values. In
certain aspects, such values are selected from the following
embodiments.
[0120] 7.4 Kinetic Properties of Anti-TNF-.alpha. Antibodies
[0121] In a specific embodiment, an anti-TNF-.alpha. antibody of
the disclosure binds to TNF-.alpha. with a k.sub.on of at least
10.sup.5 M.sup.-1s.sup.1, at least
5.times.10.sup.5M.sup.-1s.sup.-1, at least
10.sup.6M.sup.-1s.sup.-1, at least 5.times.10.sup.6
M.sup.-1s.sup.-1, at least 10.sup.7 M.sup.-1s.sup.-1, at least
5.times.10.sup.7 M.sup.-1s.sup.-1, at least 10.sup.8
M.sup.-1s.sup.-1, or with a k.sub.on of any range between any pair
of the foregoing values (e.g., 5.times.10.sup.5 to 5.times.10.sup.6
M.sup.-1s.sup.-1 or 10.sup.7 to 10.sup.8 M.sup.-1s.sup.-1).
[0122] In another embodiment, an anti-TNF-.alpha. antibody of the
disclosure binds to TNF-.alpha. with a k.sub.off rate of
5.times.10.sup.-1 s.sup.-1 or less, 10.sup.-1 s.sup.-1 or less,
5.times.10.sup.-2s.sup.-1 or less, 10.sup.-2s.sup.-1 or less,
5.times.10.sup.-3s.sup.-1 or less, 10.sup.-3s.sup.-1 or less,
5.times.10.sup.-4 s.sup.-1 or less, 10.sup.-4 s.sup.-1 or less,
5.times.10.sup.-5 s.sup.-1 or less, 10.sup.-5 s.sup.-1 or less,
5.times.10.sup.-6s.sup.-1 or less, 10.sup.-6s.sup.-1 or less,
5.times.10.sup.-7 s.sup.-1 or less, 10.sup.-7s.sup.1 or less,
5.times.10.sup.-8s.sup.-1 or less, 10.sup.-8s.sup.-1 or less,
5.times.10.sup.-9s.sup.-1 or less, 10.sup.-9s.sup.-1 or less,
5.times.10.sup.-10 s.sup.-1 or less, 10.sup.-10 s.sup.-1 or less,
or with a k.sub.off rate of any range between any pair of the
foregoing values (e.g., 5.times.10.sup.-4 to 10.sup.-6s.sup.-1, or
5.times.10.sup.-5 to 5.times.10.sup.-8s.sup.-1).
[0123] In another embodiment, an anti-TNF-.alpha. antibody of the
disclosure binds to TNF-.alpha. with a K.sub.A (k.sub.on/k.sub.off)
of at least 10.sup.11 nM.sup.-1, at least 5.times.10.sup.11
nM.sup.-1, at least 10.sup.12 nM.sup.-1, at least 5.times.10.sup.12
nM.sup.-1, at least 10.sup.13 nM.sup.-1, at least 5.times.10.sup.13
nM.sup.-1, at least 10.sup.14 nM.sup.-1, at least 5.times.10.sup.14
nM.sup.-1, at least 10.sup.15 nM.sup.-1, at least 5.times.10.sup.15
nM.sup.-1, at least 10.sup.16 nM.sup.-1, at least 5.times.10.sup.16
nM.sup.-1, at least 10.sup.17 nM.sup.-1, at least 5.times.10.sup.17
nM.sup.-1, at least 10.sup.18 nM.sup.-1, at least 5.times.10.sup.18
nM.sup.-1, at least 10.sup.19 nM.sup.-1, at least 5.times.10.sup.19
nM.sup.-1, at least 10.sup.20 nM.sup.-1, at least 5.times.10.sup.20
nM.sup.-1, at least 10.sup.21 nM.sup.-1, at least 5.times.10.sup.21
nM.sup.-1, at least 10.sup.22 nM.sup.-1, at least 5.times.10.sup.22
nM.sup.-1, at least 10.sup.23 nM.sup.-1, at least 5.times.10.sup.23
nM.sup.-1, at least 10.sup.24 nM.sup.-1, at least 5.times.10.sup.24
nM.sup.-1, or with a K.sub.A of any range between any pair of the
foregoing values (e.g., 5.times.10.sup.14 to 10.sup.22 nM.sup.-1,
or 10.sup.11 to 5.times.10.sup.18 nM.sup.-1).
[0124] In other embodiments, an anti-TNF-.alpha. antibody of the
disclosure binds to TNF-.alpha. with a K.sub.D (k.sub.off/k.sub.on)
of 5.times.10.sup.7 nM or less, 10.sup.7 nM or less,
5.times.10.sup.6 nM or less, 10.sup.6 nM or less, 5.times.10.sup.5
nM or less, 10.sup.5 nM or less, 5.times.10.sup.4 nM or less,
10.sup.4 nM or less, 5.times.10.sup.3 nM or less, 10.sup.3 nM or
less, 5.times.10.sup.2 nM or less, 100 nM or less, 90 nM or less,
80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 20 nM
or less, 15 nM or less, 10 nM or less, 5 nM or less, 3.8 nM or
less, 2 nM or less, 1.5 nM or less, 1 nM or less, 5.times.10.sup.-1
nM or less, 10.sup.-1 nM or less, 5.times.10.sup.-2 nM or less,
10.sup.-2 nM or less, 5.times.10.sup.-3 nM or less, 10.sup.-3 nM or
less, 5.times.10.sup.-4 nM or less, 10.sup.-4 nM or less,
5.times.10.sup.-5 nM or less, 10.sup.-5 nM or less,
5.times.10.sup.-6 nM or less, 10.sup.-6 nM or less, or with a
K.sub.D of any range between any pair of the foregoing values
(e.g., 5.times.10.sup.7 to 50 nM, or 15 nM to 5.times.10.sup.-3
nM).
[0125] In certain specific embodiments, an TNF-.alpha. antibody of
the disclosure binds to TNF-.alpha. with a K.sub.D
(k.sub.off/k.sub.on) between approximately 0.1 nM and approximately
1 nM, or approximately 0.1 nM and approximately 2 nM, or
approximately 0.1 nM and approximately 3 nM, or approximately 0.1
nM and approximately 4 nM, or approximately 0.1 nM and
approximately 5 nM, or approximately 0.1 nM and approximately 6 nM,
or approximately 0.1 nM and approximately 7 nM, or approximately
0.1 nM and approximately 8 nM, or approximately 0.1 nM and
approximately 9 nM, or approximately 0.1 nM and approximately 10
nM, or between approximately 0.01 nM and approximately 0.1 nM, or
between approximately 0.01 nM and approximately 1 nM, or between
approximately 0.01 nM and approximately 2 nM, or between
approximately 0.01 nM and approximately 3 nM, or between
approximately 0.01 nM and approximately 4 nM, or between
approximately 0.01 nM and approximately 5 nM, or between
approximately 0.01 nM and approximately 6 nM, or between
approximately 0.01 nM and approximately 7 nM, or between
approximately 0.01 nM and approximately 8 nM, or between
approximately 0.01 nM and approximately 9 nM, or between
approximately 0.6 nM and approximately 1.1 nM, or between
approximately 0.7 nM and approximately 1.2 nM, or between
approximately 0.5 and approximately 5 nM. In other specific
embodiments, an anti-TNF-.alpha. antibody binds to TNF-.alpha. with
a K.sub.D (k.sub.off/k.sub.on) of about 5 nM, about 3.5 nM, about
1.5 nM, about 1 nM, about 0.5 nM, about 0.1 nM, about 0.05 nM or
about 0.01 nM. In specific embodiments, the K.sub.D
(k.sub.off/k.sub.on) value is determined by assays well known in
the art or described herein, e.g., ELISA, isothermal titration
calorimetry (ITC), BIAcore, or fluorescent polarization assay.
[0126] In some embodiments, an anti-TNF-.alpha. antibody of the
disclosure binds to TNF-.alpha. and inhibits the binding of
TNF-.alpha. to p55, p75 or both at an IC.sub.50 value of less than
5.times.10.sup.7 nM, less than 10.sup.7 nM, less than
5.times.10.sup.6 nM, less than 10.sup.6 nM, less than
5.times.10.sup.5 nM, less than 10.sup.5 nM, less than
5.times.10.sup.4 nM, less than 10.sup.4 nM, less than
5.times.10.sup.3 nM, less than 10.sup.3 nM, less than
5.times.10.sup.2 nM, less than 100 nM, less than 90 nM, less than
80 nM, less than 70 nM, 65 nM, less than 60 nM, less than 50 nM,
less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM,
less than 15 nM, less than 12 nM, less than 10 nM, less than 5 nM,
less than 1 nM, less than 5.times.10.sup.-1 nM, less than 10.sup.-1
nM, less than 5.times.10.sup.-2 nM, less than 10.sup.-2 nM, less
than 5.times.10.sup.-3 nM, less than 10.sup.-3 nM, less than
5.times.10.sup.-4 nM, or less than 10.sup.-4 nM, or with an
IC.sub.50 of any range between any pair of the foregoing values
(e.g., 5.times.10.sup.7 to 50 nM, or 15 nM to 5.times.10.sup.-3
nM). IC.sub.50 can be measured according to methods well known in
the art or described herein, e.g., ELISA.
[0127] In other embodiments, an anti-TNF-.alpha. antibody of the
disclosure binds to TNF-.alpha. and neutralizes TNF-.alpha. at an
IC.sub.50 value of less than 5.times.10.sup.7 nM, less than
10.sup.7 nM, less than 5.times.10.sup.6 nM, less than 10.sup.6 nM,
less than 5.times.10.sup.5 nM, less than 10.sup.5 nM, less than
5.times.10.sup.4 nM, less than 10.sup.4 nM, less than
5.times.10.sup.3 nM, less than 10.sup.3 nM, less than
5.times.10.sup.2 nM, less than 100 nM, less than 90 nM, less than
80 nM, less than 70 nM, 65 nM, less than 60 nM, less than 50 nM,
less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM,
less than 15 nM, less than 12 nM, less than 10 nM, less than 5 nM,
less than 1 nM, less than 5.times.10.sup.-1 nM, less than 10.sup.-1
nM, less than 5.times.10.sup.-2 nM, less than 10.sup.-2 nM, less
than 5.times.10.sup.-3 nM, less than 10.sup.-3 nM, less than
5.times.10.sup.-4 nM, or less than 10.sup.-4 nM, or with an
IC.sub.50 of any range between any pair of the foregoing values
(e.g., 5.times.10.sup.7 to 50 nM, or 15 nM to 5.times.10.sup.-3
nM). An exemplary neutralization assay that can be used to measure
the IC.sub.50 of an anti-TNF-.alpha. antibody is described in
Section 7.5 below.
[0128] In certain specific embodiments, an anti-TNF-.alpha.
antibody binds to TNF-.alpha. and inhibits the binding of
TNF-.alpha. to p55, p75 or both, or inhibits TNF-.alpha. activity
in a TNF-.alpha. neutralization assay, at an IC.sub.50 value of
between approximately 1 nM and approximately 10 nM, between
approximately 1 nM and approximately 15 nM, between approximately 1
nM and approximately 20 nM, between approximately 1 nM and
approximately 25 nM, between approximately 1 nM and approximately
30 nM, between approximately 1 nM and approximately 40 nM, between
approximately 1 nM and approximately 50 nM, between approximately
10 nM and approximately 10.sup.2 nM, between approximately 10.sup.2
nM and approximately 10.sup.3 nM, between approximately 10 nM and
approximately 10.sup.4 nM, between approximately 10.sup.4 nM and
approximately 10.sup.5 nM, between approximately 10.sup.5 nM and
approximately 10.sup.6 nM, or between approximately 10.sup.6 nM and
approximately 10.sup.7 nM.
[0129] In other specific embodiments, an anti-TNF-.alpha. antibody
binds to TNF-.alpha. and inhibits the binding of TNF-.alpha. to
p55, p75 or both, or inhibits TNF-.alpha. activity in a TNF-.alpha.
neutralization assay, at an IC.sub.50 value of between
approximately 5 nM and approximately 10 nM, between approximately 5
nM and approximately 15 nM, between approximately 10 nM and
approximately 15 nM, between approximately 10 nM and approximately
20 nM, between approximately 10 nM and approximately 30 nM, between
approximately 10 nM and approximately 40 nM, between approximately
10 nM and approximately 50 nM, between approximately 1 nM and
approximately 100 nM, between approximately 10 nM and approximately
100 nM, between approximately 20 nM and approximately 100 nM,
between approximately 30 nM and approximately 100 nM, between
approximately 40 nM and approximately 100 nM, between approximately
50 nM and approximately 100 nM, between approximately 15 nM and
approximately 25 nM, or between approximately 15 nM and
approximately 20 nM.
[0130] In certain aspects of the foregoing embodiments, the
IC.sub.50 is measured in the presence of TNF-.alpha. at a
concentration of 0.001 .mu.M, 0.005 .mu.M, 0.01 .mu.M, 0.05 .mu.M,
0.1 .mu.M, 0.5 .mu.M, 1 .mu.M, 10 .mu.M, 20 .mu.M, 30 .mu.M, 40
.mu.M, 50 .mu.M, 60 .mu.M, 70 .mu.M, 80 .mu.M, 90 .mu.M, 100 .mu.M,
200 .mu.M, 300 .mu.M, 400 .mu.M, 500 .mu.M, 600 .mu.M, 700 .mu.M,
800 .mu.M, 900 .mu.M, 1000 .mu.M or at a concentration of any range
between any pair of the foregoing values (e.g., 0.01 to 50 .mu.M,
or 10 .mu.M to 100 .mu.M).
[0131] In certain embodiments, the kinetic properties of an
antibody of the disclosure are comparable to, or improved relative
to, the D2E7 antibody in a comparable assay. For example, in
certain embodiments, an anti-TNF-.alpha. antibody of the disclosure
binds to TNF-.alpha. with a k.sub.on rate ranging from 0.2.times.
to 5.times. of the k.sub.on of D2E7, for example a k.sub.on of
0.2.times. of the k.sub.on of D2E7, a k.sub.on of 0.3.times. of the
k.sub.on of D2E7, a k.sub.on of 0.4.times. of the k.sub.on of D2E7,
a k.sub.on of 0.5.times. of the k.sub.on of D2E7, a k.sub.on of
0.6.times. of the k.sub.on of D2E7, a k.sub.on of 0.7.times. of the
k.sub.on of D2E7, a k.sub.on of 0.8.times. of the k.sub.on of D2E7,
a k.sub.on of 0.9.times. of the k.sub.on of D2E7, a k.sub.on of
1.times. of the k.sub.on of D2E7, a k.sub.on of 1.1.times. of the
k.sub.on of D2E7, a k.sub.on of 1.2.times. of the k.sub.on of D2E7,
a k.sub.on of 1.3.times. of the k.sub.on of D2E7, a k.sub.on of
1.4.times. of the k.sub.on of D2E7, a k.sub.on of 1.5.times. of the
k.sub.on of D2E7, a k.sub.on of 1.75.times. of the k.sub.on of
D2E7, a k.sub.on of 2.times. of the k.sub.on of D2E7, a k.sub.on of
2.25.times. of the k.sub.on of D2E7, a k.sub.on of 2.5.times. of
the k.sub.on of D2E7, a k.sub.on of 2.75.times. of the k.sub.on of
D2E7, a k.sub.on of 3.times. of the k.sub.on of D2E7, a k.sub.on of
3.5.times. of the k.sub.on of D2E7, a k.sub.on of 4.times. of the
k.sub.on of D2E7, a k.sub.on of 4.5.times. of the k.sub.on of D2E7,
a k.sub.on of 5.times. of the k.sub.on of D2E7, or a k.sub.on
ranging between any pair of the foregoing values, e.g., a k.sub.on
of 0.7.times.-1.5.times. of the k.sub.on of D2E7, a k.sub.on of
0.9.times.-1.3.times. of the k.sub.on of D2E7, a k.sub.on of
0.8.times.-2.times. of the k.sub.on of D2E7, a k.sub.on of
0.9.times.-3.times. of the k.sub.on of D2E7, etc.
