U.S. patent application number 15/466113 was filed with the patent office on 2017-07-13 for anti-tigit antibodies.
This patent application is currently assigned to Merck Sharp & Dohme Corp.. The applicant listed for this patent is Merck Sharp & Dohme Corp.. Invention is credited to Laurence Fayadat-Dilman, Drake LaFace, Linda Liang, Gopalan Raghunathan, Wolfgang Seghezzi, Sybil M. G. Williams.
Application Number | 20170198042 15/466113 |
Document ID | / |
Family ID | 53938463 |
Filed Date | 2017-07-13 |
United States Patent
Application |
20170198042 |
Kind Code |
A1 |
Williams; Sybil M. G. ; et
al. |
July 13, 2017 |
ANTI-TIGIT ANTIBODIES
Abstract
The present invention relates to anti-TIGIT antibodies, as well
as use of these antibodies in the treatment of diseases such as
cancer and infectious disease.
Inventors: |
Williams; Sybil M. G.;
(Wayland, MA) ; LaFace; Drake; (Half Moon Bay,
CA) ; Fayadat-Dilman; Laurence; (Sunnyvale, CA)
; Raghunathan; Gopalan; (San Diego, CA) ; Liang;
Linda; (Mountain View, CA) ; Seghezzi; Wolfgang;
(Mountain View, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merck Sharp & Dohme Corp. |
Rahway |
NJ |
US |
|
|
Assignee: |
Merck Sharp & Dohme
Corp.
Rahway
NJ
|
Family ID: |
53938463 |
Appl. No.: |
15/466113 |
Filed: |
March 22, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15121624 |
Aug 25, 2016 |
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PCT/US2015/045447 |
Aug 17, 2015 |
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15466113 |
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62126733 |
Mar 2, 2015 |
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62038912 |
Aug 19, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2317/21 20130101; A61P 31/00 20180101; C07K 2317/74 20130101;
A61K 39/3955 20130101; A61K 2039/507 20130101; A61K 45/06 20130101;
C07K 2317/56 20130101; C07K 2317/73 20130101; C07K 2317/76
20130101; C07K 2317/92 20130101; C07K 16/2803 20130101; A61P 37/02
20180101; C07K 16/28 20130101; C07K 2317/52 20130101; A61P 37/04
20180101; C07K 2317/34 20130101; C07K 2317/565 20130101; C07K
2317/24 20130101; C07K 16/2818 20130101; A61P 43/00 20180101; A61P
35/00 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 45/06 20060101 A61K045/06; A61K 39/395 20060101
A61K039/395 |
Claims
1. An antibody or antigen binding fragment thereof that binds to
human TIGIT, wherein the antibody or antigen binding fragment is
selected from the group consisting of: a. an antibody or antigen
binding fragment comprising: a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO:1, a heavy chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO:2, a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO:3, 79, 80, 81, 82, 83 or 140, a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO:4, a light chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO:5, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 41,
and a light chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO:6, 74, 75, 76, 77, 78 or 142; b. an antibody
or antigen binding fragment comprising: a heavy chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO:57, a
heavy chain variable region CDR2 comprising the amino acid sequence
of SEQ ID NO:58, a heavy chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO:59, a light chain variable region
CDR1 comprising the amino acid sequence of SEQ ID NO:60, a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO:61, and a light chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO:62; and c. an antibody or antigen
binding fragment comprising: a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO:88, a heavy chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO:89, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 134, 135 or 147, a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO:90, 153, 154, 155,
156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 or 167, a
light chain variable region CDR1 comprising the amino acid sequence
of SEQ ID NO:91, a light chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO:92, 112, 113, 114, 115, 116, 117,
118, 119, 120, 121, 122 or 148, and a light chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO:93.
2. The antibody or antigen binding fragment of claim 1, wherein the
antibody comprises a heavy chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO:1, a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO:2, a
heavy chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO:3, a light chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO:4, a light chain variable region
CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a light
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO:6.
3. The antibody or antigen binding fragment of claim 1, wherein the
antibody comprises a heavy chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO:88, a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO:89, 134
or 135, a heavy chain variable region CDR3 comprising the amino
acid sequence of SEQ ID NO:90, a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO:91, a light chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO:92, and a light chain variable region CDR3 comprising the amino
acid sequence of SEQ ID NO:93.
4. The antibody or antigen binding fragment of claim 1, wherein the
antibody comprises a heavy chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO:88, a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 134, a
heavy chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO:90, a light chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO:91, a light chain variable region
CDR2 comprising the amino acid sequence of SEQ ID NO:92, and a
light chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO:93.
5. An antibody or antigen binding fragment thereof that binds to
human TIGIT comprising a light chain immunoglobulin, a heavy chain
immunoglobulin or both a light chain and a heavy chain
immunoglobulin selected from the group consisting of: a. an
antibody or antigen binding fragment thereof comprising a variable
heavy chain comprising the amino acid sequence of SEQ ID NO:7
and/or a variable light chain comprising the amino acid sequence of
SEQ ID NO:8; b. an antibody or antigen binding fragment thereof
comprising a variable heavy chain comprising the amino acid
sequence of SEQ ID NO:63 and/or a variable light chain comprising
the amino acid sequence of SEQ ID NO:64; c. an antibody or antigen
binding fragment thereof comprising a variable heavy chain
comprising the amino acid sequence of SEQ ID NO: 94 and/or a
variable light chain comprising the amino acid sequence of SEQ ID
NO:95; d. an antibody or antigen binding fragment thereof
comprising a variable heavy chain selected from the group
consisting of SEQ ID NOs: 9-24, 37-47 and 63 and/or a variable
light chain selected from the group consisting of any one of SEQ ID
NOs: 25-30, 48-52 and 64; e. an antibody or antigen binding
fragment thereof comprising a variable heavy chain selected from
the group consisting of SEQ ID NOs: 124-129 and/or a variable light
chain selected from the group consisting of any one of SEQ ID NOs:
130-133; f. an antibody or antigen binding fragment thereof
comprising a variable heavy chain comprising the amino acid
sequence of SEQ ID NO:128 and/or a variable light chain comprising
the amino acid sequence of SEQ ID NO:132; g. an antibody or antigen
binding fragment thereof comprising a variable heavy chain
comprising the amino acid sequence of SEQ ID NO:127 and/or a
variable light chain comprising the amino acid sequence of SEQ ID
NO:130; and h. an antibody or antigen binding fragment thereof
comprising a variable heavy chain comprising the amino acid
sequence of SEQ ID NO:128 and/or a variable light chain comprising
the amino acid sequence of SEQ ID NO:133. i. an antibody or antigen
binding fragment thereof comprising a variable heavy chain
comprising the amino acid sequence of SEQ ID NO:143 and/or a
variable light chain comprising the amino acid sequence of SEQ ID
NO:145; j. an antibody or antigen binding fragment thereof
comprising a variable heavy chain comprising the amino acid
sequence of SEQ ID NO:149 and/or a variable light chain comprising
the amino acid sequence of SEQ ID NO:151; k. an antibody or antigen
binding fragment thereof comprising a variable heavy chain
comprising the amino acid sequence of SEQ ID NO:144 and/or a
variable light chain comprising the amino acid sequence of SEQ ID
NO:146. l. an antibody or antigen binding fragment thereof
comprising a variable heavy chain comprising the amino acid
sequence of SEQ ID NO:150 and/or a variable light chain comprising
the amino acid sequence of SEQ ID NO:152; m. an antibody or antigen
binding fragment thereof comprising a variable heavy chain
comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity any one
of SEQ ID NOs: 7, 9-24, 37-47 and 63 and/or a variable light chain
comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity to any
one of SEQ ID NOs: 8, 25-30, 48-52 and 64; n. an antibody or
antigen binding fragment thereof comprising a variable heavy chain
comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity any one
of SEQ ID NOs: 7, 9-24, 37-47 and 63 and/or a variable light chain
comprising at least 90%, 95%, 95%, 96%, 97%, 98% or 99% identity to
any one of SEQ ID NOs: 8, 25-30, 48-52 and 64, wherein any sequence
variations occur in the framework regions of the antibody; o. an
antibody or antigen binding fragment thereof comprising a variable
heavy chain comprising at least 90%, 95%, 96%, 97%, 98% or 99%
identity any one of SEQ ID NOs: 124-129 and/or a variable light
chain comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity
to any one of SEQ ID NOs: 130-133; and p. an antibody or antigen
binding fragment thereof comprising a variable heavy chain
comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity any one
of SEQ ID NOs: 124-129 and/or a variable light chain comprising at
least 90%, 95%, 95%, 96%, 97%, 98% or 99% identity to any one of
SEQ ID NOs: 130-133, wherein any sequence variations occur in the
framework regions of the antibody; q. an antibody or antigen
binding fragment thereof comprising a variable heavy chain
comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity SEQ ID
NO:128 and/or a variable light chain comprising at least 90%, 95%,
96%, 97%, 98% or 99% identity to any one of SEQ ID NOs: 132; r. an
antibody or antigen binding fragment thereof comprising a variable
heavy chain comprising at least 90%, 95%, 96%, 97%, 98% or 99%
identity SEQ ID NO:128 and/or a variable light chain comprising at
least 90%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID
NOs: 132, wherein any sequence variations occur in the framework
regions of the antibody; s. an antibody or antigen binding fragment
thereof comprising a variable heavy chain comprising at least 90%,
95%, 96%, 97%, 98% or 99% identity SEQ ID NO:127 and/or a variable
light chain comprising at least 90%, 95%, 96%, 97%, 98% or 99%
identity to any one of SEQ ID NOs: 130; t. an antibody or antigen
binding fragment thereof comprising a variable heavy chain
comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity SEQ ID
NO:127 and/or a variable light chain comprising at least 90%, 95%,
96%, 97%, 98% or 99% identity to any one of SEQ ID NOs: 130,
wherein any sequence variations occur in the framework regions of
the antibody; u. an antibody or antigen binding fragment thereof
comprising a variable heavy chain comprising at least 90%, 95%,
96%, 97%, 98% or 99% identity SEQ ID NO:128 and/or a variable light
chain comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity
to any one of SEQ ID NOs: 133; v. an antibody or antigen binding
fragment thereof comprising a variable heavy chain comprising at
least 90%, 95%, 96%, 97%, 98% or 99% identity SEQ ID NO:128 and/or
a variable light chain comprising at least 90%, 95%, 96%, 97%, 98%
or 99% identity to any one of SEQ ID NOs: 133, wherein any sequence
variations occur in the framework regions of the antibody.
6. The antibody or antigen binding fragment of claim 1, wherein the
antibody or antigen binding fragment thereof binds to human TIGIT
with a KD value of about 1.times.10-9 M to about 1.times.10-12 M as
determined by surface plasmon resonance (e.g., BIACORE) or a
similar technique (e.g. KinExa or OCTET).
7. (canceled)
8. (canceled)
9. (canceled)
10. The antibody or antigen binding fragment of claim 1, which is a
humanized antibody comprising two heavy chains and two light
chains.
11. The antibody or antigen binding fragment of claim 1, which is a
humanized antibody, wherein the antibody comprises a human IgG1
constant domain and a human kappa constant domain.
12. (canceled)
13. An isolated polypeptide comprising the amino acid sequence of
any one of SEQ ID NOs: 7-30, 37-52, 63-64, 94-95 or 124-133,
136-139, 143-146, 149-151.
14. An isolated nucleic acid encoding: any one of the antibodies or
antigen binding fragments of claim 1.
15. An expression vector comprising the isolated nucleic acid of
claim 14.
16. A host cell comprising the expression vector of claim 15.
17. (canceled)
18. A composition comprising the antibody or antigen binding
fragment of claim 1 and a pharmaceutically acceptable carrier or
diluent.
19. The composition of claim 18, further comprising an agent
selected from the group consisting of: a. an anti-PD1 antibody or
an antigen binding fragment thereof; b. an anti-LAG3 antibody or an
antigen binding fragment thereof; c. an anti-VISTA antibody or an
antigen binding fragment thereof; d. an anti-BTLA antibody or an
antigen binding fragment thereof; e. an anti-TIM3 antibody or an
antigen binding fragment thereof; f. an anti-CTLA4 antibody or an
antigen binding fragment thereof; g. an anti-HVEM antibody or an
antigen binding fragment thereof; h. an anti-CD27 antibody or an
antigen binding fragment thereof; i. an anti-CD137 antibody or an
antigen binding fragment thereof; j. an anti-OX40 antibody or an
antigen binding fragment thereof; k. an anti-CD28 antibody or an
antigen binding fragment thereof; l. an anti-PDL1 antibody or an
antigen binding fragment thereof; m. an anti-PDL2 antibody or an
antigen binding fragment thereof; n. an anti-GITR antibody or an
antigen binding fragment thereof; o. an anti-ICOS antibody or an
antigen binding fragment thereof; p. an anti-SIRP.alpha. antibody
or an antigen binding fragment thereof; q. an anti-ILT2 antibody or
an antigen binding fragment thereof; r. an anti-ILT3 antibody or an
antigen binding fragment thereof; s. an anti-ILT4 antibody or an
antigen binding fragment thereof; and t. an anti-ILT5 antibody or
an antigen binding fragment thereof.
20. The composition of claim 19, wherein the anti-PD1 antibody or
an antigen binding fragment thereof is selected from the group
consisting of: pembrolizumab or an antigen binding fragment thereof
and nivolumab or an antigen binding fragment thereof.
21. (canceled)
22. A method of treating cancer, an infection or infectious disease
in a human subject, comprising administering to the subject an
effective amount of the antibody or antigen binding fragment of
claim 1, optionally in association with a further therapeutic agent
or therapeutic procedure.
23. (canceled)
24. A vaccine comprising the antibody or antigen binding fragment
of claim 1 and an antigen.
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to anti-TIGIT antibodies, as
well as use of these antibodies in the treatment of diseases such
as cancer and infectious disease.
BACKGROUND OF THE INVENTION
[0002] A key factor for enabling tumor immunotherapy emerged from
discoveries that inhibitory immune modulatory receptors (IMRs),
that generally function as immune checkpoints to maintain
self-tolerance, are central to the ability of tumor
microenvironments to evade immunity. Blockade of inhibitory IMRs
appears to unleash potent tumor-specific immune responses more
effectively than direct stimulation of tumor-immunity with
activating cytokines or tumor vaccines, and this approach has the
potential to transform human cancer therapy. An important
implication and opportunity now arises for the potential to develop
new antibody antagonists for other IMRs and to combine antagonist
antibodies to more than one IMR in order to increase the proportion
of responders in oncology clinical trials, as well as, expand upon
oncology indications in which tumor immunotherapy treatments are
effective.
[0003] Significantly, inhibitory IMRs and ligands that regulate
cellular immunity are commonly overexpressed on tumor cells and
tumor associated macrophages (TAMs). Notably, overexpression of
PD-L1 in tumors is associated with tumor specific T cell exhaustion
and a poor prognosis. Blockade of PD-1/PD-L1 ligation in clinical
trials resulted in durable tumor regression responses in a
substantial proportion of patients. A recent report demonstrated
that co-expression of PD-1 and another inhibitory IMR (TIM-3) in
melanoma patient derived tumor-specific CD8+ T cells was associated
with more dysfunctional T cell exhaustion phenotypes compared to
cells expressing either IMR alone. Moreover, several reports using
pre-clinical tumor models demonstrated blockade of multiple IMRs,
including PD-1, TIM-3, LAG-3 and CTLA-4 more effectively induced
anti-tumor responses than antagonizing PD-1 alone. These results
underscore the importance of further investigating IMR
pathways.
[0004] TIGIT (T cell immunoreceptor with Ig and ITIM domains) is an
immunomodulatory receptor expressed primarily on activated T cells
and NK cells. TIGIT is also known as VSIG9; VSTM3; and WUCAM. Its
structure shows one extracellular immunoglobulin domain, a type 1
transmembrane region and two ITIM motifs. TIGIT forms part of a
co-stimulatory network that consists of positive (CD226) and
negative (TIGIT) immunomodulatory receptors on T cells, and ligands
expressed on APCs (CD155 and CD112).
[0005] An important feature in the structure of TIGIT is the
presence of an immunoreceptor tyrosine-based inhibition motif
(ITIM) in its cytoplasmic tail domain. As with PD-1 and CTLA-4, the
ITIM domain in the cytoplasmic region of TIGIT is predicted to
recruit tyrosine phosphatases, such as SHP-1 and SHP-2, and
subsequent de-phosphorylation of tyrosine residues with in the
immunoreceptor tyrosine-base activation motifs (ITAM) on T cell
receptor (TCR) subunits. Hence, ligation of TIGIT by
receptor-ligands CD155 and CD112 expressed by tumor cells or TAMS
may contribute to the suppression of TCR-signaling and T cell
activation, which is essential for mounting effective anti-tumor
immunity. Thus, an antagonist antibody specific for TIGIT could
inhibit the CD155 and CD112 induced suppression of T cell responses
and enhance anti-tumor immunity. It is an object of the present
invention to obtain an anti-TIGIT antibody that can be used for the
treatment of cancer, either alone or in combination with other
reagents.
SUMMARY OF THE INVENTION
[0006] The invention provides anti-TIGIT antibodies and antigen
binding fragments thereof comprising the structural and functional
features specified below.
[0007] In one embodiment, the invention provides an antibody or
antigen binding fragment thereof that binds to human TIGIT,
comprising: a heavy chain variable region CDR3 comprising the amino
acid sequence of SEQ ID NO: 3, 79, 80, 81, 82, 83, 59, 90, 140,
153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,
166 or 167. In one embodiment, the antibody or antigen binding
fragment thereof optionally has at least one of the following
characteristics: (i) binds to human TIGIT with a KD value of about
1.times.10-9 M to about 1.times.10-12 M as determined by surface
plasmon resonance (e.g., BIACORE) or a similar technique (e.g.
KinExa or OCTET); (ii) cross-reacts with cynomolgous and rhesus
TIGIT; (iii) blocks binding of human TIGIT to human CD155 and human
CD112; (iv) increases T cell activation; (v) stimulates
antigen-specific T-cell production of IL-2 and IFN.gamma.; (vi)
blocks induction of T cell suppression of activation induced by
TIGIT ligation with cognate ligands CD and CD112.
[0008] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof that binds to human TIGIT,
comprising: a light chain variable region CDR3 comprising the amino
acid sequence of SEQ ID NO: 6, 62, 74, 75, 76, 77, 78 or 93. In one
embodiment, the antibody optionally has at least one of the
following characteristics: (i) binds to human TIGIT with a KD value
of about 1.times.10-9 M to about 1.times.10-12 M as determined by
surface plasmon resonance (e.g., BIACORE) or a similar technique
(e.g. KinExa or OCTET); (ii) cross-reacts with cynomolgous and
rhesus TIGIT; (iii) blocks binding of human TIGIT to human CD155
and human CD112; (iv) increases T cell activation; (v) stimulates
antigen-specific T-cell production of IL-2 and IFN.gamma.; (vi)
blocks induction of T cell suppression of activation induced by
TIGIT ligation with cognate ligands CD and CD112.
[0009] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof that binds to human TIGIT
comprising: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 140. In one embodiment, the antibody or
antigen binding fragment comprises: (i) a heavy chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 1;
(ii) a heavy chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 2; and (iii) a heavy chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 79, 80,
81, 82 or 83. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 2; and (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 3. In one
embodiment, the antibody or antigen binding fragment thereof
optionally has at least one of the following characteristics: (i)
binds to human TIGIT with a KD value of about 1.times.10-9 M to
about 1.times.10-12 M as determined by surface plasmon resonance
(e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); (ii)
cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocks
binding of human TIGIT to human CD155 and human CD112; (iv)
increases T cell activation; (v) stimulates antigen-specific T-cell
production of IL-2 and IFN.gamma.; (vi) blocks induction of T cell
suppression of activation induced by TIGIT ligation with cognate
ligands CD and CD112.
[0010] In another embodiment, the invention provides an antibody or
antigen binding fragment that binds to human TIGIT comprising: (i)
a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 57; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 58; and (iii) a
heavy chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO: 59. In one embodiment, the antibody or antigen
binding fragment thereof optionally has at least one of the
following characteristics: (i) binds to human TIGIT with a KD value
of about 1.times.10-9 M to about 1.times.10-12 M as determined by
surface plasmon resonance (e.g., BIACORE) or a similar technique
(e.g. KinExa or OCTET); (ii) cross-reacts with cynomolgous and
rhesus TIGIT; (iii) blocks binding of human TIGIT to human CD155
and human CD112; (iv) increases T cell activation; (v) stimulates
antigen-specific T-cell production of IL-2 and IFN.gamma.; (vi)
blocks induction of T cell suppression of activation induced by
TIGIT ligation with cognate ligands CD155 and CD112.