[0132] In embodiments, an anti-TNF-.alpha. antibody of the
disclosure binds to TNF-.alpha. with a k.sub.off rate ranging from
0.2.times. to 5.times. of the k.sub.off of D2E7, for example a
k.sub.off of 0.2.times. of the k.sub.off of D2E7, a k.sub.off of
0.3.times. of the k.sub.off of D2E7, a k.sub.off of 0.4.times. of
the k.sub.off of D2E7, a k.sub.off of 0.5.times. of the k.sub.off
of D2E7, a k.sub.off of 0.6.times. of the k.sub.off of D2E7, a
k.sub.off of 0.7.times. of the k.sub.off of D2E7, a k.sub.off of
0.8.times. of the k.sub.off of D2E7, a k.sub.off of 0.9.times. of
the k.sub.off of D2E7, a k.sub.off of 1.times. of the k.sub.off of
D2E7, a k.sub.off of 1.1.times. of the k.sub.off of D2E7, a
k.sub.off of 1.2.times. of the k.sub.off of D2E7, a k.sub.off of
1.3.times. of the k.sub.off of D2E7, a k.sub.off of 1.4.times. of
the k.sub.off of D2E7, a k.sub.off of 1.5.times. of the k.sub.off
of D2E7, a k.sub.off of 1.75.times. of the k.sub.off of D2E7, a
k.sub.off of 2.times. of the k.sub.off of D2E7, a k.sub.off of
2.25.times. of the k.sub.off of D2E7, a k.sub.off of 2.5.times. of
the k.sub.off of D2E7, a k.sub.off of 2.75.times. of the k.sub.off
of D2E7, a k.sub.off of 3.times. of the k.sub.off of D2E7, a
k.sub.off of 3.5.times. of the k.sub.off of D2E7, a k.sub.off of
4.times. of the k.sub.off of D2E7, a k.sub.off of 4.5.times. of the
k.sub.off of D2E7, a k.sub.off of 5.times. of the k.sub.off of
D2E7, or a k.sub.off ranging between any pair of the foregoing
values, e.g., a k.sub.off of 0.7.times.-1.5.times. of the k.sub.off
of D2E7, a k.sub.off of 0.9.times.-1.3.times. of the k.sub.off of
D2E7, a k.sub.off of 0.8.times.-2.times. of the k.sub.off of D2E7,
a k.sub.off of 0.9.times.-3.times. of the k.sub.off of D2E7,
etc.
[0133] In other embodiments, an anti-TNF-.alpha. antibody of the
disclosure binds to TNF-.alpha. with a K.sub.A (k.sub.on/k.sub.off)
ranging from 0.04.times. to 25.times. of the K.sub.A of D2E7, for
example a K.sub.A of 0.04.times. of the K.sub.A of D2E7, a K.sub.A
of 0.1.times. of the K.sub.A of D2E7, a K.sub.A of 0.25.times. of
the K.sub.A of D2E7, a K.sub.A of 0.5.times. of the K.sub.A of
D2E7, a K.sub.A of 0.6.times. of the K.sub.A of D2E7, a K.sub.A of
0.7.times. of the K.sub.A of D2E7, a K.sub.A of 0.8.times. of the
K.sub.A of D2E7, a K.sub.A of 0.9.times. of the K.sub.A of D2E7, a
K.sub.A of 1.times. of the K.sub.A of D2E7, a K.sub.A of 1.1.times.
of the K.sub.A of D2E7, a K.sub.A of 1.25.times. of the K.sub.A of
D2E7, a K.sub.A of 1.5.times. of the K.sub.A of D2E7, a K.sub.A of
1.75.times. of the K.sub.A of D2E7, a K.sub.A of 2.times. of the
K.sub.A of D2E7, a K.sub.A of 2.5.times. of the K.sub.A of D2E7, a
K.sub.A of 3.times. of the K.sub.A of D2E7, a K.sub.A of 4.times.
of the K.sub.A of D2E7, a K.sub.A of 4.times.% of the K.sub.A of
D2E7, a K.sub.A of 5.times. of the K.sub.A of D2E7, a K.sub.A of
7.5.times. of the K.sub.A of D2E7, a K.sub.A of 10.times. of the
K.sub.A of D2E7, a K.sub.A of 12.5.times. of the K.sub.A of D2E7, a
K.sub.A of 15.times. of the K.sub.A of D2E7, a K.sub.A of 20.times.
of the K.sub.A of D2E7, a K.sub.A of 25.times. of the K.sub.A of
D2E7, or a K.sub.A ranging between any pair of the foregoing
values, e.g., a K.sub.A of 0.7.times.-1.25.times. of the K.sub.A of
D2E7, a K.sub.A of 0.9.times.-1.5.times. of the K.sub.A of D2E7, a
K.sub.A of 0.9.times.-2.times. of the K.sub.A of D2E7, a K.sub.A of
0.8.times.-1.75.times. of the K.sub.A of D2E7, a K.sub.A of
0.9.times.-5.times. of the K.sub.A of D2E7, or any value or range
that can be calculated from the k.sub.on and k.sub.off rates
disclosed herein.
[0134] In other embodiments, an anti-TNF-.alpha. antibody of the
disclosure binds to TNF-.alpha. a K.sub.D (k.sub.off/k.sub.on)
ranging from ranging from 0.04.times. to 25.times. of the K.sub.D
of D2E7, for example a K.sub.D of 0.04.times. of the K.sub.D of
D2E7, a K.sub.D of 0.1.times. of the K.sub.D of D2E7, a K.sub.D of
0.25.times. of the K.sub.D of D2E7, a K.sub.D of 0.5.times. of the
K.sub.D of D2E7, a K.sub.D of 0.6.times. of the K.sub.D of D2E7, a
K.sub.D of 0.7.times. of the K.sub.D of D2E7, a K.sub.D of
0.8.times. of the K.sub.D of D2E7, a K.sub.D of 0.9.times. of the
K.sub.D of D2E7, a K.sub.D of 1.times. of the K.sub.D of D2E7, a
K.sub.D of 1.1.times. of the K.sub.D of D2E7, a K.sub.D of
1.25.times. of the K.sub.D of D2E7, a K.sub.D of 1.5.times. of the
K.sub.D of D2E7, a K.sub.D of 1.75.times. of the K.sub.D of D2E7, a
K.sub.D of 2.times. of the K.sub.D of D2E7, a K.sub.D of 2.5.times.
of the K.sub.D of D2E7, a K.sub.D of 3.times. of the K.sub.D of
D2E7, a K.sub.D of 4.times. of the K.sub.D of D2E7, a K.sub.D of
4.times.% of the K.sub.D of D2E7, a K.sub.D of 5.times. of the
K.sub.D of D2E7, a K.sub.D of 7.5.times. of the K.sub.D of D2E7, a
K.sub.D of 10.times. of the K.sub.D of D2E7, a K.sub.D of
12.5.times. of the K.sub.D of D2E7, a K.sub.D of 15.times. of the
K.sub.D of D2E7, a K.sub.D of 20.times. of the K.sub.D of D2E7, a
K.sub.D of 25.times. of the K.sub.D of D2E7, or a K.sub.D ranging
between any pair of the foregoing values, e.g., a K.sub.D of
0.7.times.-1.25.times. of the K.sub.D of D2E7, a K.sub.D of
0.9.times.-1.5.times. of the K.sub.D of D2E7, a K.sub.D of
0.9.times.-2.times. of the K.sub.D of D2E7, a K.sub.D of
0.8.times.-1.75.times. of the K.sub.D of D2E7, a K.sub.D of
0.9.times.-5.times. of the K.sub.D of D2E7, or any value or range
that can be calculated from the k.sub.on and k.sub.off rates
disclosed herein.
[0135] In some embodiments, an anti-TNF-.alpha. antibody of the
disclosure binds to TNF-.alpha. and inhibits the binding of
TNF-.alpha. to p55, p75 or both at an IC.sub.50 value ranging from
50% to 200% of the IC.sub.50 of D2E7, for example an IC.sub.50 of
50% of the IC.sub.50 of D2E7, an IC.sub.50 of 60% of the IC.sub.50
of D2E7, an IC.sub.50 of 70% of the IC.sub.50 of D2E7, an IC.sub.50
of 75% of the IC.sub.50 of D2E7, an IC.sub.50 of 80% of the
IC.sub.50 of D2E7, an IC.sub.50 of 90% of the IC.sub.50 of D2E7, an
IC.sub.50 of 95% of the IC.sub.50 of D2E7, an IC.sub.50 of 100% of
the IC.sub.50 of D2E7, an IC.sub.50 of 110% of the IC.sub.50 of
D2E7, an IC.sub.50 of 120% of the IC.sub.50 of D2E7, an IC.sub.50
of 125% of the IC.sub.50 of D2E7, an IC.sub.50 of 130% of the
IC.sub.50 of D2E7, an IC.sub.50 of 140% of the IC.sub.50 of D2E7,
an IC.sub.50 of 150% of the IC.sub.50 of D2E7, an IC.sub.50 of 160%
of the IC.sub.50 of D2E7, an IC.sub.50 of 170% of the IC.sub.50 of
D2E7, an IC.sub.50 of 175% of the IC.sub.50 of D2E7, an IC.sub.50
of 180% of the IC.sub.50 of D2E7, an IC.sub.50 of 190% of the
IC.sub.50 of D2E7, an IC.sub.50 of 200% of the IC.sub.50 of D2E7,
or an IC.sub.50 of any range between any pair of the foregoing
values, e.g., an IC.sub.50 of 75%-125% of the IC.sub.50 of D2E7, an
IC.sub.50 of 90%-130% of the IC.sub.50 of D2E7, an IC.sub.50 of
95%-125% of the IC.sub.50 of D2E7, an IC.sub.50 of 90%-110% of the
IC.sub.50 of D2E7, an IC.sub.50 of 90%-180% of the IC.sub.50 of
D2E7, or an IC.sub.50 of 80%-175% of the IC.sub.50 of D2E7. In
other embodiments, a single CDR substitution can result in the
foregoing differences in IC.sub.50 as compared to D2E7, whereas an
anti-TNF-.alpha. antibody of the disclosure can comprise such
substitution and up to 16 additional substitutions as compared to
D2E7.
[0136] In other embodiments, an anti-TNF-.alpha. antibody of the
disclosure binds to TNF-.alpha. and neutralizes TNF-.alpha. at an
IC.sub.50 value ranging from 50% to 200% of the IC.sub.50 of D2E7,
for example an IC.sub.50 of 50% of the IC.sub.50 of D2E7, an
IC.sub.50 of 60% of the IC.sub.50 of D2E7, an IC.sub.50 of 70% of
the IC.sub.50 of D2E7, an IC.sub.50 of 75% of the IC.sub.50 of
D2E7, an IC.sub.50 of 80% of the IC.sub.50 of D2E7, an IC.sub.50 of
90% of the IC.sub.50 of D2E7, an IC.sub.50 of 95% of the IC.sub.50
of D2E7, an IC.sub.50 of 100% of the IC.sub.50 of D2E7, an
IC.sub.50 of 110% of the IC.sub.50 of D2E7, an IC.sub.50 of 120% of
the IC.sub.50 of D2E7, an IC.sub.50 of 125% of the IC.sub.50 of
D2E7, an IC.sub.50 of 130% of the IC.sub.50 of D2E7, an IC.sub.50
of 140% of the IC.sub.50 of D2E7, an IC.sub.50 of 150% of the
IC.sub.50 of D2E7, an IC.sub.50 of 160% of the IC.sub.50 of D2E7,
an IC.sub.50 of 170% of the IC.sub.50 of D2E7, an IC.sub.50 of 175%
of the IC.sub.50 of D2E7, an IC.sub.50 of 180% of the IC.sub.50 of
D2E7, an IC.sub.50 of 190% of the IC.sub.50 of D2E7, an IC.sub.50
of 200% of the IC.sub.50 of D2E7, or an IC.sub.50 of any range
between any pair of the foregoing values, e.g., an IC.sub.50 of
75%-125% of the IC.sub.50 of D2E7, an IC.sub.50 of 90%-130% of the
IC.sub.50 of D2E7, an IC.sub.50 of 95%-125% of the IC.sub.50 of
D2E7, an IC.sub.50 of 90%-110% of the IC.sub.50 of D2E7, an
IC.sub.50 of 90%-180% of the IC.sub.50 of D2E7, or an IC.sub.50 of
80%-175% of the IC.sub.50 of D2E7. In other embodiments, a single
CDR substitution can result in the foregoing differences in
IC.sub.50 as compared to D2E7, whereas an anti-TNF-.alpha. antibody
of the disclosure can comprise such substitution and up to 16
additional substitutions as compared to D2E7.
[0137] 7.5 Reduced Immunogenicity of Anti-TNF-.alpha.
Antibodies
[0138] In certain aspects, the present disclosure provides
anti-TNF-.alpha. antibodies having reduced immunogenicity as
compared to D2E7. The present disclosure also provides
anti-TNF-.alpha. antibodies having multiple amino acid
substitutions in their CDRs as compared to the CDRs of D2E7,
wherein at least one substitution reduces the immunogenicity of the
antibody as compared to D2E7. In certain embodiments, the reduced
immunogenicity results from one or more amino acid substitutions
that result in eliminating or mitigating one or more T cell
epitopes.
[0139] In certain aspects, the anti-TNF-.alpha. antibodies of the
disclosure having reduced immunogenicity have comparable or
improved biological activity as compared to D2E7, e.g., affinity
towards TNF-.alpha. or neutralization of TNF-.alpha. activity. Such
properties can be tested, for example, by the methods described in
Section 7.3 above.
[0140] In certain embodiments, the immunogenicity of an TNF-.alpha.
antibody of the disclosure is reduced relative to D2E7 antibody.
Such antibodies generally have variant sequences relative to the
heavy and/or light chain variable region in regions corresponding
to SEQ ID NO:81 and/or SEQ ID NO:82, and/or SEQ ID NO:83. The
antibodies will generally have one, two or three amino acid
substitutions in one, two or all three sequences corresponding to
SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:83, although up to four
or five substitutions one, two or all three regions are
contemplated herein.
[0141] Exemplary CDR-L1 substitutions yielding antibodies with
lower immunogenicity as compared to D2E7 are listed in Table 11.
Antibodies of the disclosure can comprise any of the substitutions
or combinations of substitutions listed in Table 11, and,
optionally, one or more additional substitutions, such as the CDR
mutations in any of Tables 12-25, singly or in combination.
[0142] As used in the present disclosure, the term "reduced
immunogenicity" indicates that the variant sequence as compared to
SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83 elicits a reduced
proliferative response in peripheral blood mononuclear cells as
compared to a peptide of SEQ ID NO:81, SEQ ID NO:82, or SEQ ID
NO:83, respectively. An exemplary proliferation assay that can be
used to evaluate the proliferative response is set forth in Section
8 below. The reduced proliferative response can be reflected in
terms of the percentage of responders, the stimulation index, or
both.
[0143] In other embodiments, as compared to a peptide having the
sequence of SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO;83, the
variant sequence results in at least 25% fewer responders, in at
least 30% fewer responders, in at least 35% fewer responders, in at
least 40% fewer responders, in at least 45% fewer responders, in at
least 50% fewer responders, in at least 60% fewer responders, in at
least 65% fewer responders, in at least 70% fewer responders, in at
least 75% fewer responders, in at least 80% fewer responders, in at
least 85% fewer responders, in at least 90% fewer responders, in at
least 95% fewer responders, 100% fewer responders, or a reduction
in responders in a range between any of the foregoing values, e.g.,
25%-75% fewer responders, 50%-90% fewer responders, 60%-100% fewer
responders, 70%-90% fewer responders, or the like.
[0144] In other embodiments, the variant sequence results in a
stimulation index that is at least 5% less, at least 10% less, at
least 15% less, at least 20% less, at least 25% less, at least 30%
less, at least 35% less, or at least 40% less than the stimulation
index elicited by a peptide of SEQ ID NO:81, SEQ ID NO:82, or SEQ
ID NO;83, respectively, or results in a stimulation index reduced
by a range between any of the foregoing values as compared to a
peptide of SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO;83, e.g.,
5%-20% less, 10%-30% less, 25%-35% less, 30%-40% less, or the
like.
[0145] Exemplary embodiments of anti-TNF-.alpha. antibodies with
reduced immunogenicity as compared to D2E7 comprise one or more of
the CDR substitutions or combinations of substitutions set forth in
Table 11.
[0146] 7.6 Antibody Conjugates
[0147] The anti-TNF-.alpha. antibodies of the disclosure include
antibody conjugates that are modified, e.g., by the covalent
attachment of any type of molecule to the antibody, such that
covalent attachment does not interfere with binding to
TNF-.alpha..
[0148] In certain aspects, an anti-TNF-.alpha. antibody of the
disclosure can be conjugated to an effector moiety or a label. The
term "effector moiety" as used herein includes, for example,
antineoplastic agents, drugs, toxins, biologically active proteins,
for example enzymes, other antibody or antibody fragments,
synthetic or naturally occurring polymers, nucleic acids (e.g., DNA
and RNA), radionuclides, particularly radioiodide, radioisotopes,
chelated metals, nanoparticles and reporter groups such as
fluorescent compounds or compounds which can be detected by NMR or
ESR spectroscopy.
[0149] In one example, anti-TNF-.alpha. antibodies can be
conjugated to an effector moiety, such as a cytotoxic agent, a
radionuclide or drug moiety to modify a given biological response.
The effector moiety can be a protein or polypeptide, such as, for
example and without limitation, a toxin (such as abrin, ricin A,
Pseudomonas exotoxin, or Diphtheria toxin), a signaling molecule
(such as .alpha.-interferon, .beta.-interferon, nerve growth
factor, platelet derived growth factor or tissue plasminogen
activator), a thrombotic agent or an anti-angiogenic agent (e.g.,
angiostatin or endostatin) or a biological response modifier such
as a cytokine or growth factor (e.g., interleukin-1 (IL-1),
interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage
colony stimulating factor (GM-CSF), granulocyte colony stimulating
factor (G-CSF), or nerve growth factor (NGF)).
[0150] In another example the effector moieties can be cytotoxins
or cytotoxic agents. Examples of cytotoxins and cytotoxic agents
include taxol, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorabicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof.
[0151] Effector moieties also include, but are not limited to,
antimetabolites (e.g. methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C5 and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, anthramycin (AMC), calicheamicins or duocarmycins),
and anti-mitotic agents (e.g., vincristine and vinblastine).
[0152] Other effector moieties can include radionuclides such as,
but not limited to, .sup.111In and .sup.90Y, Lu.sup.177,
Bismuth.sup.213, Californium.sup.252, Iridium.sup.192 and
Tungsten.sup.188/Rhenium.sup.188 and drugs such as, but not limited
to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and
suramin.
[0153] Techniques for conjugating such effector moieties to
antibodies are well known in the art (See, e.g., Hellstrom et al.,
Controlled Drug Delivery, 2nd Ed., at pp. 623-53 (Robinson et al.,
eds., 1987)); Thorpe et al., 1982, Immunol. Rev. 62:119-58 and
Dubowchik et al., 1999, Pharmacology and Therapeutics
83:67-123).
[0154] In one example, the antibody or fragment thereof is fused
via a covalent bond (e.g., a peptide bond), through the antibody's
N-terminus or the C-terminus or internally, to an amino acid
sequence of another protein (or portion thereof; for example, at
least a 10, 20 or 50 amino acid portion of the protein). The
antibody, or fragment thereof, can linked to the other protein at
the N-terminus of the constant domain of the antibody. Recombinant
DNA procedures can be used to create such fusions, for example as
described in WO 86/01533 and EP0392745. In another example the
effector molecule can increase half-life in vivo, and/or enhance
the delivery of an antibody across an epithelial barrier to the
immune system. Examples of suitable effector molecules of this type
include polymers, albumin, albumin binding proteins or albumin
binding compounds such as those described in WO 2005/117984.
[0155] In certain aspects, an anti-TNF-.alpha. antibody is
conjugated to a small molecule toxin. In certain exemplary
embodiments, an anti-TNF-.alpha. antibody of the disclosure is
conjugated to a dolastatin or a dolostatin peptidic analogs or
derivatives, e.g., an auristatin (U.S. Pat. Nos. 5,635,483 and
5,780,588). The dolastatin or auristatin drug moiety may be
attached to the antibody through its N (amino) terminus, C
(carboxyl) terminus or internally (WO 02/088172). Exemplary
auristatin embodiments include the N-terminus linked
monomethylauristatin drug moieties DE and DF, as disclosed in U.S.
Pat. No. 7,498,298, which is hereby incorporated by reference in
its entirety (disclosing, e.g., linkers and methods of preparing
monomethylvaline compounds such as MMAE and MMAF conjugated to
linkers).
[0156] In other exemplary embodiments, small molecule toxins
include but are not limited to calicheamicin, maytansine (U.S. Pat.
No. 5,208,020), trichothene, and CC1065. In one embodiment of the
disclosure, the antibody is conjugated to one or more maytansine
molecules (e.g., about 1 to about 10 maytansine molecules per
antibody molecule). Maytansine may, for example, be converted to
May-SS-Me which may be reduced to May-SH3 and reacted with an
antibody (Chari et al., 1992, Cancer Research 52: 127-131) to
generate a maytansinoid-antibody or maytansinoid-Fc fusion
conjugate. Structural analogues of calicheamicin that can also be
used include but are not limited to .gamma..sub.1.sup.1,
.gamma..sub.3.sup.1, .gamma..sub.3.sup.1
N-acetyl-.gamma..sub.1.sup.1, PSAG, and .theta..sub.1.sup.1,
(Hinman et al., 1993, Cancer Research 53:3336-3342; Lode et al.,
1998, Cancer Research 58:2925-2928; U.S. Pat. No. 5,714,586; U.S.
Pat. No. 5,712,374; U.S. Pat. No. 5,264,586; U.S. Pat. No.
5,773,001).
[0157] Antibodies of the disclosure can also be conjugated to
liposomes for targeted delivery (See, e.g., Park et al., 1997, Adv.
Pharmacol. 40:399-435; Marty & Schwendener, 2004, Methods in
Molecular Medicine 109:389-401).
[0158] In one example antibodies of the present disclosure can be
attached to poly(ethyleneglycol) (PEG) moieties. In one particular
example the antibody is an antibody fragment and the PEG moieties
can be attached through any available amino acid side-chain or
terminal amino acid functional group located in the antibody
fragment, for example any free amino, imino, thiol, hydroxyl or
carboxyl group. Such amino acids can occur naturally in the
antibody fragment or can be engineered into the fragment using
recombinant DNA methods. See, for example, U.S. Pat. No. 5,219,996.
Multiple sites can be used to attach two or more PEG molecules. PEG
moieties can be covalently linked through a thiol group of at least
one cysteine residue located in the antibody fragment. Where a
thiol group is used as the point of attachment, appropriately
activated effector moieties (for example, thiol selective
derivatives such as maleimides and cysteine derivatives) can be
used.
[0159] In a specific example, an anti-TNF-.alpha. antibody
conjugate is a modified Fab' fragment which is PEGylated, i.e., has
PEG (poly(ethyleneglycol)) covalently attached thereto, e.g.,
according to the method disclosed in EP0948544. See also
Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical
Applications, (J. Milton Harris (ed.), Plenum Press, New York,
1992); Poly(ethyleneglycol) Chemistry and Biological Applications,
(J. Milton Harris and S. Zalipsky, eds., American Chemical Society,
Washington D.C., 1997); and Bioconjugation Protein Coupling
Techniques for the Biomedical Sciences, (M. Aslam and A. Dent,
eds., Grove Publishers, New York, 1998); and Chapman, 2002,
Advanced Drug Delivery Reviews 54:531-545. PEG can be attached to a
cysteine in the hinge region. In one example, a PEG-modified Fab'
fragment has a maleimide group covalently linked to a single thiol
group in a modified hinge region. A lysine residue can be
covalently linked to the maleimide group and to each of the amine
groups on the lysine residue can be attached a
methoxypoly(ethyleneglycol) polymer having a molecular weight of
approximately 20,000 Da. The total molecular weight of the PEG
attached to the Fab' fragment can therefore be approximately 40,000
Da.
[0160] The word "label" when used herein refers to a detectable
compound or composition which can be conjugated directly or
indirectly to an anti-TNF-.alpha. antibody of the disclosure. The
label can itself be detectable (e.g., radioisotope labels or
fluorescent labels) or, in the case of an enzymatic label, can
catalyze chemical alteration of a substrate compound or composition
which is detectable. Useful fluorescent moieties include, but are
not limited to, fluorescein, fluorescein isothiocyanate, rhodamine,
5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and
the like. Useful enzymatic labels include, but are not limited to,
alkaline phosphatase, horseradish peroxidase, glucose oxidase and
the like.
[0161] Additional anti-TNF-.alpha. antibody conjugates that are
useful for, inter alia, diagnostic purposes, are described in
Section 7.7 below.
[0162] 7.7 Diagnostic Uses of Anti-TNF-.alpha. Antibodies
[0163] The anti-TNF-.alpha. antibodies of the disclosure, including
those antibodies that have been modified, e.g., by biotinylation,
horseradish peroxidase, or any other detectable moiety (including
those described in Section 7.6), can be advantageously used for
diagnostic purposes.
[0164] In particular, the anti-TNF-.alpha. antibodies can be used,
for example, but not limited to, to purify or detect TNF-.alpha.,
including both in vitro and in vivo diagnostic methods. For
example, the antibodies have use in immunoassays for qualitatively
and quantitatively measuring levels of TNF-.alpha. in biological
samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual,
Second Edition (Cold Spring Harbor Laboratory Press, 1988), which
is incorporated by reference herein in its entirety. In one
embodiment, the anti-TNF-.alpha. antibodies of the disclosure can
be used for detecting and quantitating levels of TNF-.alpha. in the
serum.
[0165] The present disclosure further encompasses antibodies or
fragments thereof conjugated to a diagnostic agent. The antibodies
can be used diagnostically, for example, to detect expression of a
target of interest in specific cells, tissues, or serum; or to
monitor the development or progression of an immunologic response
as part of a clinical testing procedure to, e.g., determine the
efficacy of a given treatment regimen. Detection can be facilitated
by coupling the antibody to a detectable substance. Examples of
detectable substances include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent
materials, radioactive materials, positron emitting metals using
various positron emission tomographies, and nonradioactive
paramagnetic metal ions. The detectable substance can be coupled or
conjugated either directly to the antibody (or fragment thereof) or
indirectly, through an intermediate (such as, for example, a linker
known in the art) using techniques known in the art. Examples of
enzymatic labels include luciferases (e.g., firefly luciferase and
bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as horseradish peroxidase (HRPO), alkaline
phosphatase, f3-galactosidase, acetylcholinesterase, glucoamylase,
lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose
oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic
oxidases (such as uricase and xanthine oxidase), lactoperoxidase,
microperoxidase, and the like. 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; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.111In or .sup.99Tc.
[0166] The disclosure provides for the detection of expression of
TNF-.alpha., comprising contacting a biological sample (cells,
tissue, or body fluid of an individual) using one or more
anti-TNF-.alpha. antibodies of the disclosure (optionally
conjugated to detectable moiety), and detecting whether or not the
sample is positive for TNF-.alpha. expression, or whether the
sample has altered (e.g., reduced or increased) expression as
compared to a control sample.
[0167] Diseases that can be diagnosed using the present methods
include, but are not limited to, the diseases described herein. In
certain embodiments, the tissue or body fluid is peripheral blood,
peripheral blood leukocytes, biopsy tissues such as lung or skin
biopsies, and tissue.
7.8 Therapeutic Methods Using Anti-TNF-.alpha. Antibodies
[0168] 7.8.1 Clinical Benefits
[0169] The TNF-.alpha. antibodies of the present disclosure are
useful for treating disorders or symptoms of various immune and
autoimmune pathologies as well as inflammatory diseases.
TNF-.alpha.-related pathologies and diseases that can be treated
with the anti-TNF-.alpha. antibodies of the disclosure include, but
are not limited to, the following: [0170] Acute and chronic immune
and autoimmune pathologies, such as systemic lupus erythematosus,
rheumatoid arthritis, thyroidosis, graft versus host disease,
scleroderma, diabetes mellitus, Grave's disease, and the like;
[0171] Infections, including, but not limited to, sepsis syndrome,
cachexia, circulatory collapse and shock resulting from acute or
chronic bacterial infection, acute and chronic parasitic and/or
bacterial, viral or fungal infectious diseases, such as AIDS
(including sequelae such as cachexia, autoimmune disorders, AIDS
dementia complex and infections); [0172] Inflammatory diseases,
such as chronic inflammatory pathologies and vascular inflammatory
pathologies, including chronic inflammatory pathologies such as
sarcoidosis, chronic inflammatory bowel disease, ulcerative
colitis, and Crohn's pathology and vascular inflammatory
pathologies, such as, but not limited to, disseminated
intravascular coagulation, atherosclerosis, and Kawasaki's
pathology; [0173] Neurodegenerative diseases, including, but not
limited to, demyelinating diseases, such as multiple sclerosis and
acute transverse myelitis; extrapyramidal and cerebellar disorders'
such as lesions of the corticospinal system; disorders of the basal
ganglia or cerebellar disorders; hyperkinetic movement disorders
such as Huntington's Chorea and senile chorea, drug-induced
movement disorders, such as those induced by drugs which block the
CNS, dopamine receptors; hypokinetic movement disorders, such as
Parkinson's disease; Progressive supranucleo palsy, Cerebellar and
Spinocerebellar Disorders, such as astructural lesions of the
cerebellum; spinocerebellar degenerations (spinal ataxia,
Friedreich's ataxia, cerebellar cortical degenerations, multiple
systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and
Machado-Joseph); and systemic disorders (Refsum's disease,
abetalipoprotemia, ataxia, telangiectasia, and mitochondrial multi.
system disorder); demyelinating core disorders, such as multiple
sclerosis, acute transverse myelitis; disorders of the motor unit,
such as neurogenic muscular atrophies (anterior horn cell
degeneration, such as amyotrophic lateral sclerosis, infantile
spinal muscular atrophy and juvenile spinal muscular atrophy);
Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy
body disease; Senile Dementia of Lewy body type, Wernicke-Korsakoff
syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; subacute
sclerosing panencephalitis, Hallerrorden-Spatz disease,- and
Dementia pugilistica, or any subset thereof; [0174] Malignant
pathologies involving TNF-.alpha.secreting tumors or other
malignancies involving TNF-.alpha., such as, but not limited to
leukemias (acute, chronic myelocytic, chronic lymphocytic and/or
myelodyspastic syndrome); lymphomas (Hodgkin's and non-Hodgkin's
lymphomas, such as malignant lymphomas (Burkitt's lymphoma or
Mycosis fungoides), and [0175] Alcohol-induced hepatitis.