[0011] In another embodiment, the invention provides an antibody or
antigen binding fragment that binds to human TIGIT comprising: (i)
a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 147; and (iii) a
heavy chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO: 153. In one embodiment, the antibody or antigen
binding fragment thereof comprises: (i) a heavy chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 88;
(ii) a heavy chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 89, 96, 97, 98, 99, 100, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 134 or 135; and (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 90, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,
164, 165, 166 or 167. In one embodiment, the antibody or antigen
binding fragment thereof comprises: (i) a heavy chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 88;
(ii) a heavy chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 89, 134 or 135; and (iii) a heavy chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 90. In one embodiment, the antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; and (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 90. In one
embodiment, the antibody or antigen binding fragment thereof
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 134;
and (iii) a heavy chain variable region CDR3 comprising the amino
acid sequence of SEQ ID NO: 154. In one embodiment, the antibody or
antigen binding fragment thereof comprises: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 88; (ii) a heavy chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO: 134; and (iii) a heavy chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 155. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; and (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 156. In one
embodiment, the antibody or antigen binding fragment thereof
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 134;
and (iii) a heavy chain variable region CDR3 comprising the amino
acid sequence of SEQ ID NO: 157. In one embodiment, the antibody or
antigen binding fragment thereof comprises: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 88; (ii) a heavy chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO: 134; and (iii) a heavy chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 158. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; and (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 159. In one
embodiment, the antibody or antigen binding fragment thereof
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 134;
and (iii) a heavy chain variable region CDR3 comprising the amino
acid sequence of SEQ ID NO: 160. In one embodiment, the antibody or
antigen binding fragment thereof comprises: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 88; (ii) a heavy chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO: 134; and (iii) a heavy chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 161. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; and (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 162. In one
embodiment, the antibody or antigen binding fragment thereof
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 134;
and (iii) a heavy chain variable region CDR3 comprising the amino
acid sequence of SEQ ID NO: 163. In one embodiment, the antibody or
antigen binding fragment thereof comprises: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 88; (ii) a heavy chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO: 134; and (iii) a heavy chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 164. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; and (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 165. In one
embodiment, the antibody or antigen binding fragment thereof
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 134;
and (iii) a heavy chain variable region CDR3 comprising the amino
acid sequence of SEQ ID NO: 166. In one embodiment, the antibody or
antigen binding fragment thereof comprises: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 88; (ii) a heavy chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO: 134; and (iii) a heavy chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 167. In one embodiment, the antibody or antigen binding
fragment thereof optionally has at least one of the following
characteristics: (i) binds to human TIGIT with a KD value of about
1.times.10-9 M to about 1.times.10-12 M as determined by surface
plasmon resonance (e.g., BIACORE) or a similar technique (e.g.
KinExa or OCTET); (ii) cross-reacts with cynomolgous and rhesus
TIGIT; (iii) blocks binding of human TIGIT to human CD155 and human
CD112; (iv) increases T cell activation; (v) stimulates
antigen-specific T-cell production of IL-2 and IFN.gamma.; (vi)
blocks induction of T cell suppression of activation induced by
TIGIT ligation with cognate ligands CD and CD112.
[0012] In another embodiment, the invention provides an antibody or
antigen binding fragment that binds to human TIGIT comprising: (i)
a light chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 4; (ii) a light chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 141; and (iii) a
light chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO: 142. In one embodiment, the antibody or antigen
binding fragment thereof comprises: (i) a light chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 4;
(ii) a light chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 5, 65, 66, 67, 68, 69, 70, 71, 72 or; and
(iii) a light chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 6, 74, 75, 76, 77 or 78. In one embodiment,
the antibody or antigen binding fragment thereof comprises: (i) a
light chain variable region CDR1 comprising the amino acid sequence
of SEQ ID NO: 4; (ii) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 5; and (iii) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 6. In one embodiment, the antibody or antigen binding fragment
thereof optionally has at least one of the following
characteristics: (i) binds to human TIGIT with a KD value of about
1.times.10-9 M to about 1.times.10-12 M as determined by surface
plasmon resonance (e.g., BIACORE) or a similar technique (e.g.
KinExa or OCTET); (ii) cross-reacts with cynomolgous and rhesus
TIGIT; (iii) blocks binding of human TIGIT to human CD155 and human
CD112; (iv) increases T cell activation; (v) stimulates
antigen-specific T-cell production of IL-2 and IFN.gamma.; (vi)
blocks induction of T cell suppression of activation induced by
TIGIT ligation with cognate ligands CD155 and CD112.
[0013] In another embodiment, the invention provides an antibody or
antigen binding fragment that binds to human TIGIT comprising: (i)
a light chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 60; (ii) a light chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 61; and (iii) a
light chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO: 62. In one embodiment, the antibody or antigen
binding fragment thereof optionally has at least one of the
following characteristics: (i) binds to human TIGIT with a KD value
of about 1.times.10-9 M to about 1.times.10-12 M as determined by
surface plasmon resonance (e.g., BIACORE) or a similar technique
(e.g. KinExa or OCTET); (ii) cross-reacts with cynomolgous and
rhesus TIGIT; (iii) blocks binding of human TIGIT to human CD155
and human CD112; (iv) increases T cell activation; (v) stimulates
antigen-specific T-cell production of IL-2 and IFN.gamma.; (vi)
blocks induction of T cell suppression of activation induced by
TIGIT ligation with cognate ligands CD and CD112.
[0014] In another embodiment, the invention provides an antibody or
antigen binding fragment that binds to human TIGIT comprising: (i)
a light chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 91; (ii) a light chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 148; and (iii) a
light chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO: 93. In one embodiment, the antibody or antigen
binding fragment thereof comprises: (i) a light chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 91;
(ii) a light chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 92, 112, 113, 114, 115, 116, 117, 118, 119,
120, 121 or 122; and (iii) a light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 93. In one
embodiment, the antibody or antigen binding fragment thereof
comprises: (i) a light chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 91; (ii) a light chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 92;
and (iii) a light chain variable region CDR3 comprising the amino
acid sequence of SEQ ID NO: 93. In one embodiment, the antibody or
antigen binding fragment thereof optionally has at least one of the
following characteristics: (i) binds to human TIGIT with a KD value
of about 1.times.10-9 M to about 1.times.10-12 M as determined by
surface plasmon resonance (e.g., BIACORE) or a similar technique
(e.g. KinExa or OCTET); (ii) cross-reacts with cynomolgous and
rhesus TIGIT; (iii) blocks binding of human TIGIT to human CD155
and human CD112; (iv) increases T cell activation; (v) stimulates
antigen-specific T-cell production of IL-2 and IFN.gamma.; (vi)
blocks induction of T cell suppression of activation induced by
TIGIT ligation with cognate ligands CD155 and CD112.
[0015] In another embodiment, the invention provides an antibody or
antigen binding fragment that binds to human TIGIT comprising: (i)
a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 140; (iv) a light chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 141;
and (vi) a light chain variable region CDR3 comprising the amino
acid sequence of SEQ ID NO: 142. In one embodiment, the antibody or
antigen binding fragment comprising: (i) a heavy chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 1;
(ii) a heavy chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 3, 79, 80, 81, 82
or 83; (iv) a light chain variable region CDR1 comprising the amino
acid sequence of SEQ ID NO: 4; (v) a light chain variable region
CDR2 comprising the amino acid sequence of SEQ ID NO: 5, 65, 66,
67, 68, 69, 70, 71, 72 or 73; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 6, 74,
75, 76, 77 or 78. In one embodiment, the antibody or antigen
binding fragment thereof comprises: (i) a heavy chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 1;
(ii) a heavy chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO: 5; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 6. In one embodiment, the antibody or antigen binding fragment
thereof is humanized. In one embodiment, the antibody or antigen
binding fragment thereof comprises a heavy chain variable region
selected from the group consisting of SEQ ID NOs:9-24, 37-47, 143
and 144; and a light chain variable region selected from the group
consisting of SEQ ID NOs: 25-30, 48-52, 146 and 147. In one
embodiment, the antibody or antigen binding fragment thereof
optionally has at least one of the following characteristics: (i)
binds to human TIGIT with a KD value of about 1.times.10-9 M to
about 1.times.10-12 M as determined by surface plasmon resonance
(e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); (ii)
cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocks
binding of human TIGIT to human CD155 and human CD112; (iv)
increases T cell activation; (v) stimulates antigen-specific T-cell
production of IL-2 and IFN.gamma.; (vi) blocks induction of T cell
suppression of activation induced by TIGIT ligation with cognate
ligands CD and CD112.
[0016] In another embodiment, the invention provides an antibody or
antigen binding fragment that binds to human TIGIT comprising: (i)
a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 3, 79, 80, 81, 82 or 83; (iv) a light chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v)
a light chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 5, 65, 66, 67, 68, 69, 70, 71, 72 or 73; and
(vi) a light chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 6, 74, 75, 76, 77 or 78; wherein the
antibody or antigen binding fragment thereof comprises a heavy
chain variable region comprising at least 90%, 95%, 96%, 97%, 98%
or 99% identity to a heavy chain variable region selected from the
group consisting of SEQ ID NOs: 9-24 or 37-47 and a light chain
variable region comprising at least 90%, 95%, 96%, 97%, 98% or 99%
identity to a heavy chain variable region selected from the group
consisting of SEQ ID NOs: 25-30 or 48-52. In this aforementioned
embodiment, the sequence variations occur in the framework regions.
In one embodiment, the antibody binds to human TIGIT with a KD
value of about 1.times.10-9 M to about 1.times.10-12 M as
determined by surface plasmon resonance (e.g., BIACORE) or a
similar technique (e.g. KinExa or OCTET).
[0017] In another embodiment, the invention also provides an
antibody or antigen binding fragment thereof that binds to human
TIGIT comprising: (i) a heavy chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO: 2; (iii) a heavy chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v)
a light chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 5; and (vi) a light chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In one
embodiment, the antibody or antigen binding fragment thereof
comprises 1, 2 or 3 amino acid substitutions in the heavy chain
CDRs (SEQ ID NOs: 1-3) or in the light chain CDRs (SEQ ID NOs:
3-6). In one embodiment, the antibody comprises one amino acid
substitution in the heavy chain CDR of SEQ ID NO: 3, wherein the
substitution is made at position 13W, and wherein residue 13W is
substituted to: F, Y, I, V or L. In one embodiment, the antibody
comprises two amino acid substitutions in the light chain CDR of
SEQ ID NO: 5, wherein the substitutions are made at position 3-4,
and wherein residues 3N and 4S are substituted to: SN, SS, ST, TT,
SY, NQ, GS, SQ and DS. In one embodiment, the antibody comprises
one amino acid substitution in the light chain CDR of SEQ ID NO: 6,
wherein the substitution is made at position 7, and wherein residue
7W is substituted to: F, Y, I, V or L. The VH sequences of SEQ ID
NOs: 9-24 and 37-47 have the CDRs of SEQ ID NOs:1-3; and VL
sequences of SEQ ID NOs:25-30 and 48-52 have the CDRs of SEQ ID
NOs: 4-6. In some embodiments, the CDR substitutions described
above can be made in the corresponding CDRs of the VH sequences of
SEQ ID NOs: 9-24 and 37-37, and in the CDRs of the VL sequences of
SEQ ID NOs: 25-30 and 48-52. In one embodiment, the antibody binds
to human TIGIT with a KD value of about 1.times.10-9 M to about
1.times.10-12 M as determined by surface plasmon resonance (e.g.,
BIACORE) or a similar technique (e.g. KinExa or OCTET).
[0018] In another embodiment, the invention provides an antibody or
antigen binding fragment that binds to human TIGIT comprising: (i)
a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 57; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 58; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 59; (iv) a light chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO: 60; (v) a light chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO: 61; and (vi) a light chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO: 62. In one embodiment, the
antibody or antigen binding fragment thereof is humanized. In one
embodiment, the antibody or antigen binding fragment thereof
comprises 1, 2 or 3 amino acid substitutions in the heavy chain
CDRs (SEQ ID NOs: 57-59) or in the light chain CDRs (SEQ ID NOs:
60-62), and retains one or more of its functional characteristics.
In one embodiment, the antibody or antigen binding fragment thereof
optionally has at least one of the following functional
characteristics: (i) binds to human TIGIT with a KD value of about
1.times.10-9 M to about 1.times.10-12 M as determined by surface
plasmon resonance (e.g., BIACORE) or a similar technique (e.g.
KinExa or OCTET), (ii) cross-reacts with cynomolgous and rhesus
TIGIT; (iii) blocks binding of human TIGIT to human CD155 and human
CD112; (iv) increases T cell activation; (v) stimulates
antigen-specific T-cell production of IL-2 and IFN.gamma.; (vi)
blocks induction of T cell suppression of activation induced by
TIGIT ligation with cognate ligands CD155 and CD112.
[0019] In another embodiment, the invention also provides an
antibody or antigen binding fragment thereof that binds to human
TIGIT comprising: (i) a heavy chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO: 88; (ii) a heavy chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO: 147; (iii) a heavy chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO: 153; (iv) a light chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 91;
(v) a light chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 148; and (vi) a light chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In one
embodiment, the antibody or antigen binding fragment thereof
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 89,
96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,
110, 111, 134 or 135; (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 90, 154, 155, 156,
157, 158, 159, 160, 161, 162, 163, 164, 165, 166 or 167; (iv) a
light chain variable region CDR1 comprising the amino acid sequence
of SEQ ID NO: 91; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 92, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121 or 122; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 89,
134 or 135; (iii) a heavy chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO: 90; (iv) a light chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 91;
(v) a light chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 92; and (vi) a light chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In one
embodiment, the antibody or antigen binding fragment thereof
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 134;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 90; (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 91; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 92; and (vi) a light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 93. In one
embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 154; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 155; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 156; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 157; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 158; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 159; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 160; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 161; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 162; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 163; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 164; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 165; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 166; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 167; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
one embodiment, the antibody or antigen binding fragment thereof is
humanized. In one embodiment, the antibody or antigen binding
fragment thereof optionally has at least one of the following
characteristics: (i) binds to human TIGIT with a KD value of about
1.times.10-9 M to about 1.times.10-12 M as determined by surface
plasmon resonance (e.g., BIACORE) or a similar technique (e.g.
KinExa or OCTET), (ii) cross-reacts with cynomolgous and rhesus
TIGIT; (iii) blocks binding of human TIGIT to human CD155 and human
CD112; (iv) increases T cell activation; (v) stimulates
antigen-specific T-cell production of IL-2 and IFN.gamma.; (vi)
blocks induction of T cell suppression of activation induced by
TIGIT ligation with cognate ligands CD and CD112.
[0020] In one embodiment, the antibody or antigen binding fragment
thereof binds to human TIGIT and comprises: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 88; (ii) a heavy chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO: 89; (iii) a heavy chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 90;
(iv) a light chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 92; and (vi) a
light chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO: 93. In one embodiment, the antibody comprises one
amino acid substitution in the heavy chain CDR2 of SEQ ID NO: 89,
wherein the substitution is made at position 7, and wherein residue
D is substituted with: R, L, K, F, S, Y or V. In another
embodiment, the antibody comprises one amino acid substitution in
the heavy chain CDR2 of SEQ ID NO: 89, wherein the substitution is
made at position 8, and wherein residue G is substituted with: R,
N, Q, E, L K, S, Y or V. In another embodiment, the antibody
comprises one amino acid substitution in the heavy chain CDR2 of
SEQ ID NO: 89, wherein the substitution is made at position 12,
wherein the N is substituted with: A or S. In another embodiment,
the antibody comprises one amino acid substitution in the heavy
chain CDR2 of SEQ ID NO: 89, wherein the substitution is made at
position 13, wherein E is substituted with Q. In another
embodiment, the antibody comprises one amino acid substitution in
the heavy chain CDR2 of SEQ ID NO: 89, wherein the substitution is
made at position 16, wherein K is substituted with Q. In another
embodiment, the antibody comprises three amino acid substitution in
the heavy chain CDR2 of SEQ ID NO: 89, wherein amino acid residue
12 is substituted with: A or S, amino acid residue 13 is
substituted with Q and amino acid residue 16 is substituted with Q.
In one embodiment, the antibody comprises one amino acid
substitution in the heavy chain CDR3 of SEQ ID NO:90, wherein amino
acid residue 6 is substituted with: A, D, E, F, G, I, K, N, Q, R,
S, T V or Y. In another embodiment, the antibody comprises one
amino acid substitution in the light chain CDR of SEQ ID NO: 92,
wherein the substitution is made at position 1, and wherein residue
N is substituted with A, Y, W, S, T, R, H, G, I or V. In another
embodiment, the antibody comprises one amino acid substitution in
the light chain CDR of SEQ ID NO: 92, wherein the substitution is
made at position 2, and wherein residue A is substituted with N, I,
L, T, V. In one embodiment, the antibody or antigen binding
fragment thereof is humanized. In one embodiment, the antibody or
antigen binding fragment thereof optionally has at least one of the
following characteristics: (i) binds to human TIGIT with a KD value
of about 1.times.10-9 M to about 1.times.10-12 M as determined by
surface plasmon resonance (e.g., BIACORE) or a similar technique
(e.g. KinExa or OCTET), (ii) cross-reacts with cynomolgous and
rhesus TIGIT; (iii) blocks binding of human TIGIT to human CD155
and human CD112; (iv) increases T cell activation; (v) stimulates
antigen-specific T-cell production of IL-2 and IFN.gamma.; (vi)
blocks induction of T cell suppression of activation induced by
TIGIT ligation with cognate ligands CD155 and CD112.
[0021] In another embodiment, the invention also provides an
antibody or antigen binding fragment thereof that binds to human
TIGIT comprising: a heavy chain variable region comprising the
CDR1, CDR2 and CDR3 of any of SEQ ID NOs: 124-129 and/or a light
chain variable region comprising the CDR1, CDR2 and CDR3 of any of
SEQ ID NOs:130-133. In one embodiment, the invention also provides
an isolated antibody or antigen binding fragment thereof that binds
to human TIGIT comprising: a heavy chain variable region selected
from the group consisting of: SEQ ID NOs: 124-129 and/or a light
chain variable region selected from the group consisting of: SEQ ID
NOs: 130-133. In one embodiment, the D residue at position 56 of
any one of SEQ ID NOs: 124-129 can be substituted with R, L, K, F,
S, Y or V. In another embodiment, the G residue at position 57 of
any one of SEQ ID NOs: 124-129 can be substituted with R, N, Q, E,
L K, S, Y or V. In one embodiment, the W residue at position 104 of
any one of SEQ ID NOs: 124-129 can be substituted with: A, D, E, F,
G, I, K, N, Q, R, S, T, V or Y. In another embodiment, the N
residue at position 50 of any one of SEQ ID NOs: 130-133 can be
substituted with A, Y, W, S, T, I or V. In another embodiment, the
A residue at position 51 of any of SEQ ID NOs: 130-133 is
substituted with N, I, L, T or V. In one embodiment, the invention
provides an isolated antibody or antigen binding fragment thereof
that binds to human TIGIT comprising: a heavy chain variable region
comprising SEQ ID NO:128 or a light chain variable region
comprising SEQ ID NO:132. In another embodiment, the invention
provides an isolated antibody or antigen binding fragment thereof
that binds to human TIGIT comprising: a heavy chain variable region
comprising SEQ ID NO:128 or a light chain variable region
comprising SEQ ID NO:133. In another embodiment, the invention also
provides an isolated antibody or antigen binding fragment thereof
that binds to human TIGIT comprising: a heavy chain variable region
comprising SEQ ID NO:127 or a light chain variable region
comprising SEQ ID NO:130. In one embodiment, the invention provides
an isolated antibody or antigen binding fragment thereof that binds
to human TIGIT comprising: a heavy chain variable region comprising
SEQ ID NO:128 and a light chain variable region comprising SEQ ID
NO:132. In another embodiment, the invention also provides an
isolated antibody or antigen binding fragment thereof that binds to
human TIGIT comprising: a heavy chain variable region comprising
SEQ ID NO:128 and a light chain variable region comprising SEQ ID
NO:133. In another embodiment, the invention also provides an
isolated antibody or antigen binding fragment thereof that binds to
human TIGIT comprising: a heavy chain variable region comprising
SEQ ID NO:127 and a light chain variable region comprising SEQ ID
NO:130. In some embodiments, the antibody or antigen binding
fragment thereof binds to human TIGIT with a KD value of about
1.times.10-9 M to about 1.times.10-12 M as determined by surface
plasmon resonance (e.g., BIACORE) or a similar technique (e.g.
KinExa or OCTET).