[0176] In certain specific embodiments, the antibodies of the
disclosure are used to treat one or more of: [0177] Moderate to
severe rheumatoid arthritis (RA) in adults. [0178] Moderate to
severe polyarticular juvenile idiopathic arthritis (JIA) in
children 4 years of age and older. [0179] Psoriatic arthritis (PsA)
in adults. [0180] Ankylosing spondylitis (AS) in adults. [0181]
Moderate to severe Crohn's disease (CD) in adults who have not
responded well to conventional treatments. [0182] Moderate to
severe chronic plaque psoriasis (Ps) in adults.
[0183] Accordingly, the present disclosure provides methods of
treating any of the foregoing diseases in a patient in need
thereof, comprising: administering to the patient an
anti-TNF-.alpha. antibody of the disclosure. Optionally, said
administration is repeated, e.g., after one day, two days, three
days, five days, one week, two weeks, three weeks, one month, five
weeks, six weeks, seven weeks, eight weeks, two months, or three
months. The repeated administration can be at the same dose or at a
different dose. The administration can be repeated once, twice,
three times, four times, five times, six times, seven times, eight
times, nine times, ten times, or more. For example, according to
certain dosage regimens a patient receives anti-TNF-.alpha. therapy
for a prolonged period of time, e.g., 6 months, 1 year or more. The
amount of anti-TNF-.alpha. antibody administered to the patient is
in certain embodiments a therapeutically effective amount. As used
herein, a "therapeutically effective" amount of TNF-.alpha.
antibody can be administered as a single dose or over the course of
a therapeutic regimen, e.g., over the course of a week, two weeks,
three weeks, one month, three months, six months, one year, or
longer. Exemplary therapeutic regimens are described in Section
7.11 below.
[0184] According to the present disclosure, treatment of a disease
encompasses the treatment of patients already diagnosed as having
any form of the disease at any clinical stage or manifestation; the
delay of the onset or evolution or aggravation or deterioration of
the symptoms or signs of the disease; and/or preventing and/or
reducing the severity of the disease.
[0185] A "subject" or "patient" to whom the anti-TNF-.alpha.
antibody of the disclosure is administered is preferably a mammal
such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.)
or a primate (e.g., monkey or human). In certain embodiments, the
subject or patient is a human. In certain aspects, the human is a
pediatric patient. In other aspects, the human is an adult
patient.
[0186] 7.9 Pharmaceutical Compositions and Routes of
Administration
[0187] Compositions comprising an anti-TNF-.alpha. antibody of the
disclosure and, optionally one or more additional therapeutic
agents, such as the combination therapeutic agents described in
Section 7.10 below, are provided herein. The compositions will
usually be supplied as part of a sterile, pharmaceutical
composition that will normally include a pharmaceutically
acceptable carrier. This composition can be in any suitable form
(depending upon the desired method of administering it to a
patient).
[0188] The anti-TNF-.alpha. antibodies of the disclosure can be
administered to a patient by a variety of routes such as orally,
transdermally, subcutaneously, intranasally, intravenously,
intramuscularly, intraocularly, topically, intrathecally and
intracerebroventricularly. The most suitable route for
administration in any given case will depend on the particular
antibody, the subject, and the nature and severity of the disease
and the physical condition of the subject.
[0189] For treatment of indications described herein, the effective
dose of an anti-TNF-.alpha. antibody of the disclosure can range
from about 0.001 to about 75 mg/kg per single (e.g., bolus)
administration, multiple administrations or continuous
administration, or to achieve a serum concentration of 0.01-5000
.mu.g/mL serum concentration per single (e.g., bolus)
administration, multiple administrations or continuous
administration, or any effective range or value therein depending
on the condition being treated, the route of administration and the
age, weight and condition of the subject. In a certain embodiment,
each dose can range from about 0.5 .mu.g to about 50 .mu.g per
kilogram of body weight, for example from about 3 .mu.g to about 30
.mu.g per kilogram body weight. The antibody can be formulated as
an aqueous solution and administered by subcutaneous injection.
[0190] Pharmaceutical compositions can be conveniently presented in
unit dose forms containing a predetermined amount of an
anti-TNF-.alpha. antibody of the disclosure per dose. Such a unit
can contain for example but without limitation 5 mg to 5 g, for
example 10 mg to 1 g, or 20 to 50 mg. Pharmaceutically acceptable
carriers for use in the disclosure can take a wide variety of forms
depending, e.g., on the condition to be treated or route of
administration.
[0191] Therapeutic formulations of the anti-TNF-.alpha. antibodies
of the disclosure can be prepared for storage as lyophilized
formulations or aqueous solutions by mixing the antibody having the
desired degree of purity with optional pharmaceutically-acceptable
carriers, excipients or stabilizers typically employed in the art
(all of which are referred to herein as "carriers"), i.e.,
buffering agents, stabilizing agents, preservatives, isotonifiers,
non-ionic detergents, antioxidants, and other miscellaneous
additives. See, Remington's Pharmaceutical Sciences, 16th edition
(Osol, ed. 1980). Such additives must be nontoxic to the recipients
at the dosages and concentrations employed.
[0192] Buffering agents help to maintain the pH in the range which
approximates physiological conditions. They can be present at
concentration ranging from about 2 mM to about 50 mM. Suitable
buffering agents for use with the present disclosure include both
organic and inorganic acids and salts thereof such as citrate
buffers (e.g., monosodium citrate-disodium citrate mixture, citric
acid-trisodium citrate mixture, citric acid-monosodium citrate
mixture, etc.), succinate buffers (e.g., succinic acid-monosodium
succinate mixture, succinic acid-sodium hydroxide mixture, succinic
acid-disodium succinate mixture, etc.), tartrate buffers (e.g.,
tartaric acid-sodium tartrate mixture, tartaric acid-potassium
tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.),
fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,
fumaric acid-disodium fumarate mixture, monosodium
fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g.,
gluconic acid-sodium glyconate mixture, gluconic acid-sodium
hydroxide mixture, gluconic acid-potassium glyuconate mixture,
etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture,
oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate
mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate
mixture, lactic acid-sodium hydroxide mixture, lactic
acid-potassium lactate mixture, etc.) and acetate buffers (e.g.,
acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide
mixture, etc.). Additionally, phosphate buffers, histidine buffers
and trimethylamine salts such as Tris can be used.
[0193] Preservatives can be added to retard microbial growth, and
can be added in amounts ranging from 0.2%-1% (w/v). Suitable
preservatives for use with the present disclosure include phenol,
benzyl alcohol, meta-cresol, methyl paraben, propyl paraben,
octadecyldimethylbenzyl ammonium chloride, benzalconium halides
(e.g., chloride, bromide, and iodide), hexamethonium chloride, and
alkyl parabens such as methyl or propyl paraben, catechol,
resorcinol, cyclohexanol, and 3-pentanol. Isotonicifiers sometimes
known as "stabilizers" can be added to ensure isotonicity of liquid
compositions of the present disclosure and include polhydric sugar
alcohols, for example trihydric or higher sugar alcohols, such as
glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
Stabilizers refer to a broad category of excipients which can range
in function from a bulking agent to an additive which solubilizes
the therapeutic agent or helps to prevent denaturation or adherence
to the container wall. Typical stabilizers can be polyhydric sugar
alcohols (enumerated above); amino acids such as arginine, lysine,
glycine, glutamine, asparagine, histidine, alanine, ornithine,
L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic
sugars or sugar alcohols, such as lactose, trehalose, stachyose,
mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol,
glycerol and the like, including cyclitols such as inositol;
polyethylene glycol; amino acid polymers; sulfur containing
reducing agents, such as urea, glutathione, thioctic acid, sodium
thioglycolate, thioglycerol, .alpha.-monothioglycerol and sodium
thio sulfate; low molecular weight polypeptides (e.g., peptides of
10 residues or fewer); proteins such as human serum albumin, bovine
serum albumin, gelatin or immunoglobulins; hydrophylic polymers,
such as polyvinylpyrrolidone monosaccharides, such as xylose,
mannose, fructose, glucose; disaccharides such as lactose, maltose,
sucrose and trisaccacharides such as raffinose; and polysaccharides
such as dextran. Stabilizers can be present in the range from 0.1
to 10,000 weights per part of weight active protein.
[0194] Non-ionic surfactants or detergents (also known as "wetting
agents") can be added to help solubilize the therapeutic agent as
well as to protect the therapeutic protein against
agitation-induced aggregation, which also permits the formulation
to be exposed to shear surface stressed without causing
denaturation of the protein. Suitable non-ionic surfactants include
polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), Pluronic
polyols, polyoxyethylene sorbitan monoethers (TWEEN.RTM.-20,
TWEEN.RTM.-80, etc.). Nonionic surfactants can be present in a
range of about 0.05 mg/mL to about 1.0 mg/mL, for example about
0.07 mg/mL to about 0.2 mg/mL.
[0195] Additional miscellaneous excipients include bulking agents
(e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g.,
ascorbic acid, methionine, vitamin E), and cosolvents. Further
formulations suitable for the anti-TNF-.alpha. antibodies of the
disclosure are disclosed in U.S. Pat. App. No. 2004/0033228 A1, the
contents of which are incorporated by reference herein in their
entirety.
[0196] The formulation herein can also contain a combination
therapeutic agent in addition to the anti-TNF-.alpha. antibody of
the disclosure. Examples of suitable combination therapeutic agents
are provided in Section 7.10 below.
[0197] The dosing schedule for subcutaneous administration can vary
from once every six months, five months, four months, three months,
two months, once a month to biweekly, weekly, or daily depending on
a number of clinical factors, including the type of disease,
severity of disease, and the patient's sensitivity to the
anti-TNF-.alpha. antibody.
[0198] The dosage of an anti-TNF-.alpha. antibody of the disclosure
to be administered of will vary according to the particular
antibody, the type of autoimmune or inflammatory disease, the
subject, and the nature and severity of the disease, the physical
condition of the subject, the therapeutic regimen (e.g., whether a
combination therapeutic agent is used), and the selected route of
administration; the appropriate dosage can be readily determined by
a person skilled in the art.
[0199] For the treatment and/or prophylaxis of autoimmune or
inflammatory disease in humans and animals, pharmaceutical
compositions comprising anti-TNF-.alpha. antibodies can be
administered to patients (e.g., human subjects) at therapeutically
or prophylactically effective dosages (e.g., dosages which result
in inhibition of an autoimmune or inflammatory disease and/or
relief of autoimmune or inflammatory disease symptoms) using any
suitable route of administration, such as injection and other
routes of administration known in the art for antibody-based
clinical products.
[0200] It will be recognized by one of skill in the art that the
optimal quantity and spacing of individual dosages of an
anti-TNF-.alpha. antibody of the disclosure will be determined by
the nature and extent of the condition being treated, the form,
route and site of administration, and the age and condition of the
particular subject being treated, and that a physician will
ultimately determine appropriate dosages to be used. This dosage
can be repeated as often as appropriate. If side effects develop
the amount and/or frequency of the dosage can be altered or
reduced, in accordance with normal clinical practice.
[0201] 7.10 Combination Therapy
[0202] Described below are combinatorial methods in which the
anti-TNF-.alpha. antibodies of the disclosure can be utilized. The
combinatorial methods of the disclosure involve the administration
of at least two agents to a patient, the first of which is an
anti-TNF-.alpha. antibody of the disclosure, and the additional
agent(s) is a combination therapeutic agent. The anti-TNF-.alpha.
antibody and the combination therapeutic agent(s) can be
administered simultaneously, sequentially or separately.
[0203] The combinatorial therapy methods of the present disclosure
can result in a greater than additive effect, providing therapeutic
benefits where neither the anti-TNF-.alpha. antibody or combination
therapeutic agent administered in an amount that is alone
therapeutically effective.
[0204] In the present methods, the anti-TNF-.alpha. antibody of the
disclosure and the combination therapeutic agent can be
administered concurrently, either simultaneously or successively.
As used herein, the anti-TNF-.alpha. antibody of the disclosure and
the combination therapeutic agent are said to be administered
successively if they are administered to the patient on the same
day, for example during the same patient visit. Successive
administration can occur 1, 2, 3, 4, 5, 6, 7 or 8 hours apart. In
contrast, the anti-TNF-.alpha. antibody of the disclosure and the
combination therapeutic agent are said to be administered
separately if they are administered to the patient on the different
days, for example, the anti-TNF-.alpha. antibody of the disclosure
and the combination therapeutic agent can be administered at a
1-day, 2-day or 3-day, one-week, 2-week or monthly intervals. In
the methods of the present disclosure, administration of the
anti-TNF-.alpha. antibody of the disclosure can precede or follow
administration of the combination therapeutic agent.
[0205] As a non-limiting example, the anti-TNF-.alpha. antibody of
the disclosure and combination therapeutic agent can be
administered concurrently for a period of time, followed by a
second period of time in which the administration of the
anti-TNF-.alpha. antibody of the disclosure and the combination
therapeutic agent is alternated.
[0206] Because of the potentially synergistic effects of
administering an anti-TNF-.alpha. antibody of the disclosure and a
combination therapeutic agent, such agents can be administered in
amounts that, if one or both of the agents is administered alone,
is/are not therapeutically effective.
[0207] In certain aspects, the combination therapeutic agent is an
anti-rheumatic drug, an anti-inflammatory agent, a chemotherapeutic
agent, a radiotherapeutic, an immunosuppressive agent, or a
cytotoxic drug.
[0208] Anti-rheumatic drugs include, but are not limited to,
auranofin, azathioprine, chloroquine, D-penicillamine, gold sodium
thiomalate hydroxychloroquine, Myocrisin and sulfasalzine
methotrexate.
[0209] Anti-inflammatory agents include, but are not limited to,
dexamethasone, pentasa, mesalazine, asacol, codeine phosphate,
benorylate, fenbufen, naprosyn, diclofenac, etodolac and
indomethacin, aspirin and ibuprofen.
[0210] Chemotherapeutic agents include, but are not limited to,
radioactive molecules, toxins, also referred to as cytotoxins or
cytotoxic agents, which includes any agent that is detrimental to
the viability of cells, agents, and liposomes or other vesicles
containing chemotherapeutic compounds. Examples of suitable
chemotherapeutic agents include but are not limited to
1-dehydrotestosterone, 5-fluorouracil decarbazine,
6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin,
aldesleukin, alkylating agents, allopurinol sodium, altretamine,
amifostine, anastrozole, anthramycin (AMC)), anti-mitotic agents,
cisdichlorodiamine platinum (II) (DDP) cisplatin), diamino dichloro
platinum, anthracyclines, antibiotics, antimetabolites,
asparaginase, BCG live (intravesical), betamethasone sodium
phosphate and betamethasone acetate, bicalutamide, bleomycin
sulfate, busulfan, calcium leucouorin, calicheamicin, capecitabine,
carboplatin, lomustine (CCNU), carmustine (BSNU), Chlorambucil,
Cisplatin, Cladribine, Colchicin, conjugated estrogens,
Cyclophosphamide, Cyclothosphamide, Cytarabine, Cytarabine,
cytochalasin B, Cytoxan, Dacarbazine, Dactinomycin, dactinomycin
(formerly actinomycin), daunirubicin HCL, daunorucbicin citrate,
denileukin diftitox, Dexrazoxane, Dibromomannitol, dihydroxy
anthracin dione, Docetaxel, dolasetron mesylate, doxorubicin HCL,
dronabinol, E. coli L-asparaginase, eolociximab, emetine,
epoetin-.alpha., Erwinia L-asparaginase, esterified estrogens,
estradiol, estramustine phosphate sodium, ethidium bromide, ethinyl
estradiol, etidronate, etoposide citrororum factor, etoposide
phosphate, filgrastim, floxuridine, fluconazole, fludarabine
phosphate, fluorouracil, flutamide, folinic acid, gemcitabine HCL,
glucocorticoids, goserelin acetate, gramicidin D, granisetron HCL,
hydroxyurea, idarubicin HCL, ifosfamide, interferon .alpha.-2b,
irinotecan HCL, letrozole, leucovorin calcium, leuprolide acetate,
levamisole HCL, lidocaine, lomustine, maytansinoid, mechlorethamine
HCL, medroxyprogesterone acetate, megestrol acetate, melphalan HCL,
mercaptipurine, mesna, methotrexate, methyltestosterone,
mithramycin, mitomycin C, mitotane, mitoxantrone, nilutamide,
octreotide acetate, ondansetron HCL, paclitaxel, pamidronate
disodium, pentostatin, pilocarpine HCL, plimycin, polifeprosan 20
with carmustine implant, porfimer sodium, procaine, procarbazine
HCL, propranolol, rituximab, sargramostim, streptozotocin,
tamoxifen, taxol, teniposide, tenoposide, testolactone, tetracaine,
thioepa chlorambucil, thioguanine, thiotepa, topotecan HCL,
toremifene citrate, trastuzumab, tretinoin, valrubicin, vinblastine
sulfate, vincristine sulfate, and vinorelbine tartrate.
[0211] In yet other aspects of the disclosure, the combination
therapeutic agent is a TNF-.alpha. antagonist other than the
anti-TNF-.alpha. antibody of the disclosure. Examples of such
TNF-.alpha. antagonists include, but are not limited to, soluble
TNF-.alpha. receptors; etanercept (ENBREL.TM.; Immunex) or a
fragment, derivative or analog thereof; infliximab (REMICADE.RTM.;
Centacor) or a derivative, analog or antigen-binding fragment
thereof; IL-10, which is known to block TNF-.alpha. production via
interferon-.gamma.-activated macrophages (Oswald et al., 1992,
Proc. Natl. Acad. Sci. USA 89:8676-8680), TNFR-IgG (Ashkenazi et
al., 1991, Proc. Natl. Acad. Sci. USA 88:10535-10539); the murine
product TBP-1 (Serono/Yeda); the vaccine CytoTAb (Protherics);
antisense molecule 104838 (ISIS); the peptide RDP-58 (SangStat);
thalidomide (Celgene); CDC-801 (Celgene); DPC-333 (Dupont); VX-745
(Vertex); AGIX-4207 (AtheroGenics); ITF-2357 (Italfarmaco);
NPI-13021-31 (Nereus); SCIO-469 (Scios); TACE targeter
(Immunix/AHP); CLX-120500 (Calyx); Thiazolopyrim (Dynavax);
auranofin (Ridaura) (SmithKline Beecham Pharmaceuticals);
quinacrine (mepacrine dichlorohydrate); tenidap (Enablex); Melanin
(Large Scale Biological); and anti-p38 MAPK agents by Uriach.
[0212] Additional second therapeutic agents useful in combination
with an anti-TNF-.alpha. antibody and particular indications for
which combination therapy with such second therapeutic agents are
useful are disclosed in WO 2004/004633, which is incorporated by
reference herein in its entirety.
[0213] 7.11 Therapeutic Regimens
[0214] The present disclosure provides therapeutic regimens
involving the administration of the anti-TNF-.alpha. antibodies of
the disclosure. The therapeutic regimen will vary depending on the
patient's age, weight, and disease condition. The therapeutic
regimen can continue for 2 weeks to indefinitely. In specific
embodiments, the therapeutic regimen is continued for 2 weeks to 6
months, from 3 months to 5 years, from 6 months to 1 or 2 years,
from 8 months to 18 months, or the like. The therapeutic regimen
can be a non-variable dose regimen or a multiple-variable dose
regimen, for example as described in WO 2005/110452, which is
incorporated by reference in its entirety.
[0215] For the dosage exemplary regimens described below, the
anti-TNF-.alpha. antibody can be administered as a sterile,
preservative-free solution for subcutaneous administration.
[0216] In certain embodiments, the drug product is supplied as
either a single-use, prefilled pen within which is enclosed a 1 mL
prefilled glass syringe, or as a single-dose, 1 mL prefilled glass
syringe. For adult patients, in certain embodiments the syringe
delivers 0.8 mL of a pharmaceutically acceptable solution
comprising the anti-TNF-.alpha. antibody of the disclosure. In a
specific embodiment, in addition to the antibody the solution
contains 4.93 mg sodium chloride, 0.69 mg monobasic sodium
phosphate dihydrate, 1.22 mg dibasic sodium phosphate dihydrate,
0.24 mg sodium citrate, 1.04 mg citric acid monohydrate, 9.6 mg
mannitol, 0.8 mg polysorbate 80, and water for injection (USP) with
sodium hydroxide added as necessary to adjust pH. For pediatric
patients, in certain embodiments the syringe delivers 0.4 mL of a
pharmaceutically acceptable solution comprising the
anti-TNF-.alpha. antibody of the disclosure. In a specific
embodiment, in addition to the antibody the solution contains 2.47
mg sodium chloride, 0.34 mg monobasic sodium phosphate dihydrate,
0.61 mg dibasic sodium phosphate dihydrate, 0.12 mg sodium citrate,
0.52 mg citric acid monohydrate, 4.8 mg mannitol, 0.4 mg
polysorbate 80, and water for injection (USP) with sodium hydroxide
added as necessary to adjust pH.
[0217] For treatment rheumatoid arthritis, psoriatic arthritis, and
ankylosing spondylitis, an anti-TNF-.alpha. antibody of the
disclosure can be administered at a dose of 10 to 50 mg (e.g., 10
mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg or 50 mg) every
other week. Methotrexate, glucocorticoids, salicylates,
nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics or other
disease-modifying antirheumatics drug (DMARDs) can be continued
during treatment with the anti-TNF-.alpha. antibody of the
disclosure. In rheumatoid arthritis, some patients not taking
concomitant methotrexate can derive additional benefit from
increasing the dosing frequency from biweekly to weekly.
[0218] For treatment of juvenile idiopathic arthritis, an
anti-TNF-.alpha. antibody of the disclosure is administered at a
dose that depends on the patient's weight. In certain non-limiting
embodiments, the dose for pediatric patients weighing 15 kg (33
lbs) to under 30 kg (66 lbs) ranges from 5 to 25 mg (e.g., 5 mg,
7.5 mg, 10 mg, 12.5 mg, 15 mg, 20 mg, or mg) every other week. In
certain non-limiting embodiments, the dose for pediatric patients
weighing greater than 30 kg (66 lbs) ranges from 10 to 50 mg (e.g.,
10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg or 50 mg)
every other week. Methotrexate, glucocorticoids, salicylates,
NSAIDs or analgesics can be continued during treatment with the
anti-TNF-.alpha. antibody.
[0219] For treatment of Crohn's Disease, an anti-TNF-.alpha.
antibody of the disclosure can be administered in certain
non-limiting embodiments at a dose of 40-280 mg (e.g., 40 mg, 80
mg, 100 mg, 120 mg, 140 mg, 160 mg, 180 mg, 200 mg, 240 mg, or 280
mg) given initially (on Day 1 or divided between Day 1 and Day 2),
followed by a dose of approximately 40% to 60% (e.g., 50%) of the
initial dose two weeks later (Day 15). Two weeks later (Day 29), a
maintenance dose of 20% to 30% (e.g., 25%) of the initial dose is
administered every other week. Aminosalicylates, corticosteroids,
and/or immunomodulatory agents (e.g., 6-mercaptopurine and
azathioprine) can be continued during treatment with the
anti-TNF-.alpha. antibody.
[0220] For treatment of plaque psoriasis, an anti-TNF-.alpha.
antibody of the disclosure can be administered in certain
non-limiting embodiments at a dose of 40-160 mg (e.g., 40 mg, 80
mg, 100 mg, 120 mg, 140 mg, or 160 mg given initially followed by
half the initial dose given every other week starting one week
after the initial dose.
[0221] 7.12 Diagnostic and Pharmaceutical Kits
[0222] Encompassed by the present disclosure are pharmaceutical
kits containing the anti-TNF-.alpha. antibodies (including antibody
conjugates) of the disclosure. The pharmaceutical kit is a package
comprising the anti-TNF-.alpha. antibody of the disclosure (e.g.,
either in lyophilized form or as an aqueous solution) and one or
more of the following: [0223] A combination therapeutic agent, for
example as described in Section 7.10 above; [0224] A device for
administering the anti-TNF-.alpha. antibody, for example a pen,
needle and/or syringe; and [0225] Pharmaceutical grade water or
buffer to resuspend the antibody if the antibody is in lyophilized
form.
[0226] In certain aspects, each unit dose of the anti-TNF-.alpha.
antibody is packaged separately, and a kit can contain one or more
unit doses (e.g., two unit doses, three unit doses, four unit
doses, five unit doses, eight unit doses, ten unit doses, or more).
In a specific embodiment, the one or more unit doses are each
housed in a syringe or pen.
[0227] Diagnostic kits containing the anti-TNF-.alpha. antibodies
(including antibody conjugates) of the disclosure are also
encompassed herein. The diagnostic kit is a package comprising the
anti-TNF-.alpha. antibody of the disclosure (e.g., either in
lyophilized form or as an aqueous solution) and one or more
reagents useful for performing a diagnostic assay. Where the
anti-TNF-.alpha. antibody is labeled with an enzyme, the kit can
include substrates and cofactors required by the enzyme (e.g., a
substrate precursor which provides the detectable chromophore or
fluorophore). In addition, other additives can be included, such as
stabilizers, buffers (e.g., a block buffer or lysis buffer), and
the like. In certain embodiments, the anti-TNF-.alpha. antibody
included in a diagnostic kit is immobilized on a solid surface, or
a solid surface (e.g., a slide) on which the antibody can be
immobilized is included in the kit. The relative amounts of the
various reagents can be varied widely to provide for concentrations
in solution of the reagents which substantially optimize the
sensitivity of the assay. In a specific embodiment, the antibody
and one or more reagents can be provided (individually or combined)
as dry powders, usually lyophilized, including excipients which on
dissolution will provide a reagent solution having the appropriate
concentration.
8. EXAMPLE 1
Identification of Deimmunized Variants of D2E7
[0228] 8.1 Materials & Methods
[0229] 8.1.1 Peptides
[0230] Peptides were synthesized using a multi-pin format by
Mimotopes (Adelaide, Australia). The sequences of the D2E7 light
and heavy chain V regions were synthesized as 15-mer peptides
overlapping by 12 amino acids (FIG. 1 and Table 1) for a total of
69 peptides. Peptides arrived lyophilized and were re-suspended in
DMSO (Sigma-Aldrich) at approximately 1-2 mg/mL. Stock peptides
were kept frozen at -20.degree. C.
[0231] 8.1.2 Human Peripheral Blood Mononuclear Cells
[0232] Community donor buffy coat products were purchased from the
Stanford Blood Center, Palo Alto, Calif. Buffy coat material was
diluted 1:1 (v:v) with DPBS containing no calcium or magnesium.
Diluted buffy coat material (25-35 mL) was underlayed in 50 mL
conical centrifuge tubes (Sarsted or Costar) with 12.5 mL mL of
FicollPaque-PLUS (GE Healthcare). The samples were centrifuged at
900 g for 30 minutes at room temperature. Peripheral blood
mononuclear cells (PBMC) were collected from the interface. DPBS
was added to bring the final volume to 50 mLmL and the cells were
centrifuged at 350 g for 5 minutes. Pelleted cells were resuspended
in DPBS and counted.
[0233] 8.1.3 Dendritic Cells
[0234] For isolation of dendritic cells, T75 culture flasks
(Costar) were seeded with 10.sup.8 freshly isolated PBMC in a total
volume of 30 mL AIM V media (Invitrogen). Excess PBMC were frozen
at -80.degree. C. in 90% fetal calf serum (FCS), 10% DMSO at
5.times.10.sup.7 cells/ml. T75 flasks were incubated at 37.degree.
C. in 5% CO.sub.2 for 2 hours. Nonadherent cells were removed, and
the adherent monolayer was washed with DPBS. To differentiate
dendritic cells from monocytes, 30 mL of AIM V media containing 800
units/mL of GM-CSF (R and D Systems) and 500 units/mL IL-4 (R and D
Systems) were added. Flasks were incubated for 5 days. On day 5
IL-1.alpha. (Endogen) and TNF-.alpha. (Endogen) were added to 50
pg/mL and 0.2 ng/ml. Flasks were incubated for two more days. On
day 7, dendritic cells were collected by the addition of 3 mL of
100 mM EDTA containing 0.5 to 1.0 mg Mitomycin C (Sigma-Aldrich)
for a final concentration of 10 mM EDTA and 16.5 to 33 .mu.g/mL
Mitomycin C. Alternatively, dendritic cells can be irradiated with
4,000 rads for fixation. Flasks were incubated an additional hour
at 37.degree. C. and 5% CO.sub.2. Dendritic cells were collected,
and washed in AIM V media 2-3 times.
[0235] 8.1.4 Cell Culture
[0236] On day 7, previously frozen autologous PBMC were thawed
quickly in a 37.degree. C. water bath. Cells were immediately
diluted into DPBS or AIM V media and centrifuged at 350 g for 5
minutes. CD4+ cells were enriched by negative selection using
magnetic beads (Easy-Sep CD4+ kit, Stem Cell Technologies).
Autologous CD4+ T cells and dendritic cells were cocultured at
2.times.10.sup.5 CD4+ T cells per 2.times.10.sup.4 dendritic cells
per well in 96 well round bottomed plates (Costar 9077). Peptides
were added at approximately 5 .mu.g/mL. Control wells contained the
DMSO (Sigma) vehicle alone at 0.25% v:v. Positive control wells
contained DMSO at 0.25% and tetanus toxoid (List Biologicals or
CalBioChem) at 1 .mu.g/mL. Cultures were incubated for 5 days. On
day 5, 0.25 .mu.Ci per well of tritiated thymidine (Amersham or GE
Healthcare) was added. Cultures were harvested on day 6 to
filtermats using a Packard Filtermate Cell harvester. Scintillation
counting was performed using a Wallac MicroBeta 1450 scintillation
counter (Perkin Elmer).