[0022] In another embodiment, the invention also provides an
antibody or antigen binding fragment thereof that binds to human
TIGIT comprising: (i) a heavy chain variable region comprising the
CDR1, CDR2 and CDR3 of any one of SEQ ID NOs: 124-129, wherein the
heavy chain variable region comprises at least 90%, 95%, 96%, 97%,
98% or 99% identity to any of one SEQ ID NOs: 124-129 and/or (ii) a
light chain variable region comprising the CDR1, CDR2 and CDR3 of
any one of SEQ ID NOs: 130-133, wherein the light chain variable
region comprises at least 90%, 95%, 96%, 97%, 98% or 99% identity
to any one of SEQ ID NOs: 130-133. In another embodiment, the
invention also provides an antibody or antigen binding fragment
thereof that binds to human TIGIT comprising: (i) a heavy chain
variable region comprising the CDR1, CDR2 and CDR3 of SEQ ID NOs:
128, wherein the heavy chain variable region comprises 90%, 95%,
96%, 97%, 98% or 99% identity to SEQ ID NOs: 128 and/or (ii) a
light chain variable region comprising the CDR1, CDR2 and CDR3 of
SEQ ID NOs: 132, wherein the light chain variable region comprises
at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs:
132. In another embodiment, the invention also provides an antibody
or antigen binding fragment thereof that binds to human TIGIT
comprising: (i) a heavy chain variable region comprising the CDR1,
CDR2 and CDR3 of SEQ ID NOs: 128, wherein the heavy chain variable
region comprises at least 90%, 95%, 96%, 97%, 98% or 99% identity
to SEQ ID NOs: 128 and/or (ii) a light chain variable region
comprising the CDR1, CDR2 and CDR3 of SEQ ID NOs: 133, wherein the
light chain variable region comprises at least 90%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NOs: 133. In another embodiment, the
invention also provides an antibody or antigen binding fragment
thereof that binds to human TIGIT comprising: (i) a heavy chain
variable region comprising the CDR1, CDR2 and CDR3 of SEQ ID NOs:
127, wherein the heavy chain variable region comprises at least
90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 127 and/or
(ii) a light chain variable region comprising the CDR1, CDR2 and
CDR3 of SEQ ID NOs: 130, wherein the light chain variable region
comprises at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NOs: 130. In these embodiments, the allowed sequence variations
occur in the framework regions of the variable chains. In some
embodiments, the antibody or antigen binding fragment thereof binds
to human TIGIT with a KD value of about 1.times.10-9 M to about
1.times.10-12 M as determined by surface plasmon resonance (e.g.,
BIACORE) or a similar technique (e.g. KinExa or OCTET).
[0023] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof, comprising: a variable heavy
chain comprising the amino acid sequence of SEQ ID NO: 7 and/or a
variable light chain selected comprising the amino acid sequence of
SEQ ID NO: 8, wherein the antibody or antigen binding fragment
thereof binds to human TIGIT. In one embodiment, the antibody binds
to human TIGIT with a KD value of about 1.times.10-9 M to about
1.times.10-12 M as determined by surface plasmon resonance (e.g.,
BIACORE) or a similar technique (e.g. KinExa or OCTET).
[0024] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof, comprising: a variable heavy
chain comprising the amino acid sequence of SEQ ID NO: 143 and/or a
variable light chain selected comprising the amino acid sequence of
SEQ ID NO: 145, wherein the antibody or antigen binding fragment
thereof binds to human TIGIT. In one embodiment, the antibody binds
to human TIGIT with a KD value of about 1.times.10-9 M to about
1.times.10-12 M as determined by surface plasmon resonance (e.g.,
BIACORE) or a similar technique (e.g. KinExa or OCTET).
[0025] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof, comprising: a variable heavy
chain comprising the amino acid sequence of SEQ ID NO: 144 and/or a
variable light chain selected comprising the amino acid sequence of
SEQ ID NO: 146, wherein the antibody or antigen binding fragment
thereof binds to human TIGIT. In one embodiment, the antibody binds
to human TIGIT with a KD value of about 1.times.10-9 M to about
1.times.10-12 M as determined by surface plasmon resonance (e.g.,
BIACORE) or a similar technique (e.g. KinExa or OCTET).
[0026] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof, comprising: a variable heavy
chain comprising the amino acid sequence of SEQ ID NO: 63 and/or a
variable light chain selected comprising the amino acid sequence of
SEQ ID NO: 64, wherein the antibody or antigen binding fragment
thereof binds to human TIGIT. In one embodiment, the antibody binds
to human TIGIT with a KD value of about 1.times.10-9 M to about
1.times.10-12 M as determined by surface plasmon resonance (e.g.,
BIACORE) or a similar technique (e.g. KinExa or OCTET).
[0027] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof, comprising: a variable heavy
chain comprising the amino acid sequence of SEQ ID NO: 94 and/or a
variable light chain selected comprising the amino acid sequence of
SEQ ID NO: 95, wherein the antibody or antigen binding fragment
thereof binds to human TIGIT. In one embodiment, the antibody binds
to human TIGIT with a KD value of about 1.times.10-9 M to about
1.times.10-12 M as determined by surface plasmon resonance (e.g.,
BIACORE) or a similar technique (e.g. KinExa or OCTET).
[0028] In one embodiment, the invention also provides an isolated
antibody or antigen binding fragment thereof that binds to human
TIGIT comprising: a heavy chain variable region comprising SEQ ID
NO:149 and/or a light chain variable region comprising SEQ ID
NO:151. In one embodiment, the antibody binds to human TIGIT with a
KD value of about 1.times.10-9 M to about 1.times.10-12 M as
determined by surface plasmon resonance (e.g., BIACORE) or a
similar technique (e.g. KinExa or OCTET).
[0029] In one embodiment, the invention also provides an isolated
antibody or antigen binding fragment thereof that binds to human
TIGIT comprising: a heavy chain variable region comprising SEQ ID
NO:150 and/or a light chain variable region comprising SEQ ID
NO:152. In one embodiment, the antibody binds to human TIGIT with a
KD value of about 1.times.10-9 M to about 1.times.10-12 M as
determined by surface plasmon resonance (e.g., BIACORE) or a
similar technique (e.g. KinExa or OCTET).
[0030] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof that binds to the same epitope of
human TIGIT as an antibody comprising the variable heavy chain of
SEQ ID NO: 7 and the variable light chain of SEQ ID NO: 8, wherein
the antibody or antigen binding fragment thereof has at least one
of the following characteristics: (i) binds to human TIGIT with a
KD value of about 1.times.10-9 M to about 1.times.10-12 M as
determined by surface plasmon resonance (e.g., BIACORE) or a
similar technique (e.g. KinExa or OCTET), (ii) blocks binding of
human TIGIT to human CD155 and human CD112; (iii) increases T cell
activation; (iv) stimulates antigen-specific T-cell production of
IL-2 and IFN.gamma.; (v) blocks induction of T cell suppression of
activation induced by TIGIT ligation with cognate ligands CD155 and
CD112. In one embodiment, the antibody comprises at least 80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% sequence identity with the variable
heavy chain and/or the variable light chain of any one of SEQ ID
NOs: 7-30 or 37-52. In another embodiment, the antibody or antigen
binding fragment thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29 or 30 amino acid substitutions in the variable heavy light
chain of any one of SEQ ID NOs: 7, 9-24 and 37-47 and/or the
variable light chains of any one of SEQ ID NOs: 8, 25-30 and
48-52.
[0031] In one embodiment, the invention provides an antibody or
antigen binding fragment thereof that binds to an epitope of human
TIGIT that comprises at least one of the following regions:
residues 54-57 of SEQ ID NO: 31, residues 68-70 of SEQ ID NO:31 and
residues 76-81 of SEQ ID NO: 31. In one embodiment, the invention
provides an antibody or antigen binding fragment thereof that binds
to an epitope of human TIGIT comprising residues: 54-57, 68-70 and
76-81 of SEQ ID NO:31.
[0032] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof that binds to the same epitope of
human TIGIT as an antibody comprising the variable heavy chain of
SEQ ID NO: 63 and the variable light chain of SEQ ID NO: 64,
wherein the antibody or antigen binding fragment thereof has at
least one of the following characteristics: (i) binds to human
TIGIT with a KD value of about 1.times.10-9 M to about
1.times.10-12 M as determined by surface plasmon resonance (e.g.,
BIACORE) or a similar technique (e.g. KinExa or OCTET); (ii)
cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocks
binding of human TIGIT to human CD155 and human CD112; (iv)
increases T cell activation; (v) stimulates antigen-specific T-cell
production of IL-2 and IFN.gamma.; (vi) blocks induction of T cell
suppression of activation induced by TIGIT ligation with cognate
ligands CD155 and CD112. In one embodiment, the antibody or antigen
binding fragment thereof comprises at least 80%, 85%, 90%, 95%,
96%, 97%, 98% or 99% sequence identity with the variable heavy
chain of SEQ ID NO:63 and/or the variable light chain of SEQ ID NO:
64. In another embodiment, the antibody or antigen binding fragment
thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30
amino acid substitutions in the variable heavy light chain of SEQ
ID NO:63 and/or the variable light chain of SEQ ID NO: 64. In
another embodiment, the antibody or antigen binding fragment
thereof comprises 1, 2 or 3 amino acid substitutions in the heavy
chain CDRs (SEQ ID NOs: 57-59) or in the light chain CDRs (SEQ ID
NOs: 60-62).
[0033] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof that binds to the same epitope of
human TIGIT as an antibody comprising the variable heavy chain of
SEQ ID NO: 94 and the variable light chain of SEQ ID NO:95, wherein
the antibody or antigen binding fragment thereof has at least one
of the following characteristics: (i) binds to human TIGIT with a
KD value of about 1.times.10-9 M to about 1.times.10-12 M as
determined by surface plasmon resonance (e.g., BIACORE) or a
similar technique (e.g. KinExa or OCTET); (ii) cross-reacts with
cynomolgous and rhesus TIGIT; (iii) blocks binding of human TIGIT
to human CD155 and human CD112; (iv) increases T cell activation;
(v) stimulates antigen-specific T-cell production of IL-2 and
IFN.gamma.; (vi) blocks induction of T cell suppression of
activation induced by TIGIT ligation with cognate ligands CD155 and
CD112. In one embodiment, the antibody or antigen binding fragment
thereof comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
sequence with the variable heavy chain of any one of SEQ ID NOs:94
or 124-129, and/or the variable light chain of any one of SEQ ID
NO: 95 or 130-133. In another embodiment, the antibody or antigen
binding fragment thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29 or 30 amino acid substitutions in the variable heavy light
chain of any one of SEQ ID NOs:94 or 124-129, and/or the variable
light chain of any one of SEQ ID NO: 95 or 130-133. In another
embodiment, the antibody or antigen binding fragment thereof
comprises 1, 2 or 3 amino acid substitutions in the heavy chain
CDRs (SEQ ID NOs: 88-90) and/or in the light chain CDRs (SEQ ID
NOs: 91-93). In another embodiment, the antibody or antigen binding
fragment thereof comprises heavy chain CDRs of SEQ ID NOs: 88, 134
and 90 and/or the light chain CDRs of SEQ ID NOs: 91, 92 and
93.
[0034] In one embodiment, the invention provides an antibody or
antigen binding fragment thereof that binds to an epitope of human
TIGIT that comprises at least one of the following regions:
residues 53-57, residues 60-65, residues 68-70, residues 72-81,
residues 94-95, and residues 109-119 of SEQ ID NO:31. In one
embodiment, the invention provides an antibody or antigen binding
fragment thereof that binds to an epitope of human TIGIT comprising
residues: 53-57, 60-65, 68-70, 72-81, 94-95, and 109-119 of SEQ ID
NO:31.
[0035] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof that cross-blocks the binding of
(or competes with) an antibody comprising the variable heavy chain
of SEQ ID NO: 7 and the variable light chain of SEQ ID NO: 8 to
human TIGIT, wherein the antibody or antigen binding fragment
thereof has at least one of the following characteristics: (i)
binds to human TIGIT with a KD value of about 1.times.10-9 M to
about 1.times.10-12 M as determined by surface plasmon resonance
(e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); (ii)
cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocks
binding of human TIGIT to human CD155 and human CD112; (iv)
increases T cell activation; (v) stimulates antigen-specific T-cell
production of IL-2 and IFN.gamma.; (vi) blocks induction of T cell
suppression of activation induced by TIGIT ligation with cognate
ligands CD155 and CD112. In one embodiment, the antibody or antigen
binding fragment thereof comprises at least 80%, 85%, 90%, 95%,
96%, 97%, 98% or 99% sequence identity with the variable heavy
chain or variable light chains of SEQ ID NOs: 7-30 or 37-52. In
another embodiment, the antibody or antigen binding fragment
thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30
amino acid substitutions in the variable heavy light chains of SEQ
ID NOs:7, 9-24 and 37-47 or the variable light chains of SEQ ID
NOs: 8, 25-30 and 48-52. In another embodiment, the antibody or
antigen binding fragment thereof comprises 1, 2 or 3 amino acid
substitutions in the heavy chain CDRs (SEQ ID NOs: 1-3) or in the
light chain CDRs (SEQ ID NOs: 3-6).
[0036] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof that cross-blocks the binding of
(or competes with) an antibody comprising the variable heavy chain
of SEQ ID NO: 63 and the variable light chain of SEQ ID NO: 64 to
human TIGIT, wherein the antibody or antigen binding fragment
thereof has at least one of the following functional
characteristics: (i) binds to human TIGIT with a KD value of about
1.times.10-9 M to about 1.times.10-12 M as determined by surface
plasmon resonance (e.g., BIACORE) or a similar technique (e.g.
KinExa or OCTET); (ii) cross-reacts with cynomolgous and rhesus
TIGIT; (iii) blocks binding of human TIGIT to human CD155 and human
CD112; (iv) increases T cell activation; (v) stimulates
antigen-specific T-cell production of IL-2 and IFN.gamma.(vi)
blocks induction of T cell suppression of activation induced by
TIGIT ligation with cognate ligands CD155 and CD112. In one
embodiment, the antibody comprises at least 80%, 85%, 90%, 95%,
96%, 97%, 98% or 99% sequence identity with the variable heavy
chain of SEQ ID NO:63 or the variable light chains of SEQ ID NO:
64. In another embodiment, the antibody or antigen binding fragment
thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30
amino acid substitutions in the variable heavy light chain of SEQ
ID NO: 63 or the variable light chain of SEQ ID NO: 64. In another
embodiment, the antibody or antigen binding fragment thereof
comprises 1, 2 or 3 amino acid substitutions in the heavy chain
CDRs (SEQ ID NOs: 57-59) or in the light chain CDRs (SEQ ID NOs:
60-62).
[0037] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof that cross-blocks the binding of
(or competes with) an antibody comprising the variable heavy chain
of SEQ ID NO: 94 and the variable light chain of SEQ ID NO: 95 to
human TIGIT, wherein the antibody or antigen binding fragment
thereof has at least one of the following characteristics: (i)
binds to human TIGIT with a KD value of about 1.times.10-9 M to
about 1.times.10-12 M as determined by surface plasmon resonance
(e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); (ii)
cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocks
binding of human TIGIT to human CD155 and human CD112; (iv)
increases T cell activation; (v) stimulates antigen-specific T-cell
production of IL-2 and IFN.gamma.(vi) blocks induction of T cell
suppression of activation induced by TIGIT ligation with cognate
ligands CD155 and CD112. In one embodiment, the antibody comprises
at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity
with the variable heavy chain of SEQ ID NO: 94 or the variable
light chains of SEQ ID NO: 95. In another embodiment, the antibody
or antigen binding fragment thereof comprises 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29 or 30 amino acid substitutions in the variable
heavy light chain of any one of SEQ ID NOs: 94 or 124-129, or the
variable light chain of any one of SEQ ID NOs: 95 or 130-133. In
another embodiment, the antibody or antigen binding fragment
thereof comprises 1, 2 or 3 amino acid substitutions in the heavy
chain CDRs (SEQ ID NOs: 88-90) or in the light chain CDRs (SEQ ID
NOs: 91-93). In one embodiment, the antibody or antigen binding
fragment thereof comprises: a heavy chain variable region
comprising SEQ ID NO:128 and a light chain variable region
comprising SEQ ID NO:132. In another embodiment, antibody or
antigen binding fragment thereof comprises: a heavy chain variable
region comprising SEQ ID NO:127 and a light chain variable region
comprising SEQ ID NO:130. In another embodiment, antibody or
antigen binding fragment thereof comprises: a heavy chain variable
region comprising SEQ ID NO:128 and a light chain variable region
comprising SEQ ID NO:133.
[0038] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof that binds to human TIGIT
comprising a variable heavy chain selected from the group
consisting of any one of SEQ ID NOs: 9-24 and 37-47, and/or a
variable light chain selected from the group consisting of any one
of SEQ ID NOs: 25-30 and 48-52. In one embodiment, the antibody
comprises an amino acid substitution in FR4 of the heavy chain,
wherein the substitution is made at position 122 of SEQ ID NOs:
9-24 and 37-47, wherein the residue is substituted from M to: V, L,
A, R, N, P, Q, E, G, I, H, K, F, S, T, W or Y. In one embodiment,
the antibody comprises two amino acid substitutions in FR4 of the
heavy chain, wherein the substitutions are made at positions 122
and 123 of SEQ ID NOs: 9-24 and 37-47, wherein the residues are
substituted from M and V to T and L, respectively.
[0039] In another embodiment, the invention provides an antibody or
antigen binding fragment thereof that binds to human TIGIT
comprising a variable heavy chain selected from the group
consisting of any one of SEQ ID NOs: 9-24 and 37-47, and/or a
variable light chain selected from the group consisting of any one
of SEQ ID NOs: 25-30 and 48-52. In one embodiment, the antibody
comprises an amino acid substitution in FR4 of the heavy chain,
wherein the substitution is made at position 122 of SEQ ID NOs:
9-24 and 37-47, wherein the residue is substituted from M to: V, L,
A, R, N, P, Q, E, G, I, H, K, F, S, T, W or Y. In one embodiment,
the antibody comprises two amino acid substitutions in FR4 of the
heavy chain, wherein the substitutions are made at positions 122
and 123 of SEQ ID NOs: 9-24 and 37-47, wherein the residues are
substituted from M and V to T and L, respectively.
[0040] In one embodiment, the invention relates to an isolated
antibody or antigen binding fragment that binds to human TIGIT
comprising: a heavy chain comprising the amino acid sequence of SEQ
ID NO: 7 or variant thereof comprising up to 30 amino acid
substitutions, and/or a light chain comprising the amino acid
sequence of SEQ ID NO: 8 comprising up to 12 amino acid
substitutions. In one embodiment, the heavy chain comprises the
amino acid sequence of SEQ ID NO: 7 comprising amino acid
substitutions at one or more positions selected from the group
consisting of: 6, 9, 12, 15, 16, 17, 23, 25, 37, 39, 40, 43, 44,
45, 48, 67, 68, 70, 71, 79, 81, 83, 87, 88, 92, 94, 119, 122 and
123. In one embodiment, the heavy chain comprises the amino acid
sequence of SEQ ID NO:7 comprising amino acid substitutions at one
or more positions selected from the group consisting of: 6, 9, 12,
15, 16, 17, 23, 25, 37, 39, 40, 43, 44, 45, 48, 67, 68, 70, 71, 79,
81, 83, 87, 88, 92, 94, 110, 119, 122 and 123, wherein: the amino
acid at position 6 can be E or Q, the amino acid at position 9 can
be P or A, the amino acid at position 12 can be V or L, the amino
acid at position 15 can be S or P, the amino acid at position 16
can be Q or E or G, the amino acid at position 17 can be S or T,
the amino acid at position 23 can be S or T, the amino acid at
position 25 can be T or S, the amino acid at position 37 can be I
or V, the amino acid at position 39 can be K or Q, the amino acid
at position 40 can be F or P, the amino acid at position 43 can be
N or K, the amino acid at position 44 can be K or G, the amino acid
at position 45 can be M or L, the amino acid at position 48 can be
M or I, the amino acid at position 67 can be I or V, the amino acid
at position 68 can be S or T, the amino acid at position 70 can be
T or S, the amino acid at position 71 can be R or V, the amino acid
at position 79 can be F or S, the amino acid at position 81 can be
Q or K, the amino acid at position 83 can be H or S, the amino acid
at position 87 can be T or A, the amino acid at position 88 can be
D or A, the amino acid at position 92 can be T or V, the amino acid
at position 94 can be S or Y, the amino acid at position 110 can be
W, F, Y, I, V or L, the amino acid at position 119 can be P or Q,
the amino acid at position 122 can be M, V, L, A, R, N, P, Q, E, G,
I, H, K, F, S, T, W or Y, and the amino acid at position 123 can be
V, T or L. In one embodiment, the light chain comprises the amino
acid sequence of SEQ ID NO: 8 comprising amino acid substitutions
at one or more positions selected from the group consisting of: 10,
21, 22, 40, 42, 46, 52, 53, 58, 77, 83, 87, 95 and 106. In one
embodiment, the light chain comprises the amino acid sequence of
SEQ ID NO: 8 comprising amino acid substitutions at one or more
positions selected from the group consisting of: 10, 21, 22, 40,
42, 46, 52, 53, 58, 77, 83, 87, 95 and 106 wherein: the amino acid
at position 10 can be L or S, the amino acid at position 21 can be
L or I, the amino acid at position 22 can be N or T, the amino acid
at position 40 can be L or P, the amino acid at position 42 can be
E or K, the amino acid at position 46 can be F or L, the amino acid
at position 52 can be N, S, T, G or D, the amino acid at position
53 can be S, N, T, Y or Q, the amino acid at position 58 can be I
or V, the amino acid at position 77 can be G or S, the amino acid
at position 83 can be V or F, the amino acid at position 87 can be
F or Y, the amino acid at position 95 can be W, F, Y, I, V or L,
and the amino acid at position 105 can be L or I.