[0237] 8.1.5 Data Analyses
[0238] Average background CPM values were calculated by averaging
individual results from 6 to 12 replicates. The CPM values of the
four positive control wells were averaged. Replicate or triplicate
wells for each peptide were averaged. Stimulation index values for
the positive control and the peptide wells were calculated by
dividing the average experimental CPM values by the average control
values. In order to be included in the dataset, a stimulation index
of greater than 3.0 in the tetanus toxoid positive control wells
was required. A response was noted for any peptide resulting in a
stimulation index of 2.95 or greater. Peptides were tested using
peripheral blood samples from a group of 81 donors. Responses to
all peptides were compiled. For each peptide tested, the percentage
of the donor set that responded with a stimulation index of 2.95 or
greater was calculated. In addition, the average stimulation index
for all donors was calculated.
[0239] 8.1.6 HLA Genotype Analysis
[0240] HLA DRB1 and HLA DQB1 alleles were determined for each donor
using the commercially available Dynal RELI typing kits
(Invitrogen, UK). Low stringency SSO results are reported. HLA
associations were determined for responsiveness to any given
peptide using a Chi-squared analysis (one degree of freedom). Where
an allele was present in both of the responder and non-responder
populations, a relative risk value was reported.
[0241] 8.1.7 Competition ELISA of D2E7 Variant Antibodies
[0242] TNF-.alpha. was adhered onto a microwell plate, by
contacting the plate with a solution of TNF-.alpha. at a
concentration of 1 .mu.g/mL in PBS over night at 4.degree. C. The
plate was washed in 0.1% Tween 20 in PBS and blocked in Superblock
(Thermo Scientific, Rockford, Ill.). A mixture of sub-saturating
amount of biotinylated D2E7 (80 ng/mL) and unlabeled D2E7 (the
"reference" antibody) or competing anti-TNF-.alpha. antibody (the
"test" antibody) antibody in serial dilution (at a concentration of
2.8 .mu.g/mL, 8.3 .mu.g/mL, or 25 .mu.g/mL) in ELISA buffer (e.g.,
1% BSA and 0.1% Tween 20 in PBS) was added to wells and plates were
incubated for 1 hour with gentle shaking The plate was washed, 1
.mu.g/mL HRP-conjugated Streptavidin diluted in ELISA buffer was
added to each well and the plates incubated for 1 hour. Plates were
washed and bound antibodies were detected by addition of TMB (Biofx
Laboratories Inc., Owings Mills, Md.). The reaction was terminated
by addition of stop buffer (e.g., Bio FX Stop Reagents, Biofx
Laboratories Inc., Owings Mills, Md.) and the absorbance was
measured at 650 nm using microplate reader (e.g., VERSAmax,
Molecular Devices, Sunnyvale, Calif.). The IC.sub.50 values were
calculated for each antibody. The experiment was performed three
times, and average results are shown as a percent of the parent
antibody binding result.
[0243] 8.1.8 Bioassay
[0244] 3.times.10.sup.4 murine L929 cells were plated into
individual wells of a flat bottomed 96-well microtiter plate. The
cells were incubated overnight at 37.degree. C. in a humidified 5%
CO.sub.2 incubator. The next day, serial dilutions of the
anti-TNF-.alpha. antibody (e.g., 0.712 .mu.g/mL, 0.949 .mu.g/mL,
1.27 .mu.g/mL, 1.69 .mu.g/mL, 2.25 .mu.g/mL or 3 .mu.g/mL) were
prepared in 25 .mu.L of serum-free medium and added to cells (such
that the final concentration in 150 .mu.L culture was 119 ng/mL,
158 ng/mL, 211 ng/mL, 282 ng/mL, 375 ng/mL or 500 ng/mL). After a
2-hour incubation at 37.degree. C. in 5% CO.sub.2, 254, of a 240
ng/mL solution of TNF-.alpha. were added, for a final concentration
of 40 ng/mL, and the cells were further incubated for 48 hours at
37.degree. C. in 5% CO.sub.2. The wells were scored for
cytotoxicity as compared to control plates, which treated with
TNF-.alpha. but incubated with an isotype control antibody or with
the parent antibody, D2E7) using a CellTiter-Blue viability assay
(Promega, Madison, Wis.). IC.sub.50 values were determined and
expressed as percent of the parental D2E7 result.
[0245] 8.1.9 Kinetic Analysis of D2E7 Variants by BIAcore
[0246] Binding affinities of anti-TNF-.alpha. antibodies were
measured by using a BIAcore 2000 and 3000 surface plasmon resonance
system (BIAcore, GE Healthcare, Piscataway, N.J.). Polyclonal goat
anti-human Fc antibody (Jackson Immunoresearch) was first
immobilized to the biosensor surface using standard BIAcore amine
coupling reagents (N-ethyl-N'-dimethylamino-propylcarbodiimide,
EDC; N-hydroxysuccinimide, NHS; and ethanolamine HCl, pH 8.5),
followed by the capture of anti-TNF-.alpha. antibodies (D2E7 and
D2E7 variants) on parallel surfaces at a low flow rate of 5
.mu.L/min. R.sup.L was kept low to achieve a low Rmax of 25-60 RU.
No capture of the antibody was made on the reference surface to
serve as a negative control. Subsequently, TNF-.alpha. was injected
to all flow cells at a flow rate of 80 .mu.L/min for three minutes
to monitor association followed by a 30-minute flow of HBS-P
running buffer (10 mM HEPES, 150 mM sodium chloride, 0.005% P-20,
pH 7.4) to monitor the dissociation phase. At each cycle,
TNF-.alpha. (R&D systems, Minneapolis, Minn.), in 6 different
concentrations of TNF ranging between 0 nM and 128 and at four-fold
increments, was injected over the surface. The surface was
regenerated with 1.5% H.sub.3PO.sub.4 at a flow rate of 100
.mu.L/min in two brief pulses at the end of each cycle.
[0247] The binding kinetics of each TNF-.alpha. and antibody pair
were calculated from a global analysis of sensorgram data collected
from the different concentrations of TNF-.alpha. using the
BlAevaluate program. Double referencing was applied in each
analysis to eliminate background responses from the reference
surface and buffer only control (0 nM of TNF-.alpha.). The
dissociation constants (K.sub.D), the association rate constants
(k.sub.on) and the dissociation rate constants (k.sub.off) of each
binding pair was obtained by simultaneously fitting the association
and dissociation phases of the sensorgram using the 1:1 Langmuir
binding with mass transfer model. Each set of experiments was
performed 3 separate times.
[0248] 8.2 Results
[0249] 8.2.1 Identification of CD4+ T Cell Epitopes in the D2E7 VH
and VL Regions
[0250] CD4+ T cell epitope peptides were identified by an analysis
of the percent responses to the peptides within the set of 81
donors. The average percent response and standard deviation were
calculated for all peptides tested describing the D2E7 heavy chain
and light chain. A response rate greater than or equal to the
average background response plus three standard deviations was
considered a potential CD4+ T cell epitope. For the D2E7 light
chain V region, 32 peptides were tested (FIG. 2) which resulted in
an average background percent response of 5.09+3.53%. Three
standard deviations above background was determined to be 15.68%.
One peptide at position 8 displayed this level of response in the
D2E7 light chain peptide dataset, with a response rate of 17.28%
(FIG. 2). In addition, the peptide at position 11 displayed a very
high response rate of 12.35%. For the D2E7 heavy chain V region, 37
peptides were tested (FIG. 3). The average background percent
response was 2.64+2.04%. Three standard deviations above background
was 8.78%. One peptide within the D2E7 heavy chain dataset, #20,
achieved a percent response of 8.64% (FIG. 3).
[0251] The average stimulation index was calculated for all
peptides in the dataset. Light chain peptide #8 had a high average
stimulation index of 1.97+0.08 s.e.m. The peptide at position #11
returned an average stimulation index of 1.63+0.32 s.e.m. Peptide
#27 in the light chain dataset had an average SI of 1.83. This is
due to a single donor with an unusually high stimulation index of
29 to this peptide. Heavy chain peptide #20 had an average
stimulation index value of 1.34+0.05 s.e.m. All of these values are
significantly higher than the average stimulation index for all
peptides in the two datasets (1.02+0.02 for all 68 heavy chain and
light chain peptides).
[0252] These data indicate that there are two major CD4+ T cell
epitope regions in D2E7 (Table 2). In the VH region, an epitope is
found at peptide position 20 that encompasses the junction of
framework 2 and CDR2. In Table 2, the CDR-derived amino acids are
underlined. In the light chain, a large region that can contain
more than one CD4+ T cell epitope includes peptides #8 and #11.
These peptides span a section of framework 1, CDR1 and framework 2
of the light chain.
[0253] 8.2.2 HLA Associations with Responses to the VL Epitope
Peptides
[0254] The HLA class II genotypes of all 81 donors in the peptide
dataset were determined using a low-stringency SSO PCR-based
method. Associations between the presence of a particular HLA
allele and responses to the two VL peptides were determined by chi
squared analysis. Fischer P values and relative risks were
determined for all HLA types and both peptides (Table 3). There
were no significant correlations between any HLA DR or DQ type and
a response to VL peptide #8 (T22-Y36). This result suggests that
the peptide is capable of binding to HLA class II molecules in a
broadly promiscuous manner. CD4+ T cell proliferative responses to
the VL peptide #11 (N31-K45) were tightly associated with the
presence of HLA-DQ2 (p=0.003; relative risk=7.7). As HLA-DR3 is in
linkage disequilibrium with HLA-DQ2, the association between a
response to this peptide and HLA-DR3 was present but did not reach
statistical significance (p=0.10; relative risk 3.3). In addition
to HLA-DQ2, as association was found between HLA-DR12 and a
response to N31-K45 (p=0.03; relative risk 5.2). The HLA responses
to the VH peptide #20 were not tested as there were too few total
responders. Since the responders to the two VL peptides were
discrete it can be concluded that they represent two separate
peptide epitopes. Therefore, the D2E7 VH and VL region contains
three prominent peptide epitope regions.
[0255] 8.2.3 Identification of Reduced Immunogenicity Variants
[0256] Alanine scan modifications: A twenty-one amino acid sequence
of the D2E7 light chain encompasses the epitopes at T22-Y36 and
N31-K45. The twenty one amino acid sequence selected was C23-K45.
Alanine modifications were incorporated at each amino acid (Table
4). A set of 99 donors was tested with the variant peptides (FIG.
4). The parent 21-mer was created 4 times within the peptide set.
These four replicates serve as a control for the reproducibility of
the assay. The average parent peptide response was 8.3%, with a CV
% of 30%. Therefore, variant peptides with an average percent of
less than 5.8% could be considered to have a reduced rate of
response. The most reduced variants were C23A (2.02%) and P40A
(3.03%, see FIG. 4). The cysteine at position 23 is invariant, and
is therefore not a good candidate for modification in the whole
protein. Due to the unique nature of proline residues a
modification of this residue is also not likely to yield a
functional variant antibody. The third candidate would be Y32A
(4.04%). Additionally, there are a number of variants that resulted
in an average response rate of 5.05%. These changes could also be
effective but would need to be tested as whole protein molecules
for both reduced immunogenicity and functional activity.
[0257] A set of alanine-modified peptides based on the sequence of
the D2E7 VH epitope peptide were also tested (data not shown). The
response rate of the parent unmodified peptide in the replicate
test was very low. Therefore this peptide was no longer
studied.
[0258] Antigen Binding Study: The CDR-L1 region of the D2E7
antibody was subjected to comprehensive mutational analysis. Based
on antigen-binding studies performed in conjunction with the
mutational analyses, a set of candidate amino acid substitutions
within the CDR-L1 region was identified that did not significantly
reduce the affinity of the antibody to TNF-.alpha. (Table 5).
Several variant antibodies containing the candidate CDR-L1
substitutions were analyzed using BIAcore and ELISA (Table 6).
Peptides were generated containing amino acid modifications within
the CDR-L1 region that had the property of altering the amino acid
sequence while retaining the affinity of the overall antibody
molecule (Table 7). The modified epitope peptides were tested as
single amino-acid modifications, or as double modifications. The
double modifications contained a glutamine or a glycine at position
32, or a serine or glycine at position 34. A total of 79 peptides
were tested including two syntheses of the parent 21-mer peptide. A
total of 102 donors were tested with the variant peptides and the
results are shown in FIG. 5. The average percent response of the
parent peptides was 10.3+2.1%. For a percent response rate to be
less than 3 standard deviations from the parent the response rate
would be less than 4%. The average stimulation index for the parent
peptides was 1.49+0.15. For a stimulation index to reach three
standard deviations below the parent response it would be 1.03 or
lower.
[0259] Subsequent affinity measurements of the Y32G and Y32Q
mutations showed that this amino acid modification had a negative
impact on antigen binding. Therefore, all peptides carrying this
modification were removed from the analysis. A total of 10 peptides
were selected for further study. All selected peptides had a
response rate less than 4%. However, none of the peptides
demonstrated stimulation indexes 3 standard deviations below the
average parent response (Table 8).
[0260] 8.2.4 Affinity And Bioactivity Testing Of Modified D2E7
Variant Antibodies
[0261] Ten variant D2E7 VL region constructs were cloned along with
the unmodified VH region into a human IgG.sub.1-containing plasmid,
expressed in 293T/17 cell lines by transient transfection, and
antibodies purified by Protein A or Protein G affinity. The
purified antibodies were tested for TNF-.alpha. binding in a
competition ELISA assay. All ten variants competed for TNF-.alpha.
binding with the unmodified D2E7 antibody. However, there was a
range of affinities displayed, from approximately equivalent
affinity of the Q27R+A34S variant, to a 10.times. reduction in
affinity of the N31 S+A34S variant (FIG. 6).
[0262] A TNF-.alpha. toxicity bioassay was performed. L292 cells
were seeded into 96 well plates, and a constant concentration of
TNF-.alpha. was added to the culture medium. The variant antibodies
were titrated into the medium. An EC.sub.50 value was determined
for each variant (Table 9). Similarly, the variant Q27R+A34S
displayed an EC.sub.50 value approximately equivalent to the parent
D2E7 antibody.
[0263] Finally, affinity of the antibodies for TNF-.alpha. was
determined by BIAcore analysis (Table 10). Of the ten variants
tested, the Q27H+ A34S, Q27R+A34S and G28S+A34S variants all
displayed association and dissociation rates similar to D2E7. The
final affinity values for the variants were in the 130 .mu.M range
as compared to D2E7 with a measured affinity in these experiments
of 114 .mu.M.
9. EXAMPLE 2
Identification of Variants of D2E7 with Increased Affinity to
TNF-.alpha.
[0264] The D2E7 antibody was subjected to comprehensive mutational
analysis to identify mutants that had increased affinity to
TNF-.alpha. as compared to D2E7. The increased affinity of
candidate mutants to TNF-.alpha. was analyzed by ELISA and BIAcore
to confirm their characteristics as compared to D2E7.
[0265] 9.1 Materials & Methods
[0266] 9.1.1 Competition Elisa
[0267] Competition ELISA assays were done as described in Section
8.1.7. ELISA was repeated twice and average fold improvement in
IC.sub.50 is shown as WT/x.
[0268] 9.1.2 BIAcore
[0269] BIAcore assays were done as described in Section 8.1.9.
[0270] 9.2 Results
[0271] CDR variants of D2E7 that had improved K.sub.D (as measured
by BIAcore), improved ability to compete in ELISA, or both relative
to D2E7 are shown in Tables 12 and 25.
10. SPECIFIC EMBODIMENTS, CITATION OF REFERENCES
[0272] All publications, patents, patent applications and other
documents cited in this application are hereby incorporated by
reference in their entireties for all purposes to the same extent
as if each individual publication, patent, patent application or
other document were individually indicated to be incorporated by
reference for all purposes.
[0273] While various specific embodiments have been illustrated and
described, it will be appreciated that various changes can be made
without departing from the spirit and scope of the invention(s).
Sequence CWU 1
1
3431363DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 1gaggtgcagc tggtggagtc
tgggggaggc ttggtacagc ccggcaggtc cctgagactc 60tcctgtgcgg cctctggatt
cacctttgat gattatgcca tgcactgggt ccggcaagct 120ccagggaagg
gcctggaatg ggtctcagct atcacttgga atagtggtca catagactat
180gcggactctg tggagggccg attcaccatc tccagagaca acgccaagaa
ctccctgtat 240ctgcaaatga acagtctgag agctgaggat acggccgtat
attactgtgc gaaagtctcg 300taccttagca ccgcgtcctc ccttgactat
tggggccaag gtaccctggt caccgtctcg 360agt 3632121PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Thr Trp Asn Ser Gly His Ile
Asp Tyr Ala Asp Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Pro
Arg Thr Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Ser Tyr Leu
Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu
Val Thr Val Ser Ser 115 1203321DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 3gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtagggga cagagtcacc 60atcacttgtc gggcaagtca gggcatcaga aattacttag
cctggtatca gcaaaaacca 120gggaaagccc ctaagctcct gatctatgct
gcatccactt tgcaatcagg ggtcccatct 180cggttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctacagcct 240gaagatgttg
caacttatta ctgtcaaagg tataaccgtg caccgtatac ttttggccag
300gggaccaagg tggaaatcaa a 3214107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 4Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr
Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 10555PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 5Asp Tyr Ala Met His1 5617PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 6Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser
Val Glu1 5 10 15Gly712PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 7Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr1 5
10811PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 8Arg Ala Ser Gln Gly Ile Arg Asn Tyr
Leu Ala1 5 1097PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 9Ala Ala Ser Thr Leu Gln
Ser1 5109PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 10Gln Arg Tyr Asn Arg Ala
Pro Tyr Thr1 51115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 11Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 151215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 12Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser
Leu1 5 10 151315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 13Ser Gly Gly Gly Leu Val
Gln Pro Gly Arg Ser Leu Arg Leu Ser1 5 10 151415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 14Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala
Ala1 5 10 151515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 15Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe1 5 10 151615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 16Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Asp1 5 10 151715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 17Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Asp Asp Tyr Ala1 5 10 151815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 18Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met His
Trp1 5 10 151915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 19Ser Gly Phe Thr Phe Asp
Asp Tyr Ala Met His Trp Val Arg Gln1 5 10 152015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 20Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro
Gly1 5 10 152115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 21Asp Tyr Ala Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu1 5 10 152215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 22Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val1 5 10 152315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 23Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val Ser Ala Ile1 5 10 152415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 24Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Thr Trp
Asn1 5 10 152515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 25Lys Gly Leu Glu Trp Val
Ser Ala Ile Thr Trp Asn Ser Gly His1 5 10 152615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 26Glu Trp Val Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp
Tyr1 5 10 152715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 27Ser Ala Ile Thr Trp Asn
Ser Gly His Ile Asp Tyr Ala Asp Ser1 5 10 152815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 28Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu
Gly1 5 10 152915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 29Ser Gly His Ile Asp Tyr
Ala Asp Ser Val Glu Gly Arg Phe Thr1 5 10 153015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 30Ile Asp Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser
Arg1 5 10 153115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 31Ala Asp Ser Val Glu Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala1 5 10 153215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 32Val Glu Gly Arg Phe Thr Ile Ser Arg Pro Arg Thr Lys Asn
Ser1 5 10 153315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 33Arg Phe Thr Ile Ser Arg
Pro Arg Thr Lys Asn Ser Leu Tyr Leu1 5 10 153415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 34Ile Ser Arg Pro Arg Thr Lys Asn Ser Leu Tyr Leu Gln Met
Asn1 5 10 153515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 35Asp Asn Ala Lys Asn Ser
Leu Tyr Leu Gln Met Asn Ser Leu Arg1 5 10 153615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 36Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp1 5 10 153715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 37Leu Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val1 5 10 153815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 38Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys1 5 10 153915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 39Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ala Lys Val1 5 10 154015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 40Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Ser Tyr
Leu1 5 10 154115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 41Thr Ala Val Tyr Tyr Cys
Ala Lys Val Ser Tyr Leu Ser Thr Ala1 5 10 154215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 42Tyr Tyr Cys Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser
Leu1 5 10 154315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 43Ala Lys Val Ser Tyr Leu
Ser Thr Ala Ser Ser Leu Asp Tyr Trp1 5 10 154415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 44Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly Gln
Gly1 5 10 154515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 45Ser Thr Ala Ser Ser Leu
Asp Tyr Trp Gly Gln Gly Thr Leu Val1 5 10 154615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 46Ser Ser Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser1 5 10 154715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 47Ser Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser1 5 10 154815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 48Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val1 5 10 154915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 49Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly Asp Arg1 5 10 155015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 50Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
Ile1 5 10 155115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 51Ser Leu Ser Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Arg1 5 10 155215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 52Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln1 5 10 155315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 53Gly Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly Ile Arg1 5 10 155415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 54Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr
Leu1 5 10 155515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 55Thr Cys Arg Ala Ser Gln
Gly Ile Arg Asn Tyr Leu Ala Trp Tyr1 5 10 155615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 56Ala Ser Gln Gly Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln
Lys1 5 10 155715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 57Gly Ile Arg Asn Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys1 5 10 155815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 58Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys1 5 10 155915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 59Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile1 5 10 156015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 60Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala
Ala1 5 10 156115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 61Pro Gly Lys Ala Pro Lys
Leu Leu Ile Tyr Ala Ala Ser Thr Leu1 5 10 156215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 62Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser
Gly1 5 10 156315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 63Leu Leu Ile Tyr Ala Ala
Ser Thr Leu Gln Ser Gly Val Pro Ser1 5 10 156415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 64Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser1 5 10 156515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 65Ser Thr Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser Gly1 5 10 156615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 66Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr1 5 10 156715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 67Val Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 156815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 68Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile1 5 10 156915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 69Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu1 5 10 157015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 70Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
Glu1 5 10 157115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 71Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu Asp Val Ala1 5 10 157215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 72Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr
Tyr1 5 10 157315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 73Ser Ser Leu Gln Pro Glu
Asp Val Ala Thr Tyr Tyr Cys Gln Arg1 5 10 157415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 74Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn
Arg1 5 10 157515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide" 75Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro
Tyr1 5 10 157615PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 76Asp Val Ala Thr Tyr Tyr
Cys Gln Arg Tyr Asn Arg Ala Pro Tyr1 5 10 157715PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 77Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr Thr Phe
Gly1 5 10 157815PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 78Cys Gln Arg Tyr Asn Arg
Ala Pro Tyr Thr Phe Gly Gln Gly Thr1 5 10 157915PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 79Tyr Asn Arg Ala Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val
Glu1 5 10 158015PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 80Arg Ala Pro Tyr Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys1 5 10 158115PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 81Ile Asp Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser
Arg1 5 10 158215PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 82Thr Cys Arg Ala Ser Gln
Gly Ile Arg Asn Tyr Leu Ala Trp Tyr1 5 10 158315PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 83Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys1 5 10 158423PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 84Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 208523PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 85Ala Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 208623PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 86Cys Ala Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 208723PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 87Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 208823PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 88Cys Arg Ala Ala Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 208923PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 89Cys Arg Ala Ser Ala Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 209023PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 90Cys Arg Ala Ser Gln Ala
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 209123PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 91Cys Arg Ala Ser Gln Gly
Ala Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 209223PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 92Cys Arg Ala Ser Gln Gly
Ile Ala Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 209323PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 93Cys Arg Ala Ser Gln Gly
Ile Arg Ala Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 209423PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 94Cys Arg Ala Ser Gln Gly
Ile Arg Asn Ala Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 209523PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 95Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Ala Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 209623PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 96Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 209723PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 97Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Ala Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 209823PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 98Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Ala Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 209923PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 99Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Ala Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 2010023PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 100Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Ala1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 2010123PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 101Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Ala Pro Gly Lys Ala
Pro Lys 2010223PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 102Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Ala Gly Lys Ala
Pro Lys 2010323PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 103Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Ala Lys Ala
Pro Lys 2010423PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 104Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Ala Ala
Pro Lys 2010523PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 105Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Lys 2010623PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 106Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Ala Lys 2010723PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 107Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln1 5 10 15Lys Pro Gly Lys Ala
Pro Ala 2010824PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 108Thr Cys Arg Ala Ser Gln
Gly Ile Arg Asn Tyr Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys
Ala Pro Lys 2010924PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 109Thr Cys Arg Ala Ser
Asn Gly Ile Arg Asn Tyr Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly
Lys Ala Pro Lys 2011024PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 110Thr Cys Arg Ala Ser Gly Gly Ile Arg Asn Tyr Leu Ala Trp
Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2011124PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 111Thr Cys Arg Ala Ser His Gly Ile Arg
Asn Tyr Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2011224PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 112Thr Cys Arg Ala Ser Ser Gly Ile Arg
Asn Tyr Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2011324PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 113Thr Cys Arg Ala Ser Arg Gly Ile Arg
Asn Tyr Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2011424PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 114Thr Cys Arg Ala Ser Gln Ser Ile Arg
Asn Tyr Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2011524PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 115Thr Cys Arg Ala Ser Gln His Ile Arg
Asn Tyr Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2011624PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 116Thr Cys Arg Ala Ser Gln Asn Ile Arg
Asn Tyr Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2011724PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 117Thr Cys Arg Ala Ser Gln Gly Val Arg
Asn Tyr Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2011824PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 118Thr Cys Arg Ala Ser Gln Gly Thr Arg
Asn Tyr Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2011924PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 119Thr Cys Arg Ala Ser Gln Gly Ile Gln
Asn Tyr Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2012024PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 120Thr Cys Arg Ala Ser Gln Gly Ile Arg
Gly Tyr Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2012124PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 121Thr Cys Arg Ala Ser Gln Gly Ile Arg
Thr Tyr Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Phe Gly Lys Ala Pro Lys
2012224PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 122Thr Cys Arg Ala Ser Gln Gly Ile Arg
Ser Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2012324PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 123Thr Cys Arg Ala Ser Gln Gly Ile Arg
Asn Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2012424PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 124Thr Cys Arg Ala Ser Gln Gly Ile Arg
Asn Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2012524PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 125Thr Cys Arg Ala Ser Gln Gly Ile Arg
Asn Tyr Leu Ser Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2012624PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 126Thr Cys Arg Ala Ser Asn Gly Ile Arg
Asn Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2012724PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 127Thr Cys Arg Ala Ser Gly Gly Ile Arg
Asn Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2012824PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 128Thr Cys Arg Ala Ser His Gly Ile Arg
Asn Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2012924PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 129Thr Cys Arg Ala Ser Ser Gly Ile Arg
Asn Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2013024PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 130Thr Cys Arg Ala Ser Arg Gly Ile Arg
Asn Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2013124PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 131Thr Cys Arg Ala Ser Gln Ser Ile Arg
Asn Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2013224PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 132Thr Cys Arg Ala Ser Gln His Ile Arg
Asn Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2013324PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 133Thr Cys Arg Ala Ser Gln Asn Ile Arg
Asn Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2013424PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 134Thr Cys Arg Ala Ser Gln Gly Val Arg
Asn Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2013524PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 135Thr Cys Arg Ala Ser Gln Gly Thr Arg
Asn Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2013624PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 136Thr Cys Arg Ala Ser Gln Gly Ile Gln
Asn Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2013724PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 137Thr Cys Arg Ala Ser Gln Gly Ile Arg
Gly Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2013824PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 138Thr Cys Arg Ala Ser Gln Gly Ile Arg
Thr Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2013924PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 139Thr Cys Arg Ala Ser Gln Gly Ile Arg
Ser Gln Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2014024PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic
peptide" 140Thr Cys Arg Ala Ser Gln Gly Ile Arg Ser Gln Leu Gly Trp
Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2014124PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 141Thr Cys Arg Ala Ser Gln Gly Ile Arg
Ser Gln Leu Ser Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2014224PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 142Thr Cys Arg Ala Ser Asn Gly Ile Arg
Asn Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2014324PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 143Thr Cys Arg Ala Ser Gly Gly Ile Arg
Asn Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2014424PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 144Thr Cys Arg Ala Ser His Gly Ile Arg
Asn Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2014524PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 145Thr Cys Arg Ala Ser Ser Gly Ile Arg
Asn Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2014624PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 146Thr Cys Arg Ala Ser Arg Gly Ile Arg
Asn Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2014724PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 147Thr Cys Arg Ala Ser Gln Ser Ile Arg
Asn Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2014824PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 148Thr Cys Arg Ala Ser Gln His Ile Arg
Asn Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2014924PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 149Thr Cys Arg Ala Ser Gln Asn Ile Arg
Asn Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2015024PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 150Thr Cys Arg Ala Ser Gln Gly Val Arg
Asn Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2015124PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 151Thr Cys Arg Ala Ser Gln Gly Thr Arg
Asn Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2015224PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 152Thr Cys Arg Ala Ser Gln Gly Ile Gln