[0041] In another embodiment, the invention relates to an antibody
or antigen binding fragment that binds to human TIGIT comprising: a
heavy chain comprising the amino acid sequence of SEQ ID NO: 9
and/or a light chain comprising the amino acid sequence of SEQ ID
NO: 25, wherein each of the variable chains can comprise 1, 2, 3,
4, 5, 6, 7 or 8 amino acid substitutions. In one embodiment, the
antibody can comprise mutations at positions 27, 48, 67, 71, 83,
110, 122 and 123 of SEQ ID NO: 9. For example, with respect to SEQ
ID NO: 9: the amino acid at position 27 can be G or S; the amino
acid at position 48 can be I or M; the amino acid at position 67
can be V or I; the amino acid at position 71 can be V or R; the
amino acid at position 83 can be S or H; the amino acid at position
110 can be W, F, Y, I, V or L; the amino acid at position 122 can
be M, V, L, A, R, N, P, Q, E, G, I, H, K, F, S, T, W or Y; and the
amino acid at position 123 can be V, T or L. In another embodiment,
the antibody can comprise mutations at positions 46, 52, 53, 58 and
95 of SEQ ID NO: 25. For example, with respect to SEQ ID NO: 25,
the amino acid at position 46 can be L or F; the amino acid at
position 52 can be N, S, T, G or D, the amino acid at position 53
can be S, N, T, Y or Q; the amino acid at position 58 can be I or
V; the amino acid at position 58 can be V or I; and the amino acid
at position 95 can be W, F, Y, I, V or L.
[0042] In one embodiment, the invention relates to an antibody or
antigen binding fragment that binds to human TIGIT comprising: a
heavy chain comprising the amino acid sequence of SEQ ID NO: 94 or
variant thereof comprising up to 30 amino acid substitutions, and a
light chain comprising the amino acid sequence of SEQ ID NO: 95 or
a variant thereof comprising up to 18 amino acid substitutions. In
one embodiment, the heavy chain comprises a variant of the amino
acid sequence of SEQ ID NO: 94 comprising amino acid substitutions
at one or more positions selected from the group consisting of: 5,
9, 11, 12, 16, 20, 38, 40, 44, 56, 57, 61, 62, 65, 67, 68, 72, 74,
76, 79, 85, 87, 89, 91, 92, 104 and 111. In one embodiment, the
heavy chain comprises a variant of the amino acid sequence of SEQ
ID NO: 94 comprising amino acid substitutions at one or more
positions selected from the group consisting of: 5, 9, 11, 12, 16,
20, 38, 40, 44, 56, 57, 61, 62, 65, 67, 68, 72, 74, 76, 79, 85, 87,
89, 91, 92, 104 and 111, wherein: the amino acid at position 5 can
be Q or V, the amino acid at position 9 can be P or A, the amino
acid at position 11 can be V or L, the amino acid at position 12
can be V or K, the amino acid at position 16 can be S or A, the
amino acid at position 20 can be M or V, the amino acid at position
38 can be K or R, the amino acid at position 40 can be K or A, the
amino acid at position 44 can be G or R, the amino acid at position
56 can be D, R, L, K, F, S, Y or V, the amino acid at position 57
can be G, R, N, Q, E, L K, S, Y or V, the amino acid at position 61
can be N, A or S, the amino acid at position 62 can be E or Q, the
amino acid at position 65 can be K or Q, the amino acid at position
67 can be R or K, the amino acid at position 68 can be A or V, the
amino acid at position 72 can be S or R, the amino acid at position
74 can be K or T, the amino acid at position 76 can be S, I, A or
T, the amino acid at position 79 can be A or V, the amino acid at
position 85 can be R or S, the amino acid at position 87 can be T
or R, the amino acid at position 89 can be D or E, the amino acid
at position 91 can be S or T, the amino acid at position 92 can be
A or V, the amino acid at position 104 can be W, A, D, E, F, G, I,
K, N, Q, R, S, T, V or Y, and the amino acid at position 111 can be
A or Q. In one embodiment, the light chain comprises a variant of
the amino acid sequence of SEQ ID NO: 95 comprising amino acid
substitutions at one or more positions selected from the group
consisting of: 9, 17, 18, 40, 43, 45, 48, 50, 51, 70, 72, 74, 76,
83, 84, 100, 103 and 106. In one embodiment, the light chain
comprises a variant of the amino acid sequence of SEQ ID NO: 95
comprising amino acid substitutions at one or more positions
selected from the group consisting of: 9, 17, 18, 40, 43, 45, 48,
50, 51, 70, 72, 74, 76, 83, 84, 100, 103 and 106, wherein the amino
acid at position 9 can be A or S, the amino acid at position 17 can
be E or D, the amino acid at position 18 can be T or R, the amino
acid at position 40 can be Q or P, the amino acid at position 43
can be S, A or V, the amino acid at position 45 can be Q or K, the
amino acid at position 48 can be V or I, the amino acid at position
50 can be N, A, Y, W, S, T, I or V, the amino acid at position 51
can be A, N, I, L, T or V, the amino acid at position 70 can be Q
or D, the amino acid at position 72 can be S or T, the amino acid
at position 74 can be K or T, the amino acid at position 76 can be
N or S, the amino acid at position 83 can be F or V, the amino acid
at position 84 can be G or A, the amino acid at position 100 can be
A or Q, the amino acid at position 103 can be T or R and the amino
acid at position at position 106 can be L or I.
[0043] In one embodiment, the invention relates to an antibody or
antigen binding fragment that binds to human TIGIT comprising: a
heavy chain comprising the amino acid sequence of SEQ ID NO: 124 or
variant thereof comprising up to 10 amino acid substitutions,
and/or a light chain comprising the amino acid sequence of SEQ ID
NO: 130 or a variant thereof comprising up to 5 amino acid
substitutions. In another embodiment, the invention provides an
antibody or antigen binding fragment thereof that binds to human
TIGIT comprising a variable heavy chain of SEQ ID NO: 124 or a
variant thereof, wherein the variant comprises substitutions at one
or more positions selected from the group consisting of: 16, 44,
56, 57, 61, 72, 74, 76, 79, 85, 89, 92 and 104. In another
embodiment, the invention provides an antibody or antigen binding
fragment thereof that binds to human TIGIT comprising a variable
heavy chain of SEQ ID NO: 124 or a variant thereof, wherein the
variant comprises substitutions at one or more positions selected
from the group consisting of: 16, 44, 56, 57, 61, 72, 74, 76, 79,
85, 89, 92 and 104, wherein the amino acid at position 16 can be A
or S, the amino acid at position 44 can be R or G, the amino acid
at position 56 can be D, R, L, K, F, S, Y or V, the amino acid at
position 57 can be G, R, N, Q, E, L K, S, Y or V, the amino acid at
position 61 can be S or A, the amino acid at position 72 can be R
or S, the amino acid at position 74 can be T or K, the amino acid
at position 76 can be A or T or I, the amino acid at position 79
can be A or V, the amino acid at position 85 can be S or R, the
amino acid at position 89 can be E or D, the amino acid at position
92 can be A or V and the amino acid at position 104 can be W, A, D,
E, F, G, I, K, N, Q, R, S, T, V or Y. In another embodiment, the
invention provides an antibody or antigen binding fragment thereof
that binds to human TIGIT comprising a variable light chain of SEQ
ID NO: 130 or a variant thereof comprising substitutions at one or
more positions selected from the group consisting of: 43, 50, 51,
70 and 83. In another embodiment, the invention provides an
antibody or antigen binding fragment thereof that binds to human
TIGIT comprising a variable light chain of SEQ ID NO: 130 or a
variant thereof comprising substitutions at one or more positions
selected from the group consisting of: 43, 50, 51, 70 and 83,
wherein the amino acid at position 43 can be S, A or V, the amino
acid at position 50 can be N, A, Y, W, S, T, I or V, the amino acid
at position 51 can be A, N, I, L, T or V, the amino acid at
position 70 can be Q or D, and the amino acid at position 83 can be
F or V.
[0044] In any of the above embodiments, the antibody or antigen
binding fragment thereof is isolated.
[0045] In any of the above embodiments, the antibody or antigen
binding fragment thereof is a recombinant antibody.
[0046] In any of the above embodiments, the antibody or antigen
binding fragment thereof is a full-length antibody.
[0047] In any of the above mentioned embodiments, the antibody or
antigen binding fragment thereof of the invention can comprise a
variable heavy region consisting of: (a) any of the variable heavy
chains described above and (b) a leader peptide (for example, the
leader peptide of SEQ ID NO: 53). In any of the above mentioned
embodiments, the antibody or antigen binding fragment thereof of
the invention can comprise a light heavy region consisting of: (a)
any of the light heavy chains described above and (b) a leader
peptide (for example, the leader peptide of SEQ ID NO: 54).
[0048] In any of the above mentioned embodiments, the antibody or
antigen binding fragment thereof of the invention is an antibody
comprising any of the variable heavy chains described above and any
human heavy chain constant domain. In one embodiment, the antibody
or antigen binding fragment thereof of the invention is of the IgG
isotype, and comprises a human IgG1, IgG2, IgG3 or IgG4 human heavy
chain constant domain. In one embodiment, the antibody or antigen
binding fragment thereof of the invention comprises a human heavy
chain IgG1 constant domain (SEQ ID NO: 86) or a variant thereof,
wherein the variant comprises up to 20 modified amino acid
substitutions. In one embodiment, the antibody or antigen binding
fragment thereof of the invention is an antibody comprising a human
heavy chain IgG1 constant domain comprising the amino acid sequence
of SEQ ID NO: 86. In one embodiment, the antibody or antigen
binding fragment thereof of the invention comprises a human heavy
chain IgG1 constant domain wherein the IgG1 constant domain is
afucosylated. In one embodiment, the antibody or antigen binding
fragment thereof of the invention comprises a human heavy chain
IgG4 constant domain or a variant thereof, wherein the variant
comprises up to 20 modified amino acid substitutions. In another
embodiment, the antibody or antigen binding fragment thereof of the
invention comprises a human heavy chain IgG4 constant domain,
wherein the amino acid at position 228 (using EU numbering scheme)
has been substituted from Ser to Pro. In one embodiment, the
antibody or antigen binding fragment thereof of the invention
comprises a human heavy chain IgG4 constant domain comprising the
amino acid sequence of SEQ ID NO: 55.
[0049] In any of the above mentioned embodiments, the antibody or
antigen binding fragment thereof of the invention can comprise any
of the variable light chains described above and human light chain
constant domain. In one embodiment, the antibody or antigen binding
fragment thereof of the invention comprises a human kappa light
chain constant domain or a variant thereof, wherein the variant
comprises up to 20 modified amino acid substitutions. In another
embodiment, the antibody or antigen binding fragment thereof of the
invention comprises a human lambda light chain constant domain or a
variant thereof, wherein the variant comprises up to 20 modified
amino acid substitutions. In one embodiment, the antibody or
antigen binding fragment thereof of the invention comprises a human
kappa light chain constant domain comprising the amino acid
sequence of SEQ ID NO: 56.
[0050] In one embodiment, the anti-TIGIT antibody of the invention
comprises a full tetrameric structure having two light chains and
two heavy chains, wherein each light chain comprises: a variable
region comprising SEQ ID NO:132 and a human kappa light chain (SEQ
ID NO:56); and each heavy chain comprises: a variable region
comprising SEQ ID NO:128, a human IgG1 constant region (SEQ ID
NO:86).
[0051] In one embodiment, the anti-TIGIT antibody of the invention
comprises a full tetrameric structure having two light chains and
two heavy chains, wherein each light chain comprises: a variable
region comprising SEQ ID NO:130 and a human kappa light chain (SEQ
ID NO:56); and each heavy chain comprises: a variable region
comprising SEQ ID NO:127, a human IgG1 constant region (SEQ ID
NO:86).
[0052] In one embodiment, the anti-TIGIT antibody of the invention
comprises a full tetrameric structure having two light chains and
two heavy chains, wherein each light chain comprises: a variable
region comprising SEQ ID NO:133 and a human kappa light chain (SEQ
ID NO:56); and each heavy chain comprises: a variable region
comprising SEQ ID NO:128, a human IgG1 constant region (SEQ ID
NO:86).
[0053] In any of the above mentioned embodiments, the anti-TIGIT
antibody or antigen binding fragment thereof of the invention can
be conjugated to at least one therapeutic agent. In one embodiment,
the wherein the therapeutic agent comprises a second antibody or
fragment thereof, an immunomodulator, a hormone, a cytotoxic agent,
an enzyme, a radionuclide, a second antibody conjugated to at least
one immunomodulator, enzyme, radioactive label, hormone, antisense
oligonucleotide, or cytotoxic agent, or a combination thereof.
[0054] The invention also provides isolated polypeptides comprising
the amino acid sequence of any one of SEQ ID NOs: 1-30, 37-52,
57-83 or 88-151, or a fragment of any said sequences.
[0055] The invention also provides isolated nucleic acids encoding
anyone of the anti-TIGIT antibodies or antigen binding fragments of
the invention. In one embodiment, the invention provides isolated
nucleic acids encoding anyone of the polypeptides of SEQ ID NOs:
1-30, 37-52, 57-64 or 88-133, wherein said polypeptides can
optionally comprise a leader sequence. The invention also provides
expression vectors comprising a nucleic acid encoding anyone of the
polypeptides of SEQ ID NOs: 1-30, 37-52, 57-83 or 88-167 (wherein
said polypeptides can optionally comprise a leader sequence). These
isolated nucleic acids and the expression vectors comprising them
may be used to express the antibodies of the invention or antigen
binding fragments thereof in recombinant host cells. Thus, the
invention also provides host cells comprising isolated nucleic
acids encoding anyone of the polypeptides of SEQ ID NOs: 1-30,
37-52, 57-83 or 88-151 (wherein said polypeptides can optionally
comprise a leader sequence). In one embodiment, the host cell is
Chinese hamster ovary cell. In one embodiment, the host cell is a
yeast cell, for example a Pichia cell or a Pichia pastoris host
cell.
[0056] The invention also provides pharmaceutical compositions
comprising an antibody or antigen binding fragment of the invention
and a pharmaceutically acceptable carrier or diluent. In one
embodiment, the composition comprises a further therapeutic agent.
In one embodiment, the further therapeutic agent is selected from
the group consisting of: an anti-PD1 antibody or an antigen binding
fragment thereof; an anti-LAG3 antibody or an antigen binding
fragment thereof; an anti-VISTA antibody or an antigen binding
fragment thereof; an anti-BTLA antibody or an antigen binding
fragment thereof; an anti-TIM3 antibody or an antigen binding
fragment thereof; an anti-CTLA4 antibody or an antigen binding
fragment thereof; an anti-HVEM antibody or an antigen binding
fragment thereof; an anti-CD27 antibody or an antigen binding
fragment thereof; an anti-CD137 antibody or an antigen binding
fragment thereof; an anti-OX40 antibody or an antigen binding
fragment thereof; an anti-CD28 antibody or an antigen binding
fragment thereof; an anti-PDL1 antibody or an antigen binding
fragment thereof; an anti-PDL2 antibody or an antigen binding
fragment thereof; an anti-GITR antibody or an antigen binding
fragment thereof; an anti-ICOS antibody or an antigen binding
fragment thereof; an anti-SIRP.alpha. antibody or an antigen
binding fragment thereof; an anti-ILT2 antibody or antigen binding
fragment thereof; an anti-ILT3 antibody or antigen binding fragment
thereof; an anti-ILT4 antibody or antigen binding fragment thereof;
and an anti-ILT5 antibody or an antigen binding fragment thereof;
an anti 4-1BB antibody or an antigen binding fragment thereof. In
one embodiment, the anti-PD1 antibody or an antigen binding
fragment thereof; is selected from the group consisting of:
pembrolizumab or an antigen binding fragment thereof and nivolumab
or an antigen binding fragment thereof. In one embodiment, the
anti-TIGIT antibody or antigen binding fragment of the invention
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 2;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 3, 79, 80, 81, 82, 83 or 140; (iv) a light
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO: 5, 65, 66, 67, 68, 69, 70, 71,
72, 73 or 141; and (vi) a light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 6, 74, 75, 76, 77,
78 or 142. In one embodiment, the anti-TIGIT antibody or antigen
binding fragment thereof comprises: (i) a heavy chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 88;
(ii) a heavy chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 89, 96, 97, 98, 99, 100, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 134, 135 or 147; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 90; (iv) a light chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO: 91; (v) a light chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO: 92, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 or
148; and (vi) a light chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO: 93. In one embodiment, the
anti-TIGIT antibody or antigen binding fragment of the invention
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 2;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 4; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 5; and (vi) a light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 6. In another
embodiment, the anti-TIGIT antibody or antigen binding fragment of
the invention comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 57; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 58; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 59; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 60; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 61; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 62. In
one embodiment, the anti-TIGIT antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 89, 134 or 135; (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 90; (iv) a light
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO: 91; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 92; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 93. In one embodiment, the anti-TIGIT antibody or antigen
binding fragment thereof comprises: (i) a heavy chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 88;
(ii) a heavy chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 134; (iii) a heavy chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) a
light chain variable region CDR1 comprising the amino acid sequence
of SEQ ID NO: 91; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 92; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 93.
[0057] In one embodiment, the invention provides a composition
comprising: (i) an anti-TIGIT antibody or antigen binding fragment
of the invention; and (ii) an anti-PD1 antibody comprising the
heavy chain sequence of SEQ ID NO: 33 and the light chain variable
sequence of SEQ ID NO: 34. In another embodiment, the invention
provides a composition comprising: (a) an anti-TIGIT antibody or
antigen binding fragment of the invention; and (b) an anti-PD1
antibody comprising the heavy chain sequence of SEQ ID NO: 35 and
the light chain variable sequence of SEQ ID NO: 36. In one
embodiment, the anti-PD1 antibody is administered prior to the
administration of an anti-TIGIT antibody. In one embodiment, the
anti-PD1 antibody is administered 4-10 days prior to the
administration of the anti-TIGIT antibody. In one embodiment,
pretreatment treatment with anti-PD1 antibody may modulate immune
cells resulting in enhanced Fc-mediated function of the anti-TIGIT
antibodies. In one embodiment, the anti-TIGIT antibody or antigen
binding fragment of the invention comprises: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 79, 80,
81, 82, 83 or 140; (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 4; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 5, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 141; and (vi) a
light chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO: 6, 74, 75, 76, 77, 78 or 142. In one embodiment, the
anti-TIGIT antibody or antigen binding fragment thereof comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 89, 96, 97, 98,
99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,
134, 135 or 147; (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 90; (iv) a light
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO: 91; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 92, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122 or 148; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 93. In one embodiment, the anti-TIGIT antibody or antigen
binding fragment of the invention comprises: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a
light chain variable region CDR1 comprising the amino acid sequence
of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 6. In another embodiment, the anti-TIGIT antibody or antigen
binding fragment of the invention comprises: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 57; (ii) a heavy chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO: 58; (iii) a heavy chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 59;
(iv) a light chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 60; (v) a light chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 61; and (vi) a
light chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO: 62. In one embodiment, the anti-TIGIT antibody or
antigen binding fragment thereof comprises: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 88; (ii) a heavy chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO: 89, 134 or 135; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 90; (iv) a light chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO: 91; (v) a light chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO: 92; and (vi) a light chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO: 93. In one embodiment, the
anti-TIGIT antibody or antigen binding fragment thereof comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 134; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 90; (iv) a light chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO: 91; (v) a light chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO: 92; and (vi) a light chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO: 93.
[0058] The invention also comprises a combination comprising an
anti-TIGIT antibody or antigen binding fragment of the invention,
in combination with one, two or more therapeutic agents; wherein
the second therapeutic agent is selected from the group consisting
of: an anti-PD1 antibody or an antigen binding fragment thereof; an
anti-LAG3 antibody or an antigen binding fragment thereof; an
anti-VISTA antibody or an antigen binding fragment thereof; an
anti-BTLA antibody or an antigen binding fragment thereof; an
anti-TIM3 antibody or an antigen binding fragment thereof; an
anti-CTLA4 antibody or an antigen binding fragment thereof; an
anti-HVEM antibody or an antigen binding fragment thereof; an
anti-CD27 antibody or an antigen binding fragment thereof; an
anti-CD137 antibody or an antigen binding fragment thereof; an
anti-OX40 antibody or an antigen binding fragment thereof; an
anti-CD28 antibody or an antigen binding fragment thereof; an
anti-PDL1 antibody or an antigen binding fragment thereof; an
anti-PDL2 antibody or an antigen binding fragment thereof; an
anti-GITR antibody or an antigen binding fragment thereof; an
anti-ICOS antibody or an antigen binding fragment thereof; an
anti-SIRP.alpha. antibody or an antigen binding fragment thereof;
an anti-ILT2 antibody or antigen binding fragment thereof; an
anti-ILT3 antibody or antigen binding fragment thereof; an
anti-ILT4 antibody or antigen binding fragment thereof; an
anti-ILT5 antibody or an antigen binding fragment thereof; and an
anti 4-1BB antibody or an antigen binding fragment thereof. In one
embodiment, the anti-TIGIT antibody or antigen binding fragment of
the invention comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 3, 79, 80, 81, 82, 83 or 140;
(iv) a light chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 5, 65, 66, 67, 68,
69, 70, 71, 72, 73 or 141; and (vi) a light chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO: 6, 74, 75,
76, 77, 78 or 142. In one embodiment, the anti-TIGIT antibody or
antigen binding fragment thereof comprises: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 88; (ii) a heavy chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO: 89, 96, 97, 98, 99, 100, 101,
102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 134, 135 or 147;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 90; (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 91; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 92, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,
122 or 148; and (vi) a light chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 93. In one embodiment, the
anti-TIGIT antibody or antigen binding fragment of the invention
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 2;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 4; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 5; and (vi) a light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 6. In another
embodiment, the anti-TIGIT antibody or antigen binding fragment of
the invention comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 57; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 58; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 59; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 60; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 61; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 62. In
one embodiment, the anti-TIGIT antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 89, 134 or 135; (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 90; (iv) a light
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO: 91; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 92; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 93. In one embodiment, the anti-TIGIT antibody or antigen
binding fragment thereof comprises: (i) a heavy chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 88;
(ii) a heavy chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 134; (iii) a heavy chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) a
light chain variable region CDR1 comprising the amino acid sequence
of SEQ ID NO: 91; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 92; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 93.
[0059] The invention also provides a vessel or injection device
comprising anyone of the anti-TIGIT antibodies or antigen binding
fragments of the invention. In one embodiment, the anti-TIGIT
antibody or antigen binding fragment of the invention comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 3, 79, 80, 81, 82, 83 or 140; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 4; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 5, 65, 66, 67, 68, 69, 70, 71, 72, 73
or 141; and (vi) a light chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO: 6, 74, 75, 76, 77, 78 or 142. In
one embodiment, the anti-TIGIT antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 89, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,
107, 108, 109, 110, 111, 134, 135 or 147; (iii) a heavy chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 90; (iv) a light chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 91; (v) a light chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 92,
112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 or 148; and
(vi) a light chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 93. In one embodiment, the anti-TIGIT
antibody or antigen binding fragment of the invention comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and
(vi) a light chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 6. In another embodiment, the anti-TIGIT
antibody or antigen binding fragment of the invention comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 57; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 58; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 59; (iv) a light chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO: 60; (v) a light chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO: 61; and (vi) a light chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO: 62. In one embodiment, the
anti-TIGIT antibody or antigen binding fragment thereof comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 89, 134 or 135;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 90; (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 91; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 92; and (vi) a light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 93. In one
embodiment, the anti-TIGIT antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 90; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO:
93.
[0060] The invention also provides a method of producing an
anti-TIGIT antibody or antigen binding fragment of the invention
comprising: culturing a host cell comprising a polynucleotide
encoding a heavy chain and/or light chain of an antibody of the
invention (or an antigen binding fragment thereof) under conditions
favorable to expression of the polynucleotide; and optionally,
recovering the antibody or antigen binding fragment from the host
cell and/or culture medium. In one embodiment, the polynucleotide
encoding the heavy chain and the polynucleotide encoding the light
chain are in a single vector. In another embodiment, the
polynucleotide encoding the heavy chain and the polynucleotide
encoding the light chain are in different vectors. In one
embodiment, the polynucleotide encoding the heavy chain and the
polynucleotide encoding the light chain encode an antibody or
antigen binding fragment comprising: (i) a heavy chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 1;
(ii) a heavy chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 3, 79, 80, 81, 82,
83 or 140; (iv) a light chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO:4; (v) a light chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO:5, 65,
66, 67, 68, 69, 70, 71, 72, 73 or 141; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO:6, 74, 75, 76, 77, 78 or 142. In another embodiment, the
polynucleotide encoding the heavy chain and the polynucleotide
encoding the light chain encode an antibody or antigen binding
fragment comprising: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO:88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 89, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,
107, 108, 109, 110, 111, 134, 135 or 147; (iii) a heavy chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 90; (iv) a light chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 91; (v) a light chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 92,
112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 or 148; and
(vi) a light chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 93. In another embodiment, the
polynucleotide encoding the heavy chain and the polynucleotide
encoding the light chain encode an antibody or antigen binding
fragment comprising: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 3; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 4; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 5; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In
another embodiment, the polynucleotide encoding the heavy chain and
the polynucleotide encoding the light chain encode an antibody or
antigen binding fragment comprising: (i) a heavy chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 57;
(ii) a heavy chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 58; (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 59; (iv) a light
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO: 60; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 61; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO:62. In another embodiment, the polynucleotide encoding the heavy
chain and the polynucleotide encoding the light chain encode an
antibody or antigen binding fragment comprising: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 88; (ii) a heavy chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO: 89, 134 or 135; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 90; (iv) a light chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO: 91; (v) a light chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO: 92; and (vi) a light chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO: 93. In another embodiment, the
polynucleotide encoding the heavy chain and the polynucleotide
encoding the light chain encode an antibody or antigen binding
fragment comprising: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 90; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In
another embodiment, the polynucleotide encoding the heavy chain and
the polynucleotide encoding the light chain encode an antibody or
antigen binding fragment comprising: a heavy chain variable region
comprising SEQ ID NO:128 and a light chain variable region
comprising SEQ ID NO:132. In another embodiment, the polynucleotide
encoding the heavy chain and the polynucleotide encoding the light
chain encode an antibody or antigen binding fragment comprising: a
heavy chain variable region comprising SEQ ID NO:127 and a light
chain variable region comprising SEQ ID NO:130. In another
embodiment, the polynucleotide encoding the heavy chain and the
polynucleotide encoding the light chain encode an antibody or
antigen binding fragment comprising: a heavy chain variable region
comprising SEQ ID NO:128 and a light chain variable region
comprising SEQ ID NO:133.
[0061] The invention also provides a method of treating cancer in a
subject in need thereof, comprising administering to the subject an
effective amount of an anti-TIGIT antibody or antigen binding
fragment of the invention, optionally in association with a further
therapeutic agent or therapeutic procedure. In one embodiment, the
subject been treated is a human subject. In one embodiment, the
further therapeutic agent is selected from the group consisting of:
an anti-PD1 antibody or an antigen binding fragment thereof; an
anti-LAG3 antibody or an antigen binding fragment thereof; an
anti-VISTA antibody or an antigen binding fragment thereof; an
anti-BTLA antibody or an antigen binding fragment thereof; an
anti-TIM3 antibody or an antigen binding fragment thereof; an
anti-CTLA4 antibody or an antigen binding fragment thereof; an
anti-HVEM antibody or an antigen binding fragment thereof; an
anti-CD27 antibody or an antigen binding fragment thereof; an
anti-CD137 antibody or an antigen binding fragment thereof; an
anti-OX40 antibody or an antigen binding fragment thereof; an
anti-CD28 antibody or an antigen binding fragment thereof; an
anti-PDL1 antibody or an antigen binding fragment thereof; an
anti-PDL2 antibody or an antigen binding fragment thereof; an
anti-GITR antibody or an antigen binding fragment thereof; an
anti-ICOS antibody or an antigen binding fragment thereof; an
anti-SIRP.alpha. antibody or an antigen binding fragment thereof;
an anti-ILT2 antibody or antigen binding fragment thereof; an
anti-ILT3 antibody or antigen binding fragment thereof; an
anti-ILT4 antibody or antigen binding fragment thereof; an
anti-ILT5 antibody or an antigen binding fragment thereof; and an
anti-4-1BB antibody or an antigen binding fragment thereof. In one
embodiment, the anti-PD1 antibody or an antigen binding fragment
thereof is selected from the group consisting of: pembrolizumab or
an antigen binding fragment thereof and nivolumab or an antigen
binding fragment thereof. In one embodiment, the anti-TIGIT
antibody or antigen binding fragment of the invention comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 3, 79, 80, 81, 82, 83 or 140; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO:4; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO:5, 65, 66, 67, 68, 69, 70, 71, 72, 73 or
141; and (vi) a light chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO:6, 74, 75, 76, 77, 78 or 142. In
one embodiment, the anti-TIGIT antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO:88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 89, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,
107, 108, 109, 110, 111, 134, 135 or 147; (iii) a heavy chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 90; (iv) a light chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 91; (v) a light chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 92,
112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 or 148; and
(vi) a light chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 93. In one embodiment, the anti-TIGIT
antibody or antigen binding fragment of the invention comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and
(vi) a light chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 6. In another embodiment, the anti-TIGIT
antibody or antigen binding fragment of the invention comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 57; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 58; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 59; (iv) a light chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO: 60; (v) a light chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO: 61; and (vi) a light chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO: 62. In one embodiment, the
anti-TIGIT antibody or antigen binding fragment thereof comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 89, 134 or 135;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 90; (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 91; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 92; and (vi) a light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 93. In one
embodiment, the anti-TIGIT antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 90; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO:
93.
[0062] The invention also provides a method of treating cancer in a
subject in need thereof, comprising administering to the subject an
effective amount of an anti-TIGIT antibody or antigen binding
fragment of the invention, and further administering an anti-PD1
antibody or an antigen binding fragment thereof. In one embodiment,
the anti-PD1 antibody or an antigen binding fragment thereof is
selected from the group consisting of: pembrolizumab or an antigen
binding fragment thereof and nivolumab or an antigen binding
fragment thereof. In one embodiment, the anti-TIGIT antibody or
antigen binding fragment of the invention comprises: (i) a heavy
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 3, 79, 80, 81, 82, 83 or 140; (iv) a light chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO:4; (v)
a light chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO:5, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 141;
and (vi) a light chain variable region CDR3 comprising the amino
acid sequence of SEQ ID NO:6, 74, 75, 76, 77, 78 or 142. In one
embodiment, the anti-TIGIT antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 89, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,
107, 108, 109, 110, 111, 134, 135 or 147; (iii) a heavy chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 90; (iv) a light chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 91; (v) a light chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 92,
112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 or 148; and
(vi) a light chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 93. In one embodiment, the anti-TIGIT
antibody or antigen binding fragment of the invention comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and
(vi) a light chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 6. In another embodiment, the anti-TIGIT How
antibody or antigen binding fragment of the invention comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 57; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 58; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 59; (iv) a light chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO: 60; (v) a light chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO: 61; and (vi) a light chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO: 62. In one embodiment, the
anti-TIGIT antibody or antigen binding fragment thereof comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 89, 134 or 135;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 90; (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 91; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 92; and (vi) a light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 93. In one
embodiment, the anti-TIGIT antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 90; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO:
93.
[0063] The invention also provides a method of treating an
infection or infectious disease in a subject, comprising
administering to the subject an effective amount of an antibody or
antigen binding fragment of the invention, optionally in
association with a further therapeutic agent or therapeutic
procedure. In one embodiment, the subject been treated is a human
subject. In one embodiment, the further therapeutic agent is
selected from the group consisting of: an anti-PD1 antibody or an
antigen binding fragment thereof; an anti-LAG3 antibody or an
antigen binding fragment thereof; an anti-VISTA antibody or an
antigen binding fragment thereof; an anti-BTLA antibody or an
antigen binding fragment thereof; an anti-TIM3 antibody or an
antigen binding fragment thereof; an anti-CTLA4 antibody or an
antigen binding fragment thereof; an anti-HVEM antibody or an
antigen binding fragment thereof; an anti-CD27 antibody or an
antigen binding fragment thereof; an anti-CD137 antibody or an
antigen binding fragment thereof; an anti-OX40 antibody or an
antigen binding fragment thereof; an anti-CD28 antibody or an
antigen binding fragment thereof; an anti-PDL1 antibody or an
antigen binding fragment thereof; an anti-PDL2 antibody or an
antigen binding fragment thereof; an anti-GITR antibody or an
antigen binding fragment thereof; an anti-ICOS antibody or an
antigen binding fragment thereof; an anti-SIRP.alpha. antibody or
an antigen binding fragment thereof; an anti-ILT2 antibody or
antigen binding fragment thereof; an anti-ILT3 antibody or antigen
binding fragment thereof; an anti-ILT4 antibody or antigen binding
fragment thereof; an anti-ILT5 antibody or an antigen binding
fragment thereof; and an anti-4-1BB antibody or an antigen binding
fragment thereof. In one embodiment, the anti-PD1 antibody or an
antigen binding fragment thereof; is selected from the group
consisting of: pembrolizumab or an antigen binding fragment thereof
and nivolumab or an antigen binding fragment thereof. In one
embodiment, the anti-TIGIT antibody or antigen binding fragment of
the invention comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 3, 79, 80, 81, 82, 83 or 140;
(iv) a light chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO:4; (v) a light chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO:5, 65, 66, 67, 68,
69, 70, 71, 72, 73 or 141; and (vi) a light chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO:6, 74, 75, 76,
77, 78 or 142. In one embodiment, the anti-TIGIT antibody or
antigen binding fragment thereof comprises: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO:88; (ii) a heavy chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 89, 96, 97, 98, 99, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 134, 135 or 147; (iii) a
heavy chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO: 90; (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 91; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 92, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,
122 or 148; and (vi) a light chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 93. In one embodiment, the
anti-TIGIT antibody or antigen binding fragment of the invention
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 2;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 4; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 5; and (vi) a light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 6. In another
embodiment, the anti-TIGIT antibody or antigen binding fragment of
the invention comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 57; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 58; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO:59; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 60; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 61; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 62. In
one embodiment, the anti-TIGIT antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO:88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 89, 134 or 135; (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 90; (iv) a light
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO: 91; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 92; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 93. In one embodiment, the anti-TIGIT antibody or antigen
binding fragment thereof comprises: (i) a heavy chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 88;
(ii) a heavy chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 134; (iii) a heavy chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) a
light chain variable region CDR1 comprising the amino acid sequence
of SEQ ID NO: 91; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 92; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 93.
[0064] The invention also provides a vaccine comprising an antibody
or antigen binding fragment of the invention. In one embodiment,
the anti-TIGIT antibody or antigen binding fragment of the
invention comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 3, 79, 80, 81, 82, 83 or
1401; (iv) a light chain variable region CDR1 comprising the amino
acid sequence of SEQ ID NO: 4; (v) a light chain variable region
CDR2 comprising the amino acid sequence of SEQ ID NO: 5, 65, 66,
67, 68, 69, 70, 71, 72, 73 or 141; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO:6, 74,
75, 76, 77, 78 or 142. In one embodiment, the anti-TIGIT antibody
or antigen binding fragment thereof comprises: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 88; (ii) a heavy chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO: 89, 96, 97, 98, 99, 100, 101,
102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 134, 135 or 147;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 90; (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 91; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 92, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,
122 or 148; and (vi) a light chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 93. In one embodiment, the
anti-TIGIT antibody or antigen binding fragment of the invention
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 2;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 4; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 5; and (vi) a light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 6. In another
embodiment, the anti-TIGIT antibody or antigen binding fragment of
the invention comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 57; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 58; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 59; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 60; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 61; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 62. In
one embodiment, the anti-TIGIT antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 89, 134 or 135; (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 90; (iv) a light
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO: 91; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 92; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 93. In one embodiment, the anti-TIGIT antibody or antigen
binding fragment thereof comprises: (i) a heavy chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 88;
(ii) a heavy chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 134; (iii) a heavy chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) a
light chain variable region CDR1 comprising the amino acid sequence
of SEQ ID NO: 91; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 92; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 93. In one embodiment, the vaccine further comprises an
antigen.
[0065] The invention also provides a method for detecting the
presence of a TIGIT peptide or a fragment thereof in a sample
comprising contacting the sample with an antibody or antigen
binding fragment thereof of the invention and detecting the
presence of a complex between the antibody or fragment and the
peptide; wherein detection of the complex indicates the presence of
the TIGIT peptide. In one embodiment, the anti-TIGIT antibody or
antigen binding fragment of the invention comprises: (i) a heavy
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 3, 79, 80, 81, 82, 83 or 140; (iv) a light chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO:4; (v)
a light chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO:5, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 141;
and (vi) a light chain variable region CDR3 comprising the amino
acid sequence of SEQ ID NO: 6, 74, 75, 76, 77, 78 or 142. In one
embodiment, the antibody or antigen binding fragment thereof
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 89,
96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,
110, 111, 134, 135 or 147; (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 153; (iv) a light
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO: 91; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 92, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122 or 148; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 93. In one embodiment, the antibody or antigen binding fragment
of the invention comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 3; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 4; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 5; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In
another embodiment, the antibody or antigen binding fragment of the
invention comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 57; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 58; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 59; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 60; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 61; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 62. In
one embodiment, the antibody or antigen binding fragment thereof
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 89,
134 or 135; (iii) a heavy chain variable region CDR3 comprising the
amino acid sequence of SEQ ID NO: 90; (iv) a light chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 91;
(v) a light chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 92; and (vi) a light chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In one
embodiment, the anti-TIGIT antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 90; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO:
93.
[0066] The invention also provides a method of increasing the
activity of an immune cell, comprising contacting the immune cell
with any one of the antibodies or antigen binding fragments of the
invention. In one embodiment, the invention provides a method of
increasing the activity of an immune cell, comprising administering
to a subject in need thereof an effective amount of an antibody or
antigen binding fragments of the invention. In one embodiment, the
method is used for: the treatment of cancer, the treatment of an
infection or infectious disease, or as a vaccine adjuvant. In one
embodiment, the increase in activity of an immune cell can be
detected by measuring the proliferation of the immune cell. For
example, an increase in activity of a T cell can be detected by
measuring the proliferation of the T cell. In one embodiment, the
increase in activity of an immune cell can be detected by measuring
T cell activation ex vivo in a sample derived from the subject. In
one embodiment, the increase in T cell activity is determined by:
(i) measuring mixed lymphocyte reactions or direct anti-CD3 mAb
stimulation of T cell receptor (TCR) signaling to induce production
of a cytokine selected from the group consisting of: IL-2,
TNF.alpha., IL-17, IFN.gamma., IL-1.beta., GM-CSF, RANTES, IL-6,
IL-8, IL-5 and IL-13; (ii) measuring SEB induced production of one
or more cytokines selected from the group consisting of: IL-2,
TNF.alpha., IL-17, IFN.gamma., GM-CSF, RANTES, IL-6, IL-8, IL-5 and
IL-13; or (iii) measuring TT induced production of a cytokine
selected from the group consisting of: IL-2, TNF.alpha., IL-17,
IFN.gamma., GM-CSF, RANTES, IL-6, IL-8, IL-5 and IL-13. In one
embodiment, the anti-TIGIT antibody or antigen binding fragment of
the invention comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 3, 79, 80, 81, 82, 83 or 140;
(iv) a light chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 5, 65, 66, 67, 68,
69, 70, 71, 72, 73 or 141; and (vi) a light chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO: 6, 74, 75,
76, 77, 78 or 142. In one embodiment, the anti-TIGIT antibody or
antigen binding fragment thereof comprises: (i) a heavy chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 88; (ii) a heavy chain variable region CDR2 comprising the
amino acid sequence of SEQ ID NO: 89, 96, 97, 98, 99, 100, 101,
102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 134, 135 or 147;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 90; (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 91; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 92, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,
122 or 148; and (vi) a light chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 93. In one embodiment, the
anti-TIGIT antibody or antigen binding fragment of the invention
comprises: (i) a heavy chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 2;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 4; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 5; and (vi) a light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 6. In another
embodiment, the anti-TIGIT antibody or antigen binding fragment of
the invention comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 57; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 58; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 59; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 60; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 61; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 62. In
one embodiment, the anti-TIGIT antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 89, 134 or 135; (iii) a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 90; (iv) a light
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO: 91; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 92; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 93. In one embodiment, the anti-TIGIT antibody or antigen
binding fragment thereof comprises: (i) a heavy chain variable
region CDR1 comprising the amino acid sequence of SEQ ID NO: 88;
(ii) a heavy chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 134; (iii) a heavy chain variable region
CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) a
light chain variable region CDR1 comprising the amino acid sequence
of SEQ ID NO: 91; (v) a light chain variable region CDR2 comprising
the amino acid sequence of SEQ ID NO: 92; and (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 93.