Asn Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2015324PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 153Thr Cys Arg Ala Ser Gln Gly Ile Arg
Gly Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2015424PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 154Thr Cys Arg Ala Ser Gln Gly Ile Arg
Thr Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2015524PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 155Thr Cys Arg Ala Ser Gln Gly Ile Arg
Ser Ser Leu Ala Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2015624PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 156Thr Cys Arg Ala Ser Gln Gly Ile Arg
Asn Ser Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2015724PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 157Thr Cys Arg Ala Ser Gln Gly Ile Arg
Asn Ser Leu Ser Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2015824PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 158Thr Cys Arg Ala Ser Asn Gly Ile Arg
Asn Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2015924PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 159Thr Cys Arg Ala Ser Gly Gly Ile Arg
Asn Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2016024PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 160Thr Cys Arg Ala Ser His Gly Ile Arg
Asn Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2016124PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 161Thr Cys Arg Ala Ser Ser Gly Ile Arg
Asn Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2016224PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 162Thr Cys Arg Ala Ser Arg Gly Ile Arg
Asn Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2016324PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 163Thr Cys Arg Ala Ser Gln Ser Ile Arg
Asn Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2016424PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 164Thr Cys Arg Ala Ser Gln His Ile Arg
Asn Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2016524PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 165Thr Cys Arg Ala Ser Gln Asn Ile Arg
Asn Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2016624PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 166Thr Cys Arg Ala Ser Gln Gly Val Arg
Asn Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2016724PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 167Thr Cys Arg Ala Ser Gln Gly Thr Arg
Asn Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Phe Lys
2016824PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 168Thr Cys Arg Ala Ser Gln Gly Ile Gln
Asn Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2016924PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 169Thr Cys Arg Ala Ser Gln Gly Ile Arg
Gly Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2017024PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 170Thr Cys Arg Ala Ser Gln Gly Ile Arg
Thr Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2017124PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 171Thr Cys Arg Ala Ser Gln Gly Ile Arg
Ser Tyr Leu Gly Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2017224PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 172Thr Cys Arg Ala Ser Asn Gly Ile Arg
Asn Tyr Leu Ser Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2017324PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 173Thr Cys Arg Ala Ser Gly Gly Ile Arg
Asn Tyr Leu Ser Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2017424PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 174Thr Cys Arg Ala Ser His Gly Ile Arg
Asn Tyr Leu Ser Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2017524PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 175Thr Cys Arg Ala Ser Ser Gly Ile Arg
Asn Tyr Leu Ser Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2017624PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 176Thr Cys Arg Ala Ser Gln Gly Ile Gln
Asn Tyr Leu Ser Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2017724PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 177Thr Cys Arg Ala Ser Gln Gly Ile Arg
Thr Tyr Leu Ser Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2017824PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 178Thr Cys Arg Ala Ser Arg Gly Ile Arg
Asn Tyr Leu Ser Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2017924PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 179Thr Cys Arg Ala Ser Gln Ser Ile Arg
Asn Tyr Leu Ser Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2018024PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 180Thr Cys Arg Ala Ser Gln Gly Ile Arg
Ser Tyr Leu Ser Trp Tyr Gln1 5 10 15Gln Lys Pro Gly Lys Ala Pro Lys
2018111PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 181Arg Ala Ser Gln Gly Ile Arg Asn Tyr
Leu Ala1 5 1018211PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 182Arg Ala Ser Gln Gly Ile
Arg Asn Tyr Leu Ala1 5 1018311PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 183Arg Ala Ser Gln Gly Ile Gln Asn Tyr Leu Ala1 5
1018411PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 184Arg Ala Ser Gln Gly Ile Arg Asn Tyr
Leu Ser1 5 1018511PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 185Arg Ala Ser Gln Gly Ile
Gln Asn Tyr Leu Ser1 5 1018611PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 186Arg Ala Ser Gln Gly Ile Arg Thr Tyr Leu Ala1 5
1018711PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 187Arg Ala Ser Gln Gly Ile Arg Thr Tyr
Leu Ser1 5 1018811PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 188Arg Ala Ser Gln Gly Thr
Arg Asn Tyr Leu Ala1 5 1018911PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 189Arg Ala Ser Gln Gly Ile Arg Asn Tyr Leu Gly1 5
1019011PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 190Arg Ala Ser Gln Gly Thr Arg Asn Tyr
Leu Gly1 5 1019111PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 191Arg Ala Ser Gly Gly Ile
Arg Asn Tyr Leu Ala1 5 1019211PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 192Arg Ala Ser Gly Gly Ile Arg Asn Tyr Leu Ser1 5
1019311PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 193Arg Ala Ser Gly Gly Ile Arg Asn Tyr
Leu Gly1 5 1019411PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 194Arg Ala Ser Gln Gly Ile
Arg Thr Tyr Leu Gly1 5 1019511PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 195Arg Ala Ser His Gly Ile Arg Asn Tyr Leu Ala1 5
1019611PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 196Arg Ala Ser His Gly Ile Arg Asn Tyr
Leu Ser1 5 1019711PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 197Arg Ala Ser Arg Gly Ile
Arg Asn Tyr Leu Ala1 5 1019811PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 198Arg Ala Ser Arg Gly Ile Arg Asn Tyr Leu Ser1 5
1019911PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 199Arg Ala Ser Gln Ser Ile Arg Asn Tyr
Leu Ala1 5 1020011PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 200Arg Ala Ser Gln Ser Ile
Arg Asn Tyr Leu Ser1 5 1020111PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 201Arg Ala Ser Gln Gly Ile Arg Ser Tyr Leu Ser1 5
1020210PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 202Arg Ala Ser Gln Gly Thr Arg Tyr Leu
Ser1 5 1020311PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 203Arg Ala Ser Gln Gly Ile
Arg Thr Tyr Leu Gly1 5 102045PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 204Asp Tyr Ala Met His1 52055PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 205Asp Tyr Thr Met His1 52065PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 206Glu Tyr Ala Met His1 52075PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 207Gln His Ala Leu His1 52085PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 208Gln His Ala Met His1 52095PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 209His Tyr Ala Leu His1 52105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 210Gln Tyr Ala Met His1 52115PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 211His Tyr Ala Met His1 52125PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 212Tyr Tyr Ala Met His1 521317PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 213Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser
Val Glu1 5 10 15Gly21418PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 214Pro Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp
Ser Val1 5 10 15Glu Gly21515PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 215Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu
Gly1
5 10 1521612PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 216Val Ser Tyr Leu Ser Thr
Ala Ser Ser Leu Asp Tyr1 5 1021713PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 217Pro Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa1 5
1021813PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 218Pro Ala Ser Tyr Leu Ser Thr Ala Ser
Ser Leu Asp Tyr1 5 1021913PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 219Pro Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Tyr1 5
1022012PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 220Val Ser Tyr Leu Ser Thr Ala Ser Ser
Leu Asp Asn1 5 1022112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 221Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa1 5
1022212PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 222Ala Ser Tyr Leu Ser Thr Ser Ser Ser
Leu Asp Tyr1 5 1022312PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 223Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Gln Tyr1 5
1022412PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 224Ala Ser Tyr Leu Ser Thr Ala Ser Ser
Leu Gln Tyr1 5 1022512PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 225Val Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Tyr1 5
1022612PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 226Ala Ser Tyr Leu Ser Thr Ala Ser Ser
Leu Glu Tyr1 5 1022712PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 227Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asn1 5
1022812PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 228Ala Ser Tyr Leu Ser Thr Ser Ser Ser
Leu Asp Lys1 5 1022912PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 229Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asp1 5
1023012PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 230Ala Ser Tyr Leu Ser Thr Ser Phe Ser
Leu Asp Tyr1 5 1023112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 231Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Ala1 5
1023212PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 232Ala Ser Tyr Leu Ser Thr Ser Ser Ser
Leu Glu Tyr1 5 1023312PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 233Val Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asn1 5
1023412PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 234Val Ser Tyr Leu Ser Thr Ala Ser Ser
Ala Asp Tyr1 5 1023512PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 235Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Ala Tyr1 5
1023612PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 236Val Ala Tyr Leu Ser Thr Ala Ser Ser
Leu Asp Tyr1 5 1023712PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 237Val Ser Ala Leu Ser Thr Ala Ser Ser Leu Asp Tyr1 5
1023812PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 238Val Ser Tyr Ala Ser Thr Ala Ser Ser
Leu Asp Tyr1 5 1023912PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 239Val Ser Tyr Leu Ala Thr Ala Ser Ser Leu Asp Tyr1 5
1024012PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 240Val Ser Tyr Leu Ser Ala Ala Ser Ser
Leu Asp Tyr1 5 1024112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 241Val Ser Tyr Leu Ser Thr Ala Ala Ser Leu Asp Tyr1 5
1024212PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 242Val Ser Tyr Leu Ser Thr Ala Ser Ala
Leu Asp Tyr1 5 1024312PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 243Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Gln Xaa1 5
1024412PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 244Val Ser Tyr Leu Ser Thr Ala Ser Ser
Leu Gln Tyr1 5 1024512PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 245Val Ser Ala Ser Thr Gly Pro Ser Val Phe Pro Leu1 5
1024612PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 246Val His Tyr Leu Ser Thr Ala Ser Ser
Leu Asp Tyr1 5 1024712PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 247Val His Tyr Leu Ser Thr Ala Ser Gln Leu His His1 5
1024812PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 248Val Gln Tyr Leu Ser Thr Ala Ser Ser
Leu Asp Tyr1 5 1024912PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 249Val Lys Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr1 5
1025012PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 250Val Pro Tyr Leu Ser Thr Ala Ser Ser
Leu Asp Tyr1 5 1025112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 251Val Ser Tyr Leu Ser Thr Ala Ser Pro Leu Asp Tyr1 5
1025212PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 252Val Ser Tyr Leu Ser Thr Ala Ser Ser
Leu Pro Tyr1 5 1025312PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 253Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Pro1 5
1025412PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 254Val Ser Tyr Leu Ser Thr Ala Ser Ser
Leu Asp Ser1 5 1025512PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 255Ala Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr1 5
1025612PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 256Ala Ser Tyr Leu Ser Thr Ser Ser Ser
Leu His Tyr1 5 1025712PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 257Ala Ser Phe Leu Ser Thr Ser Ser Ser Leu Glu Tyr1 5
1025811PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 258Arg Ala Ser Gln Gly Ile Arg Asn Tyr
Leu Ala1 5 1025912PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 259Pro Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala1 5 1026011PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 260Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly1 5
1026111PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 261Arg Ala Ser Gln Gly Ile Ser Asn Tyr
Leu Ala1 5 1026211PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 262Arg Ala Arg Gln Gly Ile
Ser Asn Tyr Leu Ala1 5 1026312PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 263Arg Ala Ser Gln Ser Ile Arg Ser Asn Tyr Leu Ala1 5
1026411PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 264Arg Ala Ser Gln Glu Ile Arg Asn Tyr
Leu Ala1 5 1026511PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 265His Ala Ser Gln Lys Ile
Arg Asn Tyr Leu Ala1 5 1026611PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 266Arg Ala Ser Leu Gly Ile Arg Asn Tyr Leu Ala1 5
1026711PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 267Arg Ala Ser Gln Gly Ile Ser Ser Tyr
Leu Ala1 5 1026811PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 268Arg Ala Ser Ser Gly Ile
Arg Asn Tyr Leu Ala1 5 1026911PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 269His Ala Ser Arg Arg Ile Arg Asn Tyr Leu Ala1 5
1027011PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 270His Ala Ser Arg Arg Ile Leu Asn Tyr
Leu Ala1 5 1027111PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 271His Ala Ser Arg Lys Ile
Leu Asn Tyr Leu Ala1 5 1027211PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 272His Ala Ser Arg Lys Ile Arg Asn Tyr Leu Ala1 5
1027311PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 273His Ala Ser Gln Lys Ile Arg Asn Tyr
Leu Ala1 5 1027411PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 274His Ala Ser Lys Arg Ile
Arg Asn Tyr Leu Ala1 5 1027511PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 275His Ala Ser Lys Lys Ile Arg Asn Tyr Leu Ala1 5
1027611PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 276His Ala Ser Arg Glu Ile Arg Asn Tyr
Leu Ala1 5 1027711PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 277His Ala Ser Gln Gly Ile
Arg Asn Tyr Leu Ala1 5 1027811PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 278Gly Ala Ser Gln Gly Ile Arg Asn Tyr Leu Ala1 5
1027911PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 279Arg Ala Ser Lys Gly Ile Arg Asn Tyr
Leu Ala1 5 1028011PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 280Arg Ala Ser Gln Lys Ile
Arg Asn Tyr Leu Ala1 5 1028111PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 281Arg Ala Ser Gln Gly Lys Arg Asn Tyr Leu Ala1 5
1028211PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 282Arg Ala Ser Gln Gly Leu Arg Asn Tyr
Leu Ala1 5 1028311PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 283Arg Ala Ser Tyr Gly Ile
Arg Asn Tyr Leu Ala1 5 102847PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 284Ala Ala Ser Thr Leu Gln Ser1 52858PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 285Pro Ala Ala Ser Thr Leu Gln Ser1 52867PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 286Ala Ala Ser Ser Leu Leu His1 52877PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 287Ala Ala Ser Ser Leu Gln Pro1 52887PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 288Ala Ala Ser Ser Leu Leu Arg1 52897PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 289Ala Ala Ser Ser Leu Leu Lys1 52907PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 290Ala Ala Ser Ser Leu Gln Gln1 52917PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 291Ala Ala Ser Ser Leu Leu Pro1 52927PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 292Ala Ala Ser Ser Leu Leu Gln1 52937PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 293Ala Ala Ser Thr Leu Leu Lys1 52947PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 294Ala Ala Ser Ala Leu Gln Ser1 52957PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 295Ala Ala Ser Thr Phe Gln Ser1 52967PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 296Ala Ala Ser Thr Leu Gln Lys1 52977PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 297Ala Ala Leu Thr Leu Gln Ser1 52987PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 298Ala Ala Ser Thr Leu Leu Ser1 52997PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 299Ala Ala Ser Thr Leu Gln Pro1 53007PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 300Ala Ala Ser Ser Leu Gln Ser1 53017PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 301Ala Ala Tyr Thr Leu Gln Ser1 53029PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 302Gln Arg Tyr Asn Arg Ala Pro Tyr Thr1
530310PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 303Pro Gln Lys Tyr Asn Arg Ala Pro Tyr
Thr1 5 1030410PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 304Pro Gln Arg Tyr Asn Arg
Ala Pro Tyr Xaa1 5 1030510PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 305Pro Gln Arg Tyr Asn Arg Ala Pro Tyr Ala1 5
103069PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 306Gln Lys Tyr Asn Arg Ala Pro Tyr Thr1
53079PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 307Gln Arg Tyr Asn Arg Ala Pro Tyr Ala1
53089PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 308Gln Arg Tyr Asn Arg Ala Pro Tyr Xaa1
53099PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 309Gln Arg Tyr Asn Arg Ala
Pro Tyr Thr1 53109PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 310Gln Lys Tyr Asn Ser Ala
Pro Tyr Ala1 53119PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 311Gln Lys Tyr Asn Arg Ala
Pro Tyr Ala1 53129PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 312Gln Lys Tyr Asn Arg Ala
Pro Tyr Thr1 53139PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 313Gln Lys Tyr Ser Ser Ala
Pro Tyr Thr1 53149PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 314Gln Lys Tyr Asn Ser Ala
Pro Tyr Thr1 53159PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 315Gln Lys Tyr Asn Ser Ala
Pro Tyr Tyr1 53169PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 316Gln Lys Tyr Asn Ser Ala
Pro Tyr Asn1 53179PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 317Gln Lys Tyr Thr Ser Ala
Pro Tyr Thr1 53189PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 318Gln Lys Tyr Asn Arg Ala
Pro Tyr Asn1 53199PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 319Gln Lys Tyr Asn Ser Ala
Ala Tyr Ser1 53209PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 320Gln Gln Tyr Asn Ser Ala
Pro Asp Thr1 53219PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 321Gln Lys Tyr Asn Ser Asp
Pro Tyr Thr1 53229PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 322Gln Lys Tyr Ile Ser Ala
Pro Tyr Thr1 53239PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 323Gln Lys Tyr Asn Arg Pro
Pro Tyr Thr1 53249PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 324Gln Arg Tyr Asn Asp Ala
Pro Tyr Thr1 53259PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 325Gln Arg Tyr Ala Arg Ala
Pro Tyr Thr1 53269PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 326Gln Arg Tyr Asn Ala Ala
Pro Tyr Thr1 53279PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 327Gln Arg Tyr Asn Arg Ala
Ala Tyr Thr1 53289PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 328Gln Arg Tyr Asn Arg Ala
Pro Ala Thr1 53299PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 329Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr1 53309PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 330Gln Ser Asp Asn Phe Ala
Thr Tyr Tyr1 53319PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 331Gln Arg Tyr Asp Lys Pro
Pro Tyr Thr1 53329PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 332Gln Arg Tyr Asn Lys Pro
Pro Tyr Thr1 53339PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 333Gln Arg Tyr Asp Arg Pro
Pro Tyr Thr1 53349PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 334Gln Arg Tyr Asp Arg Ala
Pro Tyr Thr1 53359PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 335Gln Arg Tyr Asn Lys Ala
Pro Tyr Thr1 53369PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 336Gln Arg Tyr Asn Arg Pro
Pro Tyr Thr1 53379PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 337Gln Lys Tyr Gln Arg Ala
Pro Tyr Thr1 53389PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 338Gln Lys Tyr Ser Ser Ala
Pro Tyr Ala1 53399PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 339Ala Arg Tyr Asn Arg Ala
Pro Tyr Thr1 53409PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 340Gln Ala Tyr Asn Arg Ala
Pro Tyr Thr1 53419PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 341Gln Arg Ala Asn Arg Ala
Pro Tyr Thr1 53429PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 342Gln Pro Glu Asp Val Ala
Thr Tyr Tyr1 53439PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 343Gln Pro Glu Asp Val Ala
Ala Tyr Tyr1 5
* * * * *