[0067] The invention also comprises a method of treating cancer or
infectious disease in a subject, comprising administering to the
subject an effective amount of an antagonist anti-TIGIT antibody
and an antagonist anti-PD1 antibody, wherein the anti-TIGIT
antibody has increased effector function when compared to a
parental antibody. As used herein, a "parental anti-antibody"
refers to antibody having a wild-type Fc region and/or wild type
glycosylation (i.e., glycosylation pattern resulting from
expression of the polypeptide in a non-engineered mammalian host
cell). The effector function of a parental antibody can be
increased by mutating its Fc region or by altering its
glycosylation (as discussed in further detail below). In one
embodiment, the anti-PD1 antibody is administered prior to the
administration of a parental antibody. In one embodiment, the
anti-PD1 antibody is administered 4-10 days prior to the
administration of the anti-TIGIT antibody. In one embodiment,
pretreatment treatment with anti-PD1 antibody may modulate immune
cells resulting in enhanced Fc-mediated function of the anti-TIGIT
antibodies. In one embodiment, the anti-TIGIT antibody comprises a
human IgG1 constant domain. In one embodiment, treatment with the
anti-TIGIT and anti-PD1 antibodies does not result in depletion of
Tregs. In one embodiment, the anti-PD1 antibody or an antigen
binding fragment thereof; is selected from the group consisting of:
pembrolizumab and nivolumab. In one embodiment, the anti-TIGIT
antibody or antigen binding fragment of the invention comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 3 (iv) a light chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 4; (v) a light chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and
(vi) a light chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 6. In one embodiment, the anti-TIGIT
antibody or antigen binding fragment of the invention comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 89, 134 or 135;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 90 (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 91; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 92; and (vi) a light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 93. In one
embodiment, the anti-TIGIT antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 90; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO:
93.
[0068] The invention also comprises a method of treating cancer or
infectious disease in a subject, comprising administering to the
subject an effective amount of an antagonist anti-TIGIT antibody
and an antagonist anti-PD1 antibody, wherein the anti-TIGIT
antibody is afucosylated. In one embodiment, the anti-TIGIT
antibody or antigen binding fragment of the invention comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy
chain variable region CDR3 comprising the amino acid sequence of
SEQ ID NO: 3 (iv) a light chain variable region CDR1 comprising the
amino acid sequence of SEQ ID NO: 4; (v) a light chain variable
region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and
(vi) a light chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 6. In one embodiment, the anti-TIGIT
antibody or antigen binding fragment of the invention comprises:
(i) a heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 89, 134 or 135;
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 90 (iv) a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 91; (v) a light
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 92; and (vi) a light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO: 93. In one
embodiment, the anti-TIGIT antibody or antigen binding fragment
thereof comprises: (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising
the amino acid sequence of SEQ ID NO: 90; (iv) a light chain
variable region CDR1 comprising the amino acid sequence of SEQ ID
NO: 91; (v) a light chain variable region CDR2 comprising the amino
acid sequence of SEQ ID NO: 92; and (vi) a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO:
93.
[0069] The invention also provides a method of increasing the
anti-tumor activity of an anti-TIGIT antibody comprising: obtaining
a parental anti-TIGIT antibody and increasing the effector function
of the parental anti-TIGIT antibody; wherein the activity of the
resulting anti-TIGIT antibody is increased as compared to the
parental anti-TIGIT antibody. As used herein, a "parental
anti-antibody" refers to antibody having a wild-type Fc region
and/or wild type glycosylation (i.e., glycosylation pattern
resulting from expression of the polypeptide in a non-engineered
mammalian host cell). The effector function of a parental antibody
can be increased by mutating its Fc region or by altering its
glycosylation, for example by making the antibody afucosylated (as
discussed in further detail below). In one embodiment, the effector
function of a parental anti-TIGIT antibody is increased by making
mutations in the Fc region of the parental anti-TIGIT antibody. In
another embodiment, the effector function of a parental anti-TIGIT
antibody is increased by removing the fucose residues from the
antibody, or expressing the antibody in a host cell that has been
genetically engineered to remove the activity of the enzyme that
adds fucose to glycoproteins.
BRIEF DESCRIPTION OF THE FIGURES
[0070] FIG. 1 shows binding of antibody 14A6 to human and rhesus
TIGIT (expressed in CHO-K1 cells).
[0071] FIG. 2 shows binding of antibody 28H5 to human and rhesus
TIGIT (expressed in CHO-K1 cells).
[0072] FIG. 3 shows that antibodies 14A6 and 28H5 block hCD155
interaction with hTIGIT as determined by a cell-based ELISA
blocking assay.
[0073] FIG. 4 shows binding of antibody 31C6 to human and rhesus
TIGIT (expressed in CHO-K1 cells), and also shows that antibody
31C6 blocks hCD155 interaction with hTIGIT.
[0074] FIG. 5 shows the activity of antibodies 14A6 and 28H5 in an
in vitro T cell assay.
[0075] FIG. 6 shows the activity of antibodies 14A6 and 31C6 in
vitro T cell assay.
[0076] FIG. 7 shows expression of TIGIT, CD226, CD155 and CD96 in
primary human T cell lines. At day 3, clone BC4-49 shows highest
up-regulation of TIGIT (negative) ad down-regulation of CD226
(positive)
[0077] FIGS. 8A and 8B show the activity of various anti-TIGIT
antibodies in an in vitro T cell assay. It shows that MBS43 and
14A6, 37D10 and 28H5 anti-TIGIT antibodies rescue IFN.gamma.
responses in primary human T cells.
[0078] FIGS. 9A-9D show the effect of concurrent administration of
a rat anti-mouse TIGIT antibody (GIGD7) and an anti-mouse PD-1
antibody compared to the monotherapy treatment arms on the
anti-tumor response of mice implanted with the CT26 cell line
(n=10/group). Treatment was commenced when tumors reached 75
mm3-115 mm3.
[0079] FIG. 10 shows the effect of Fc isotype on the anti-tumor
activity of an anti-TIGIT antibody (18G10) in combination with an
anti-PD-1 antibody in an animal tumor model.
[0080] FIGS. 11A-11C show the effect of Fc isotype on the
anti-tumor activity of an anti-TIGIT antibody (18G10) in
combination with an anti-PD-1 antibody in an animal tumor
model.
[0081] FIG. 12 shows the effect of Fc isotype on the anti-tumor
activity of an anti-TIGIT antibody (11A11) in combination with an
anti-PD-1 antibody in an animal tumor model.
[0082] FIGS. 13A-13C show the effect of Fc isotype on the
anti-tumor activity of an anti-TIGIT antibody (11A11) in
combination with an anti-PD-1 antibody in an animal tumor
model.
[0083] FIG. 14 shows a heat map indicating regions of human TIGIT
which are strongly or weakly protected from deuteration by the
binding of the 14A6 antibody. The amino acid sequence shown in the
heatmap corresponds to SEQ ID NO:87.
[0084] FIG. 15 shows a heat map indicating regions of human TIGIT
which are strongly or weakly protected from deuteration by the
binding of the 31C6 antibody. The amino acid sequence shown in the
heatmap corresponds to SEQ ID NO:87.
[0085] FIG. 16 shows the effect of various humanized clones of 31C6
as compared to chimera and parental antibodies in an engineered T
cell functional assay.
[0086] FIG. 17 shows the effect of various humanized clones of 31C6
as compared to chimera antibodies in a primary T cell functional
assay.
DETAILED DESCRIPTION
Abbreviations
[0087] Throughout the detailed description and examples of the
invention the following abbreviations will be used: [0088] ADCC
Antibody-dependent cellular cytotoxicity [0089] CDC
Complement-dependent cytotoxicity [0090] CDR Complementarity
determining region in the immunoglobulin variable regions, defined
using the Kabat numbering system [0091] CHO Chinese hamster ovary
[0092] ELISA Enzyme-linked immunosorbant assay [0093] FR Antibody
framework region: the immunoglobulin variable regions excluding the
CDR regions. [0094] HRP Horseradish peroxidase [0095] IFN
interferon [0096] IC50 concentration resulting in 50% inhibition
[0097] IgG Immunoglobulin G [0098] Kabat An immunoglobulin
alignment and numbering system pioneered by Elvin A. Kabat ((1991)
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.)
[0099] mAb or Mab or MAb Monoclonal antibody [0100] SEB
Staphylococcus Enterotoxin B [0101] TT Tetanus toxoid [0102] V
region The segment of IgG chains which is variable in sequence
between different antibodies. It extends to Kabat residue 109 in
the light chain and 113 in the heavy chain. [0103] VH
Immunoglobulin heavy chain variable region [0104] VK Immunoglobulin
kappa light chain variable region
Definitions
[0105] So that the invention may be more readily understood,
certain technical and scientific terms are specifically defined
below. Unless specifically defined elsewhere in this document, all
other technical and scientific terms used herein have the meaning
commonly understood by one of ordinary skill in the art to which
this invention belongs.
[0106] As used herein, including the appended claims, the singular
forms of words such as "a," "an," and "the," include their
corresponding plural references unless the context clearly dictates
otherwise.
[0107] "Administration" and "treatment," as it applies to an
animal, human, experimental subject, cell, tissue, organ, or
biological fluid, refers to contact of an exogenous pharmaceutical,
therapeutic, diagnostic agent, or composition to the animal, human,
subject, cell, tissue, organ, or biological fluid. Treatment of a
cell encompasses contact of a reagent to the cell, as well as
contact of a reagent to a fluid, where the fluid is in contact with
the cell. "Administration" and "treatment" also means in vitro and
ex vivo treatments, e.g., of a cell, by a reagent, diagnostic,
binding compound, or by another cell.
[0108] "Treat" or "treating" means to administer a therapeutic
agent, such as a composition containing any of the antibodies or
antigen-binding fragments of the present invention, internally or
externally to a subject or patient having one or more disease
symptoms, or being suspected of having a disease, for which the
agent has therapeutic activity. Typically, the agent is
administered in an amount effective to alleviate one or more
disease symptoms in the treated subject or population, whether by
inducing the regression of or inhibiting the progression of such
symptom(s) by any clinically measurable degree. The amount of a
therapeutic agent that is effective to alleviate any particular
disease symptom may vary according to factors such as the disease
state, age, and weight of the patient, and the ability of the drug
to elicit a desired response in the subject. Whether a disease
symptom has been alleviated can be assessed by any clinical
measurement typically used by physicians or other skilled
healthcare providers to assess the severity or progression status
of that symptom.
TIGIT
[0109] The term TIGIT includes human TIGIT, cynomolgous monkey
TIGIT and rhesus TIGIT as well as fragments thereof such as the
mature fragment thereof lacking the signal peptide. In an
embodiment of the invention, the amino acid sequence of human TIGIT
comprises the amino acid sequence disclosed in amino acid residues
25-244 of Genbank Accession Number NP_776160.2 (SEQ ID NO: 31).
(Amino acid residues 1-24 of SEQ ID NO:31 correspond to a leader
peptide.)
[0110] In an embodiment of the invention, the amino acid sequence
of cynomolgous monkey, e.g., Macaca fascicularis TIGIT comprises
the amino acid sequence disclosed in (SEQ ID NO: 32); see also
Genbank Accession no. XP_005548157. The amino acid sequence of
rhesus monkey TIGIT is identical to the amino acid sequence of
cynomolgous monkey TIGIT. (Amino acid residues 1-24 of SEQ ID NO:32
correspond to a leader peptide.)
Anti-TIGIT Antibodies and Antigen-Binding Fragments Thereof
[0111] The present invention provides antibodies or antigen-binding
fragments thereof that bind human TIGIT and uses of such antibodies
or fragments. In some embodiments, the anti-TIGIT antibodies are
isolated.
[0112] As used herein, an anti-TIGIT antibody or antigen-binding
fragment thereof refers to an antibody or antigen-binding fragment
thereof that specifically binds to human TIGIT. An antibody or
antigen-binding fragment thereof that "specifically binds to human
TIGIT" is an antibody or antigen-binding fragment thereof that
binds to human TIGIT with a KD of about 1 nM or a higher affinity
(e.g., 1 nM-2 pM, 1 nM, 100 pM, 10 pM or 2 pM), but does not bind
to other proteins lacking this sequence. For example, an antibody
that "specifically binds" human TIGIT does not bind to human CD226,
human CD155 and human CD112. As a further example, an antibody or
antigen-binding fragment that specifically binds to human TIGIT may
bind to a FLAG.RTM.-tagged form of human TIGIT but will not bind to
other FLAG.RTM.-tagged proteins that lack human TIGIT epitopes. In
one embodiment, the antibody of the invention which specifically
binds to human TIGIT is also cross-reactive with cynomolgus and
rhesus TIGIT. As used herein "cross-reactivity" refers to the
ability of an antibody to react with a homologous protein from
other species. Whether an antibody specifically binds to human
TIGIT can be determined using any assay known in the art. Examples
of assays known in the art to determining binding affinity include
surface plasmon resonance (e.g., BIACORE) or a similar technique
(e.g. KinExa or OCTET).
[0113] The present invention includes anti-TIGIT antibodies and
methods of use thereof. As used herein, the term "antibody" refers
to any form of antibody that exhibits the desired biological
activity. Thus, it is used in the broadest sense and specifically
covers, but is not limited to, monoclonal antibodies (including
full length monoclonal antibodies comprising two light chains and
two heavy chains), polyclonal antibodies, multispecific antibodies
(e.g., bispecific antibodies), humanized antibodies, fully human
antibodies, chimeric antibodies and camelized single domain
antibodies.
[0114] The present invention includes non-human parental (e.g.
mouse and rodent) anti-TIGIT antibodies and antigen-binding
fragments thereof and methods of use thereof. These antibodies may
be modified for an intended use, such as humanization of an
antibody for use as a human therapeutic antibody or fragment.
[0115] The present invention includes anti-TIGIT antigen-binding
fragments and methods of use thereof. As used herein, unless
otherwise indicated, "antibody fragment" or "antigen-binding
fragment" refers to antigen-binding fragments of antibodies, i.e.
antibody fragments that retain the ability to bind specifically to
the antigen bound by the full-length antibody, e.g. fragments that
retain one or more CDR regions. Examples of antigen-binding
fragments include, but are not limited to, Fab, Fab', F(ab').sub.2,
and Fv fragments; diabodies; linear antibodies; single-chain
antibody molecules, e.g., sc-Fv; nanobodies and multispecific
antibodies formed from antibody fragments.
[0116] The present invention includes anti-TIGIT Fab fragments and
methods of use thereof. A "Fab fragment" is comprised of one light
chain and the C.sub.H1 and variable regions of one heavy chain. The
heavy chain of a Fab molecule cannot form a disulfide bond with
another heavy chain molecule. An "Fab fragment" can be the product
of papain cleavage of an antibody.
[0117] The present invention includes anti-TIGIT antibodies and
antigen-binding fragments thereof which comprise an Fc region and
methods of use thereof. An "Fc" region contains two heavy chain
fragments comprising the C.sub.H1 and C.sub.H2 domains of an
antibody. The two heavy chain fragments are held together by two or
more disulfide bonds and by hydrophobic interactions of the
C.sub.H3 domains.
[0118] The present invention includes anti-TIGIT Fab' fragments and
methods of use thereof. A "Fab' fragment" contains one light chain
and a portion or fragment of one heavy chain that contains the
V.sub.H domain and the C.sub.H1 domain and also the region between
the C.sub.H1 and C.sub.H2 domains, such that an interchain
disulfide bond can be formed between the two heavy chains of two
Fab' fragments to form a F(ab').sub.2 molecule.
[0119] The present invention includes anti-TIGIT F(ab').sub.2
fragments and methods of use thereof. A "F(ab').sub.2 fragment"
contains two light chains and two heavy chains containing a portion
of the constant region between the C.sub.H1 and C.sub.H2 domains,
such that an interchain disulfide bond is formed between the two
heavy chains. A F(ab').sub.2 fragment thus is composed of two Fab'
fragments that are held together by a disulfide bond between the
two heavy chains. An "F(ab').sub.2 fragment" can be the product of
pepsin cleavage of an antibody.
[0120] The present invention includes anti-TIGIT Fv fragments and
methods of use thereof. The "Fv region" comprises the variable
regions from both the heavy and light chains, but lacks the
constant regions.
[0121] The present invention includes anti-TIGIT scFv fragments and
methods of use thereof. The term "single-chain Fv" or "scFv"
antibody refers to antibody fragments comprising the V.sub.H and
V.sub.L domains of an antibody, wherein these domains are present
in a single polypeptide chain. Generally, the Fv polypeptide
further comprises a polypeptide linker between the V.sub.H and
V.sub.L domains which enables the scFv to form the desired
structure for antigen-binding. For a review of scFv, see Pluckthun
(1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113,
Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.
See also, International Patent Application Publication No. WO
88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203.
[0122] The present invention includes anti-TIGIT domain antibodies
and methods of use thereof. A "domain antibody" is an
immunologically functional immunoglobulin fragment containing only
the variable region of a heavy chain or the variable region of a
light chain. In some instances, two or more V.sub.H regions are
covalently joined with a peptide linker to create a bivalent domain
antibody. The two V.sub.H regions of a bivalent domain antibody may
target the same or different antigens.
[0123] The present invention includes anti-TIGIT bivalent
antibodies and methods of use thereof. A "bivalent antibody"
comprises two antigen-binding sites. In some instances, the two
binding sites have the same antigen specificities. However,
bivalent antibodies may be bispecific (see below).
[0124] The present invention includes anti-TIGIT camelized single
domain antibodies and methods of use thereof. In certain
embodiments, antibodies herein also include camelized single domain
antibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem.
Sci. 26:230; Reichmann et al. (1999) J. Immunol. Methods 231:25; WO
94/04678; WO 94/25591; U.S. Pat. No. 6,005,079). In one embodiment,
the present invention provides single domain antibodies comprising
two V.sub.H domains with modifications such that single domain
antibodies are formed.
[0125] The present invention includes anti-TIGIT diabodies and
methods of use thereof. As used herein, the term "diabodies" refers
to small antibody fragments with two antigen-binding sites, which
fragments comprise a heavy chain variable domain (V.sub.H)
connected to a light chain variable domain (V.sub.L) in the same
polypeptide chain (V.sub.H-V.sub.L or V.sub.L-V.sub.H). By using a
linker that is too short to allow pairing between the two domains
on the same chain, the domains are forced to pair with the
complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, e.g.,
EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl.
Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody
variants generally see Holliger and Hudson (2005) Nat. Biotechnol.
23:1126-1136.
[0126] Typically, an antibody or antigen-binding fragment of the
invention which is modified in some way retains at least 10% of its
binding activity (when compared to the parental antibody) when that
activity is expressed on a molar basis. Preferably, an antibody or
antigen-binding fragment of the invention retains at least 20%,
50%, 70%, 80%, 90%, 95% or 100% or more of the TIGIT binding
affinity as the parental antibody. It is also intended that an
antibody or antigen-binding fragment of the invention can include
conservative or non-conservative amino acid substitutions (referred
to as "conservative variants" or "function conserved variants" of
the antibody) that do not substantially alter its biologic
activity.
[0127] The present invention includes isolated anti-TIGIT
antibodies and antigen-binding fragments thereof and methods of use
thereof. "Isolated" antibodies or antigen-binding fragments thereof
are at least partially free of other biological molecules from the
cells or cell cultures in which they are produced. Such biological
molecules include nucleic acids, proteins, lipids, carbohydrates,
or other material such as cellular debris and growth medium. An
isolated antibody or antigen-binding fragment may further be at
least partially free of expression system components such as
biological molecules from a host cell or of the growth medium
thereof. Generally, the term "isolated" is not intended to refer to
a complete absence of such biological molecules or to an absence of
water, buffers, or salts or to components of a pharmaceutical
formulation that includes the antibodies or fragments.
[0128] The present invention includes monoclonal anti-TIGIT
antibodies and antigen-binding fragments thereof as well as
monoclonal compositions comprising a plurality of isolated
monoclonal antibodies. The term "monoclonal antibody", as used
herein, refers to a population of substantially homogeneous
antibodies, i.e., the antibody molecules comprising the population
are identical in amino acid sequence except for possible naturally
occurring mutations that may be present in minor amounts. In
contrast, conventional (polyclonal) antibody preparations typically
include a multitude of different antibodies having different amino
acid sequences in their variable domains, particularly their CDRs
that are often specific for different epitopes. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al. (1975) Nature
256: 495, or may be made by recombinant DNA methods (see, e.g.,
U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al. (1991) Nature 352: 624-628 and Marks
et al. (1991) J. Mol. Biol. 222: 581-597, for example. See also
Presta (2005) J. Allergy Clin. Immunol. 116:731.
[0129] The present invention includes anti-TIGIT chimeric
antibodies (e.g., human constant domain/mouse variable domain) and
methods of use thereof. As used herein, a "chimeric antibody" is an
antibody having the variable domain from a first antibody and the
constant domain from a second antibody, where the first and second
antibodies are from different species. (U.S. Pat. No. 4,816,567;
and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81:
6851-6855). Typically, the variable domains are obtained from an
antibody from an experimental animal (the "parental antibody"),
such as a rodent, and the constant domain sequences are obtained
from human antibodies, so that the resulting chimeric antibody will
be less likely to elicit an adverse immune response in a human
subject than the parental (e.g., mouse) antibody.
[0130] The present invention includes anti-TIGIT humanized
antibodies and antigen-binding fragments thereof (e.g., rat or
mouse antibodies that have been humanized) and methods of use
thereof. The invention includes any humanized version of the 14A6
antibody (comprising SEQ ID NOs:7 and 8), the 28H5 antibody
(comprising SEQ ID NOs:63 and 64) and the 31C6 antibody (comprising
SEQ ID NOs: 94-95). As used herein, the term "humanized antibody"
refers to forms of antibodies that contain sequences from both
human and non-human (e.g., mouse or rat) antibodies. 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 hypervariable loops correspond to those of
a non-human immunoglobulin, and all or substantially all of the
framework (FR) regions are those of a human immunoglobulin
sequence. The humanized antibody may optionally comprise at least a
portion of a human immunoglobulin constant region (Fc).
[0131] In general, the basic antibody structural unit comprises a
tetramer. Each tetramer includes two identical pairs of polypeptide
chains, each pair having one "light" (about 25 kDa) and one "heavy"
chain (about 50-70 kDa). The amino-terminal portion of each chain
includes a variable region of about 100 to 110 or more amino acids
primarily responsible for antigen recognition. The carboxy-terminal
portion of the heavy chain may define a constant region primarily
responsible for effector function. Typically, human light chains
are classified as kappa and lambda light chains. Furthermore, human
heavy chains are typically classified as mu, delta, gamma, alpha,
or epsilon, and define the antibody's isotype as IgM, IgD, IgG,
IgA, and IgE, respectively. Within light and heavy chains, the
variable and constant regions are joined by a "J" region of about
12 or more amino acids, with the heavy chain also including a "D"
region of about 10 more amino acids. See generally, Fundamental
Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.
(1989).
[0132] The variable regions of each light/heavy chain pair form the
antibody binding site. Thus, in general, an intact antibody has two
binding sites. Except in bifunctional or bispecific antibodies, the
two binding sites are, in general, the same.
[0133] Typically, the variable domains of both the heavy and light
chains comprise three hypervariable regions, also called
complementarity determining regions (CDRs), located within
relatively conserved framework regions (FR). The CDRs are usually
aligned by the framework regions, enabling binding to a specific
epitope. In general, from N-terminal to C-terminal, both light and
heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3,
CDR3 and FR4. The assignment of amino acids to each domain is,
generally, in accordance with the definitions of Sequences of
Proteins of Immunological Interest, Kabat, et al.; National
Institutes of Health, Bethesda, Md.; 5.sup.th ed.; NIH Publ. No.
91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et
al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987)
J. Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature
342:878-883.
[0134] As used herein, the term "hypervariable region" refers to
the amino acid residues of an antibody or antigen-binding fragment
thereof that are responsible for antigen-binding. The hypervariable
region comprises amino acid residues from a "complementarity
determining region" or "CDR" (i.e. CDRL1, CDRL2 and CDRL3 in the
light chain variable domain and CDRH1, CDRH2 and CDRH3 in the heavy
chain variable domain). See Kabat et al. (1991) Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (defining the CDR
regions of an antibody by sequence); see also Chothia and Lesk
(1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions of an
antibody by structure). As used herein, the term "framework" or
"FR" residues refers to those variable domain residues other than
the hypervariable region residues defined herein as CDR
residues.
[0135] "Isolated nucleic acid molecule" or "isolated
polynucleotide" means a DNA or RNA of genomic, mRNA, cDNA, or
synthetic origin or some combination thereof which is not
associated with all or a portion of a polynucleotide in which the
isolated polynucleotide is found in nature, or is linked to a
polynucleotide to which it is not linked in nature. For purposes of
this disclosure, it should be understood that "a nucleic acid
molecule comprising" a particular nucleotide sequence does not
encompass intact chromosomes. Isolated nucleic acid molecules
"comprising" specified nucleic acid sequences may include, in
addition to the specified sequences, coding sequences for up to ten
or even up to twenty or more other proteins or portions or
fragments thereof, or may include operably linked regulatory
sequences that control expression of the coding region of the
recited nucleic acid sequences, and/or may include vector
sequences.
[0136] The phrase "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to use promoters, polyadenylation
signals, and enhancers.
[0137] A nucleic acid or polynucleotide is "operably linked" when
it is placed into a functional relationship with another nucleic
acid sequence. For example, DNA for a presequence or secretory
leader is operably linked to DNA for a polypeptide if it is
expressed as a preprotein that participates in the secretion of the
polypeptide; a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the sequence; or a
ribosome binding site is operably linked to a coding sequence if it
is positioned so as to facilitate translation. Generally, but not
always, "operably linked" means that the DNA sequences being linked
are contiguous, and, in the case of a secretory leader, contiguous
and in reading phase. However, enhancers do not have to be
contiguous. Linking is accomplished by ligation at convenient
restriction sites. If such sites do not exist, the synthetic
oligonucleotide adaptors or linkers are used in accordance with
conventional practice.
[0138] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that not all progeny will have precisely identical DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same function or biological activity as screened for
in the originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
[0139] As used herein, "germline sequence" refers to a sequence of
unrearranged immunoglobulin DNA sequences. Any suitable source of
unrearranged immunoglobulin sequences may be used. Human germline
sequences may be obtained, for example, from JOINSOLVER germline
databases on the website for the National Institute of Arthritis
and Musculoskeletal and Skin Diseases of the United States National
Institutes of Health. Mouse germline sequences may be obtained, for
example, as described in Giudicelli et al. (2005) Nucleic Acids
Res. 33:D256-D261.
Physical and Functional Properties of the Exemplary Anti-TIGIT
Antibodies
[0140] The present invention provides anti-TIGIT antibodies and
antigen-binding fragments thereof having specified structural and
functional features, and methods of use of the antibodies or
antigen-binding fragments thereof in the treatment or prevention of
disease (e.g., cancer or infectious disease).
[0141] An "anti-TIGIT antibody or antigen-binding fragment thereof
of the present invention" includes: any antibody or antigen-binding
fragment thereof that is discussed herein (e.g., 14A6, 28H5, 31C6
or humanized versions of the these antibodies disclosed in Table 4)
or a variant thereof (e.g., sequence variant or functional
variant); any antibody or antigen-binding fragment comprising any
one or more of the CDRs set forth in Table 4; any antibody or
antigen-binding fragment that binds to the same epitope in human
TIGIT as the antibodies discussed herein (e.g., 14A6, 28H5 or
31C6); and any antibody or antigen-binding fragment that
cross-blocks (partially or fully) or is cross-blocked (partially or
fully) by an antibody discussed herein (e.g., 14A6, 28H5 or 31C6)
for TIGIT binding.
[0142] Cross-blocking antibodies and antigen-binding fragments
thereof can be identified based on their ability to cross-compete
with an antibody of the invention in standard binding assays (e.g.,
BIACore, ELISA, flow cytometry). For example, standard ELISA assays
can be used in which a recombinant TIGIT (e.g., human TIGIT)
protein is immobilized on the plate, one of the antibodies is
fluorescently labeled and the ability of non-labeled antibodies to
compete off the binding of the labeled antibody is evaluated.
Additionally or alternatively, BIAcore analysis can be used to
assess the ability of the antibodies to cross-compete. The ability
of a test antibody to inhibit the binding of another antibody (for
example, antibody 14A6 or 28H5 or 31C6) to TIGIT (e.g., human
TIGIT) demonstrates that the test antibody can compete with another
antibody (e.g., 14A6 or 28H5 or 31C6) for binding to TIGIT (e.g.,
human TIGIT) and thus, may, in some cases, bind to the same epitope
on TIGIT (e.g., human TIGIT) as antibody 14A6 or 28H5 or 31C6 or to
an overlapping epitope.
[0143] As stated above, antibodies and fragments that bind to the
same epitope as any of the anti-TIGIT antibodies or antigen-binding
fragments thereof of the present invention also form part of the
present invention. Further, antibodies that bind to an epitope that
overlaps with the epitope bound by any of the anti-TIGIT antibodies
of the invention also form part of the present invention. There are
several methods available for mapping antibody epitopes on target
antigens, including: H/D-Ex Mass spec, X-ray crystallography,
pepscan analysis and site directed mutagenesis. For example, HDX
(Hydrogen Deuterium Exchange) coupled with proteolysis and mass
spectrometry can be used to determine the epitope of an antibody on
a specific antigen Y. HDX-MS relies on the accurate measurement and
comparison of the degree of deuterium incorporation by an antigen
when incubated in D.sub.2O on its own and in presence of its
antibody at various time intervals. Deuterium is exchanged with
hydrogen on the amide backbone of the proteins in exposed areas
whereas regions of the antigen bound to the antibody will be
protected and will show less or no exchange after analysis by
LC-MS/MS of proteolytic fragments. Example 9 exemplifies the use of
HDX to map the epitope bound by antibody 14A6.
[0144] Examples of the immunoglobulin chains of anti-TIGIT
antibodies of the invention as well as their CDRs include, but are
not limited those disclosed in Table 4 (SEQ ID NOs: 1-30, 37-52,
57-83 or 88-167). The present invention includes any polypeptide
comprising or consisting of the amino acid sequences of SEQ ID NOs:
1-30, 37-52, 57-83 or 88-167, and recombinant nucleotides encoding
such polypeptides.
[0145] The scope of the present invention includes isolated
anti-TIGIT antibodies and antigen-binding fragments thereof (e.g.,
humanized antibodies), comprising a variant of an immunoglobulin
chain set forth herein, e.g., any of SEQ ID NOs: 7-30, 37-52,
63-64, 94-95 or 124-133; wherein the variant exhibits one or more
of the following properties: (i) binds human TIGIT; (ii)
cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocks
binding of human TIGIT to human CD155 and human CD112; (iv)
increases T cell activation; (v) stimulates antigen-specific T-cell
production of IL-2 and IFN.gamma.; (vi) blocks induction of T cell
suppression of activation induced by TIGIT ligation with cognate
ligands CD155 and CD112.
[0146] In other embodiments, the invention provides antibodies or
antigen-binding fragment thereof that binds human TIGIT (e.g.,
humanized antibodies) and has V.sub.L domains and V.sub.H domains
with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence
identity with SEQ ID NOs: 7-30, 37-52, 63-64, 94-95 or 124-133;
wherein the variant exhibits the desired binding and properties,
e.g., (i) binds human TIGIT with a KD value of about 1.times.10-9 M
to about 1.times.10-12 M as determined by surface plasmon resonance
(e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); (ii)
cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocks
binding of human TIGIT to human CD155 and human CD112; (iv)
increases T cell activation; (v) stimulates antigen-specific T-cell
production of IL-2 and IFN.gamma.; (vi) blocks induction of T cell
suppression of activation induced by TIGIT ligation with cognate
ligands CD155 and CD112.
[0147] "Conservatively modified variants" or "conservative
substitution" refers to substitutions of amino acids in a protein
with other amino acids having similar characteristics (e.g. charge,
side-chain size, hydrophobicity/hydrophilicity, backbone
conformation and rigidity, etc.), such that the changes can
frequently be made without altering the biological activity of the
protein. Those of skill in this art recognize that, in general,
single amino acid substitutions in non-essential regions of a
polypeptide do not substantially alter biological activity (see,
e.g., Watson et al. (1987) Molecular Biology of the Gene, The
Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition,
substitutions of structurally or functionally similar amino acids
are less likely to disrupt biological activity. Exemplary
conservative substitutions are set forth in Table 1.
TABLE-US-00001 TABLE 1 Exemplary Conservative Amino Acid
Substitutions Original residue Conservative substitution Ala (A)
Gly; Ser Arg (R) Lys; His Asn (N) Gln; His Asp (D) Glu; Asn Cys (C)
Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln
Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; His Met (M) Leu;
Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) Thr Thr (T) Ser
Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu
[0148] Function-conservative variants of the antibodies of the
invention are also contemplated by the present invention.
"Function-conservative variants," as used herein, refers to
antibodies or fragments in which one or more amino acid residues
have been changed without altering a desired property, such an
antigen affinity and/or specificity. Such variants include, but are
not limited to, replacement of an amino acid with one having
similar properties, such as the conservative amino acid
substitutions of Table 1. Also provided are isolated polypeptides
comprising the V.sub.L domains of the anti-TIGIT antibodies of the
invention (e.g., SEQ ID NOs: 8, 25-30 and 48-52), and isolated
polypeptides comprising the V.sub.H domains (e.g., SEQ ID NOs: 7,
9-24 and 37-47) of the anti-TIGIT antibodies of the invention
having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more
amino acid substitutions. Also provided are isolated polypeptides
comprising the V.sub.L domains of the anti-TIGIT antibodies of the
invention (e.g., SEQ ID NO:64) and isolated polypeptides comprising
the V.sub.H domains (e.g., SEQ ID NO:63) of the anti-TIGIT
antibodies of the invention having up to 0, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30 or more amino acid substitutions. Also provided
are isolated polypeptides comprising the V.sub.L domains of the
anti-TIGIT antibodies of the invention (e.g., SEQ ID NOs: 95 and
130-133) and isolated polypeptides comprising the V.sub.H domains
(e.g., SEQ ID NOs: 94 and 124-129) of the anti-TIGIT antibodies of
the invention having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30 or more amino acid substitutions.
[0149] In another embodiment, provided is an antibody or
antigen-binding fragment thereof that binds TIGIT and has V.sub.L
domains and V.sub.H domains with at least 99% 98%, 97%, 96%, 95%,
90%, 85%, 80% or 75% sequence identity to one or more of the
V.sub.L domains or V.sub.H domains described herein, and exhibits
specific binding to TIGIT. In another embodiment the binding
antibody or antigen-binding fragment thereof of the present
invention comprises V.sub.L and V.sub.H domains (with and without
signal sequence) having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30 or more amino acid substitutions, and exhibits specific
binding to TIGIT.
Polynucleotides and Polypeptides
[0150] The present invention further comprises the polynucleotides
encoding any of the polypeptides or immunoglobulin chains of
anti-TIGIT antibodies and antigen-binding fragments thereof of the
invention. For example, the present invention includes the
polynucleotides encoding the amino acids described in SEQ ID NOs:
1-30, 37-52, 57-83 and 88-167, as well as polynucleotides which
hybridize thereto and, also, any polypeptide encoded by such a
hybridizing polynucleotide. In one embodiment, the invention
comprises a nucleic acid sequence comprising or consisting
essentially of SEQ ID NO:84 or SEQ ID NO:85.
[0151] In general, the polynucleotides hybridize under low,
moderate or high stringency conditions, and encode antibodies or
antigen-binding fragments thereof that maintain the ability to bind
to TIGIT (human, rhesus and/or cynomolgous monkey, e.g., Macaca
fascicularis). A first polynucleotide molecule is "hybridizable" to
a second polynucleotide molecule when a single stranded form of the
first polynucleotide molecule can anneal to the second
polynucleotide molecule under the appropriate conditions of
temperature and solution ionic strength (see Sambrook, et al.,
supra). The conditions of temperature and ionic strength determine
the "stringency" of the hybridization. Typical low stringency
hybridization conditions include 55.degree. C., 5.times.SSC, 0.1%
SDS and no formamide; or 30% formamide, 5.times.SSC, 0.5% SDS at
42.degree. C. Typical moderate stringency hybridization conditions
are 40% formamide, with 5.times. or 6.times.SSC and 0.1% SDS at
42.degree. C. High stringency hybridization conditions are 50%
formamide, 5.times. or 6.times.SSC at 42.degree. C. or, optionally,
at a higher temperature (e.g., 57.degree. C., 59.degree. C.,
60.degree. C., 62.degree. C., 63.degree. C., 65.degree. C. or
68.degree. C.). In general, SSC is 0.15M NaCl and 0.015M
Na-citrate. Hybridization requires that the two polynucleotide
contain complementary sequences, although, depending on the
stringency of the hybridization, mismatches between bases are
possible. The appropriate stringency for hybridizing
polynucleotides depends on the length of the polynucleotides and
the degree of complementation, variables well known in the art. The
greater the degree of similarity or homology between two nucleotide
sequences, the higher the stringency under which the nucleic acids
may hybridize. For hybrids of greater than 100 nucleotides in
length, equations for calculating the melting temperature have been
derived (see Sambrook, et al., supra, 9.50-9.51). For hybridization
with shorter polynucleotides, e.g., oligonucleotides, the position
of mismatches becomes more important, and the length of the
oligonucleotide determines its specificity (see Sambrook, et al.,
supra, 11.7-11.8).
[0152] In another embodiment, an isolated polynucleotide, for
example DNA, encoding the polypeptide chains of the isolated
antibodies or antigen-binding fragments set forth herein is
provided. In one embodiment, the isolated polynucleotide encodes an
antibody or antigen-binding fragment thereof comprising at least
one mature immunoglobulin light chain variable (V.sub.L) domain
according to the invention and/or at least one mature
immunoglobulin heavy chain variable (V.sub.H) domain according to
the invention. In some embodiments the isolated polynucleotide
encodes both a light chain and a heavy chain on a single
polynucleotide molecule, and in other embodiments the light and
heavy chains are encoded on separate polynucleotide molecules. In
another embodiment the polynucleotides further encodes a signal
sequence.
[0153] In one embodiment, the invention comprises an isolated
polynucleotide encoding an antibody heavy variable (V.sub.H) domain
or an antigen-binding fragment thereof comprising CDR-H1 (SEQ ID
NO:1), CDR-H2 (SEQ ID NO:2) and CDR-H3 (SEQ ID NO:3 or 79 or 80 or
81 or 82, 83 or 140).
[0154] In one embodiment, the invention comprises an isolated
polynucleotide encoding an antibody light chain variable (V.sub.L)
domain or an antigen-binding fragment thereof comprising CDR-L1
(SEQ ID NO:4), CDR-L2 (SEQ ID NO:5 or 65 or 66 or 67 or 68 or 69 or
70 or 71 or 72 or 73 or 141) and CDR-L3 (SEQ ID NO:6 or 74 or 75 or
76 or 77 or 78).
[0155] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.H) domain of SEQ ID NO: 7.
[0156] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.L) domain of SEQ ID NO: 8.
[0157] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.H) domain of any one of SEQ ID NOs: 9-24 or 37-47.
[0158] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.L) domain of any one of SEQ ID NOs: 25-30 or 48-52
[0159] In one embodiment, the invention comprises an isolated
polynucleotide encoding an antibody heavy variable (V.sub.H) domain
or an antigen-binding fragment thereof comprising CDR-H1 (SEQ ID
NO:57), CDR-H2 (SEQ ID NO:58) and CDR-H3 (SEQ ID NO:59).
[0160] In one embodiment, the invention comprises an isolated
polynucleotide encoding an antibody light chain variable (V.sub.L)
domain or an antigen-binding fragment thereof comprising CDR-L1
(SEQ ID NO:60), CDR-L2 (SEQ ID NO:61) and CDR-L3 (SEQ ID
NO:62).
[0161] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.H) domain of SEQ ID NO: 63.
[0162] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.L) domain of SEQ ID NO: 64.
[0163] In one embodiment, the invention comprises an isolated
polynucleotide encoding an antibody heavy variable (V.sub.H) domain
or an antigen-binding fragment thereof comprising CDR-H1 (SEQ ID
NO: 88), CDR-H2 (SEQ ID NO: 89, 96, 97, 98, 99, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 134 or 135) and CDR-H3 (SEQ
ID NO: 90).
[0164] In one embodiment, the invention comprises an isolated
polynucleotide encoding an antibody light chain variable (V.sub.L)
domain or an antigen-binding fragment thereof comprising CDR-L1
(SEQ ID NO: 91), CDR-L2 (SEQ ID NO: 92, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122 or 123) and CDR-L3 (SEQ ID NO:
93).
[0165] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.H) domain of SEQ ID NO: 94.
[0166] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.L) domain of SEQ ID NO: 95.
[0167] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.H) domain of any one of SEQ ID NOs: 124-129.
[0168] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.L) domain of any one of SEQ ID NOs: 130-133.
[0169] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.H) domain of SEQ ID NO: 127.
[0170] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.H) domain of SEQ ID NO: 128.
[0171] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.L) domain of SEQ ID NO: 130.
[0172] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.L) domain of SEQ ID NO: 132.
[0173] In one embodiment, the invention comprises an isolated
polynucleotide encoding the immunoglobulin heavy chain variable
(V.sub.L) domain of SEQ ID NO: 133.
[0174] This present invention also provides vectors, e.g.,
expression vectors, such as plasmids, comprising the isolated
polynucleotides of the invention, wherein the polynucleotide is
operably linked to control sequences that are recognized by a host
cell when the host cell is transfected with the vector. Also
provided are host cells comprising a vector of the present
invention and methods for producing the antibody or antigen-binding
fragment thereof or polypeptide disclosed herein comprising
culturing a host cell harboring an expression vector or a nucleic
acid encoding the immunoglobulin chains of the antibody or
antigen-binding fragment thereof in culture medium, and isolating
the antigen or antigen-binding fragment thereof from the host cell
or culture medium.
[0175] Also included in the present invention are polypeptides,
e.g., immunoglobulin polypeptides, comprising amino acid sequences
that are at least about 75% identical, 80% identical, more
preferably at least about 90% identical and most preferably at
least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to
the amino acid sequences of the antibodies provided herein when the
comparison is performed by a BLAST algorithm wherein the parameters
of the algorithm are selected to give the largest match between the
respective sequences over the entire length of the respective
reference sequences (e.g. expect threshold: 10; word size: 3; max
matches in a query range: 0; BLOSUM 62 matrix; gap costs: existence
11, extension 1; conditional compositional score matrix
adjustment).
[0176] Sequence identity refers to the degree to which the amino
acids of two polypeptides are the same at equivalent positions when
the two sequences are optimally aligned.
[0177] The following references relate to BLAST algorithms often
used for sequence analysis: BLAST ALGORITHMS: Altschul et al.
(2005) FEBS J. 272(20): 5101-5109; Altschul, S. F., et al., (1990)
J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet.
3:266-272; Madden, T. L., et al., (1996) Meth. Enzymol.
266:131-141; Altschul, S. F., et al., (1997) Nucleic Acids Res.
25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656;
Wootton, J. C., et al., (1993) Comput. Chem. 17:149-163; Hancock,
J. M. et al., (1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENT
SCORING SYSTEMS: Dayhoff, M. O., et al., "A model of evolutionary
change in proteins." in Atlas of Protein Sequence and Structure,
(1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352, Natl.
Biomed. Res. Found., Washington, D.C.; Schwartz, R. M., et al.,
"Matrices for detecting distant relationships." in Atlas of Protein
Sequence and Structure, (1978) vol. 5, suppl. 3." M. O. Dayhoff
(ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.;
Altschul, S. F., (1991) J. Mol. Biol. 219:555-565; States, D. J.,
et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc.
Natl. Acad. Sci. USA 89:10915-10919; Altschul, S. F., et al.,
(1993) J. Mol. Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S.,
et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S.,
et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A.,
et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, S. F.
"Evaluating the statistical significance of multiple distinct local
alignments." in Theoretical and Computational Methods in Genome
Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, New York.
Binding Affinity
[0178] By way of example, and not limitation, the antibodies and
antigen-binding fragments disclosed herein may bind human TIGIT
with a K.sub.D value of at least about 1.times.10.sup.-9M (i.e, a
K.sub.D value of 1.times.10.sup.-9M or lower) as determined by
surface plasmon resonance (e.g., BIACORE) or a similar technique
(e.g. KinExa or OCTET). In one embodiment, the antibodies and
antigen-binding fragments disclosed herein may bind human TIGIT
with a K.sub.D value of at least about 1.times.10.sup.-9M to about
1.times.10.sup.-12 M as determined by surface plasmon resonance
(e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET). In
one embodiment, the antibodies and antigen-binding fragments
disclosed herein may bind human TIGIT with a K.sub.D value of at
about 1.times.10.sup.-9 M to about 1.times.10.sup.-12 M as
determined by surface plasmon resonance (e.g., BIACORE) or a
similar technique (e.g. KinExa or OCTET). In one embodiment, the
antibodies and antigen-binding fragments disclosed herein may bind
human TIGIT with a K.sub.D value of at least about 50 pM (i.e, a
K.sub.D value of about 50 pM or lower) as determined by BIACORE or
a similar technique. In one embodiment, the antibodies and
antigen-binding fragments disclosed herein may bind human TIGIT
with a K.sub.D value of at least about 10 pM (i.e, a K.sub.D value
of about 10 pm lower) as determined by BIACORE or a similar
technique. In one embodiment, the antibodies and antigen-binding
fragments of the invention may bind to human TIGIT with a K.sub.D
of about 50 pM to about 1 pM as determined by BIACORE or a similar
technique.
Immune Cell Activation
[0179] In some embodiments, the antibodies or antigen binding
fragments of the invention increase the activity of an immune cell.
The increase of the activity of an immune cell can be detected
using any method known in the art. In one embodiment, the increase
in activity of an immune cell can be detected by measuring the
proliferation of the immune cell. For example, an increase in
activity of a T cell can be detected by measuring the proliferation
of the T cell or signal transduction events such as tyrosine
phosphorylation of immune receptors or downstream kinases that
transmit signals to transcriptional regulators. In other
embodiments, the increase in activity of an immune cell can be
detected by measuring CTL or NK cell cytotoxic function on specific
target cells or IFN.gamma. cytokine responses, which are associated
with stimulation of anti-tumor immunity. In yet other embodiments,
the increase in activity of an immune cell can be detected by
measuring T cell activation ex vivo in a sample derived from the
subject. In one embodiment, the increase in T cell activity is
determined by: (i) measuring SEB (Staphylococcus Enterotoxin B)
induced production of one or more pro-inflammatory cytokines
selected from the group consisting of: IL-2, TNF.alpha., IL-17,
IFN.gamma., IL-1.beta., GM-CSF, RANTES, IL-6, IL-8, IL-5 and IL-13;
or (ii) measuring mixed lymphocyte reactions or direct anti-CD3 mAb
stimulation of T cell receptor (TCR) signaling to induce production
of a cytokine selected from the group consisting of: IL-2,
TNF.alpha., IL-17, IFN.gamma., IL-1.beta., GM-CSF, RANTES, IL-6,
IL-8, IL-5 and IL-13. In certain embodiments, the anti-TIGIT
antibody or antigen binding fragment thereof of the present
invention will stimulates antigen-specific T-cell production of
IL-2 and/or IFN.gamma. by at least 1.5 fold.
[0180] The present invention includes antagonist anti-TIGIT
antibodies and antigen-binding fragments thereof and methods of use
thereof, e.g., humanized, antagonist anti-TIGIT antibodies and
fragments. An antagonist anti-TIGIT antibody or antigen-binding
fragment thereof antagonizes an activity of human TIGIT such as by
inhibiting TIGIT binding to CD155 and CD112, and inhibiting
functional ITIM signal transduction by TIGIT upon binding to CD155
and CD112. Measurement of anti-TIGIT antagonist activity can be
assessed by demonstrating blocking of T cell suppression following
TCR activation induced by TIGIT ligation with cognate ligands CD155
and CD112. Hence, in one embodiment of increased responses,
treating with antagonist anti-TIGIT antibodies are able to rescue
IL-2 responses to levels observed in T cells that are not repressed
by CD155 or CD112 induction of TIGIT. In a more preferred level of
activation, responses, following treatment with an anti-TIGIT
antagonist antibody may increase responses to a level higher than T
cell responses not repressed by CD155 or CD112.
Ability of Anti-hTIGIT Antibodies to Block Binding to hCD155 and
hCD112
[0181] In some embodiments, the anti-TIGIT antibodies or antigen
binding fragments of the invention are able to block binding of
human TIGIT to human CD155 and human CD112. The ability to block
binding of human TIGIT to human CD155 and human CD112 can be
determined using any method known in the art. In one embodiment,
the ability of the antibodies to block binding of human TIGIT to
human CD155 and human CD112 is determined using an ELISA assay as
described in Example 2.
Methods of Making Antibodies and Antigen-Binding Fragments
Thereof
[0182] Hybridoma cells that produce parental (e.g., rat or mouse)
monoclonal anti-TIGIT antibodies or antigen-binding fragments
thereof discussed herein may be produced by methods which are
commonly known in the art. Such isolated hybridomas are part of the
present invention. These methods include, but are not limited to,
the hybridoma technique originally developed by Kohler, et al.,
(1975) (Nature 256:495-497), as well as the trioma technique
(Hering, et al., (1988) Biomed. Biochim. Acta. 47:211-216 and
Hagiwara, et al., (1993) Hum. Antibod. Hybridomas 4:15), the human
B-cell hybridoma technique (Kozbor, et al., (1983) Immunology Today
4:72 and Cote, et al., (1983) Proc. Natl. Acad. Sci. U.S.A
80:2026-2030), the EBV-hybridoma technique (Cole, et al., in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96, 1985), and electric field based electrofusion using a Cyto
Pulse large chamber cull fusion electroporator (Cyto Pulse
Sciences, Inc., Glen Burnie, Md.). Preferably, mouse splenocytes
are isolated and fused with PEG or by electrofusion to a mouse
myeloma cell line based upon standard protocols. The resulting
hybridomas may then be screened for the production of
antigen-specific antibodies. For example, single cell suspensions
of splenic lymphocytes from immunized mice may by fused to
one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma
cells (ATCC, CRL 1580) with 50% PEG. Cells may be plated at
approximately 2.times.10.sup.5 cells/mL in a flat bottom microtiter
plate, followed by a two week incubation in selective medium
containing 20% fetal Clone Serum, 18% "653" conditioned media, 5%
origen (IGEN), 4 mM L-glutamine, 1 mM L-glutamine, 1 mM sodium
pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml
penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and
1.times.HAT (Sigma; the HAT is added 24 hours after the fusion).
After two weeks, cells may be cultured in medium in which the HAT
is replaced with HT. Individual wells may then be screened by ELISA
for anti-TIGIT monoclonal IgG antibodies. Once extensive hybridoma
growth occurs, medium can be observed usually after 10-14 days. The
antibody secreting hybridomas may be replated, screened again, and
if still positive for human IgG, anti-TIGIT monoclonal antibodies,
can be subcloned at least twice by limiting dilution. The stable
subclones may then be cultured in vitro to generate small amounts
of antibody in tissue culture medium for characterization.
[0183] Thus, the present invention includes methods for making an
anti-TIGIT antibody or antigen-binding fragment thereof of the
present invention comprising culturing a hybridoma cell that
expresses the antibody or fragment under condition favorable to
such expression and, optionally, isolating the antibody or fragment
from the hybridoma and/or the growth medium (e.g. cell culture
medium).
[0184] The anti-TIGIT antibodies disclosed herein may also be
produced recombinantly (e.g., in an E. coli/T7 expression system, a
mammalian cell expression system or a lower eukaryote expression
system). In this embodiment, nucleic acids encoding the antibody
immunoglobulin molecules of the invention (e.g., V.sub.H or
V.sub.L) may be inserted into a pET-based plasmid and expressed in
the E. coli/T7 system. For example, the present invention includes
methods for expressing an antibody or antigen-binding fragment
thereof or immunoglobulin chain thereof in a host cell (e.g.,
bacterial host cell such as E. coli such as BL21 or BL21DE3)
comprising expressing T7 RNA polymerase in the cell which also
includes a polynucleotide encoding an immunoglobulin chain that is
operably linked to a T7 promoter. For example, in an embodiment of
the invention, a bacterial host cell, such as a E. coli, includes a
polynucleotide encoding the T7 RNA polymerase gene operably linked
to a lac promoter and expression of the polymerase and the chain is
induced by incubation of the host cell with IPTG
(isopropyl-beta-D-thiogalactopyranoside).
[0185] There are several methods by which to produce recombinant
antibodies which are known in the art. One example of a method for
recombinant production of antibodies is disclosed in U.S. Pat. No.
4,816,567.
[0186] Transformation can be by any known method for introducing
polynucleotides into a host cell. Methods for introduction of
heterologous polynucleotides into mammalian cells are well known in
the art and include dextran-mediated transfection, calcium
phosphate precipitation, polybrene-mediated transfection,
protoplast fusion, electroporation, encapsulation of the
polynucleotide(s) in liposomes, biolistic injection and direct
microinjection of the DNA into nuclei. In addition, nucleic acid
molecules may be introduced into mammalian cells by viral vectors.
Methods of transforming cells are well known in the art. See, for
example, U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461 and
4,959,455.
[0187] Thus, the present invention includes recombinant methods for
making an anti-TIGIT antibody or antigen-binding fragment thereof
of the present invention, or an immunoglobulin chain thereof,
comprising introducing a polynucleotide encoding one or more
immunoglobulin chains of the antibody or fragment (e.g., heavy
and/or light immunoglobulin chain); culturing the host cell (e.g.,
CHO or Pichia or Pichia pastoris) under condition favorable to such
expression and, optionally, isolating the antibody or fragment or
chain from the host cell and/or medium in which the host cell is
grown.
[0188] Anti-TIGIT antibodies can also be synthesized by any of the
methods set forth in U.S. Pat. No. 6,331,415.
[0189] Eukaryotic and prokaryotic host cells, including mammalian
cells as hosts for expression of the antibodies or fragments or
immunoglobulin chains disclosed herein are well known in the art
and include many immortalized cell lines available from the
American Type Culture Collection (ATCC). These include, inter alia,
Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby
hamster kidney (BHK) cells, monkey kidney cells (COS), human
hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3
cells, HEK-293 cells and a number of other cell lines. Mammalian
host cells include human, mouse, rat, dog, monkey, pig, goat,
bovine, horse and hamster cells. Cell lines of particular
preference are selected through determining which cell lines have
high expression levels. Other cell lines that may be used are
insect cell lines, such as Sf9 cells, amphibian cells, bacterial
cells, plant cells and fungal cells. Fungal cells include yeast and
filamentous fungus cells including, for example, Pichia pastoris,
Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia
membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri),
Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia
guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica,
Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula
polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida
albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus
oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium
sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens
and Neurospora crassa. Pichia sp., any Saccharomyces sp., Hansenula
polymorpha, any Kluyveromyces sp., Candida albicans, any
Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any
Fusarium sp., Yarrowia lipolytica, and Neurospora crassa. When
recombinant expression vectors encoding the heavy chain or
antigen-binding portion or fragment thereof, the light chain and/or
antigen-binding fragment thereof 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
or fragment or chain in the host cells or secretion of the into the
culture medium in which the host cells are grown.
[0190] Antibodies and antigen-binding fragments thereof and
immunoglobulin chains can be recovered from the culture medium
using standard protein purification methods. Further, expression of
antibodies and antigen-binding fragments thereof and immunoglobulin
chains of the invention (or other moieties therefrom) from
production cell lines can be enhanced using a number of known
techniques. For example, the glutamine synthetase gene expression
system (the GS system) is a common approach for enhancing
expression under certain conditions. The GS system is discussed in
whole or part in connection with European Patent Nos. 0 216 846, 0
256 055, and 0 323 997 and European Patent Application No.
89303964.4. Thus, in an embodiment of the invention, the mammalian
host cells (e.g., CHO) lack a glutamine synthetase gene and are
grown in the absence of glutamine in the medium wherein, however,
the polynucleotide encoding the immunoglobulin chain comprises a
glutamine synthetase gene which complements the lack of the gene in
the host cell.
[0191] The present invention includes methods for purifying an
anti-TIGIT antibody or antigen-binding fragment thereof of the
present invention comprising introducing a sample comprising the
antibody or fragment to a purification medium (e.g., cation
exchange medium, anion exchange medium, hydrophobic exchange
medium, affinity purification medium (e.g., protein-A, protein-G,
protein-A/G, protein-L)) and either collecting purified antibody or
fragment from the flow-through fraction of said sample that does
not bind to the medium; or, discarding the flow-through fraction
and eluting bound antibody or fragment from the medium and
collecting the eluate. In an embodiment of the invention, the
medium is in a column to which the sample is applied. In an
embodiment of the invention, the purification method is conducted
following recombinant expression of the antibody or fragment in a
host cell, e.g., wherein the host cell is first lysed and,
optionally, the lysate is purified of insoluble materials prior to
purification on a medium.
[0192] In general, glycoproteins produced in a particular cell line
or transgenic animal will have a glycosylation pattern that is
characteristic for glycoproteins produced in the cell line or
transgenic animal. Therefore, the particular glycosylation pattern
of an antibody will depend on the particular cell line or
transgenic animal used to produce the antibody. However, all
antibodies encoded by the nucleic acid molecules provided herein,
or comprising the amino acid sequences provided herein, comprise
the instant invention, independent of the glycosylation pattern the
antibodies may have. Similarly, in particular embodiments,
antibodies with a glycosylation pattern comprising only
non-fucosylated N-glycans may be advantageous, because these
antibodies have been shown to typically exhibit more potent
efficacy than their fucosylated counterparts both in vitro and in
vivo (See for example, Shinkawa et al., J. Biol. Chem. 278:
3466-3473 (2003); U.S. Pat. Nos. 6,946,292 and 7,214,775). These
antibodies with non-fucosylated N-glycans are not likely to be
immunogenic because their carbohydrate structures are a normal
component of the population that exists in human serum IgG.
[0193] The present invention includes polyclonal anti-TIGIT
antibodies and antigen-binding fragments thereof, e.g., a
composition comprising a plurality of anti-TIGIT antibodies and
fragments, which include one or more of the anti-TIGIT antibodies
or antigen-binding fragments thereof of the present invention, and
methods of use thereof. A polyclonal antibody is an antibody which
was produced among or in the presence of one or more other,
non-identical antibodies. In general, polyclonal antibodies are
produced from collections of different B-lymphocytes, e.g., the
B-lymphocyte of an animal treated with an immunogen of interest,
which produces a population of different antibodies but which are
all directed to the immunogen. Usually, polyclonal antibodies are
obtained directly from an immunized animal, e.g., spleen, serum or
ascites fluid.
[0194] The present invention includes bispecific and bifunctional
antibodies and antigen-binding fragments having a binding
specificity for TIGIT and another antigen such as, for example,
PD-1 or PD-L1 or LAG-3, and methods of use thereof. In an
embodiment of the invention, the anti-TIGIT chains comprise any one
of the VH/VL sequences described in Table 4, and the PD1 chains
comprise the amino acid sequence of SEQ ID NOs: 33 and 34 or of SEQ
ID NOs: 35 and 36 (or an antigen binding fragment of any of said
sequences). A bispecific or bifunctional antibody is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites. Bispecific antibodies can be produced
by a variety of methods including fusion of hybridomas or linking
of Fab' fragments. See, e.g., Songsivilai, et al., (1990) Clin.
Exp. Immunol. 79: 315-321, Kostelny, et al., (1992) J Immunol.
148:1547-1553. In addition, bispecific antibodies may be formed as
"diabodies" (Holliger, et al., (1993) PNAS USA 90:6444-6448) or as
"Janusins" (Traunecker, et al., (1991) EMBO J. 10:3655-3659 and
Traunecker, et al., (1992) Int. J. Cancer Suppl. 7:51-52).
[0195] The present invention further includes anti-TIGIT
antigen-binding fragments of the anti-TIGIT antibodies disclosed
herein. The antibody fragments include F(ab).sub.2 fragments, which
may be produced by enzymatic cleavage of an IgG by, for example,
pepsin. Fab fragments may be produced by, for example, reduction of
F(ab).sub.2 with dithiothreitol or mercaptoethylamine.
[0196] Immunoglobulins may be assigned to different classes
depending on the amino acid sequences of the constant domain of
their heavy chains. There are at least five major classes of
immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these
may be further divided into subclasses (isotypes), e.g. IgG1, IgG2,
IgG3 and IgG4; IgA1 and IgA2. The invention comprises antibodies
and antigen-binding fragments of any of these classes or subclasses
of antibodies.
[0197] In one embodiment, the antibody or antigen-binding fragment
comprises a heavy chain constant region, e.g. a human constant
region, such as .gamma.1, .gamma.2, .gamma.3, or .gamma.4 human
heavy chain constant region or a variant thereof. In another
embodiment, the antibody or antigen-binding fragment comprises a
light chain constant region, e.g. a human light chain constant
region, such as lambda or kappa human light chain region or variant
thereof. By way of example, and not limitation the human heavy
chain constant region can be .gamma.4 and the human light chain
constant region can be kappa. In an alternative embodiment, the Fc
region of the antibody is .gamma.4 with a Ser228Pro mutation
(Schuurman, J et. al., Mol. Immunol. 38: 1-8, 2001).
[0198] In one embodiment, the antibody or antigen-binding fragment
comprises a heavy chain constant region of the IgG1 subtype.
[0199] In some embodiments, different constant domains may be
appended to humanized V.sub.L and V.sub.H regions derived from the
CDRs provided herein. For example, if a particular intended use of
an antibody (or fragment) of the present invention were to call for
altered effector functions, a heavy chain constant domain other
than human IgG1 may be used, or hybrid IgG1/IgG4 may be
utilized.
[0200] Although human IgG1 antibodies provide for long half-life
and for effector functions, such as complement activation and
antibody-dependent cellular cytotoxicity, such activities may not
be desirable for all uses of the antibody. In such instances a
human IgG4 constant domain, for example, may be used. The present
invention includes anti-TIGIT antibodies and antigen-binding
fragments thereof which comprise an IgG4 constant domain, e.g.,
antagonist, humanized anti-TIGIT antibodies and fragments, and
methods of use thereof. In one embodiment, the IgG4 constant domain
can differ from the native human IgG4 constant domain (Swiss-Prot
Accession No. P01861.1) at a position corresponding to position 228
in the EU system and position 241 in the KABAT system, where the
native Ser108 is replaced with Pro, in order to prevent a potential
inter-chain disulfide bond between Cys106 and Cys109 (corresponding
to positions Cys 226 and Cys 229 in the EU system and positions Cys
239 and Cys 242 in the KABAT system) that could interfere with
proper intra-chain disulfide bond formation. See Angal et al.
(1993) Mol. Imunol. 30:105. In other instances, a modified IgG1
constant domain which has been modified to increase half-life or
reduce effector function can be used.
Antibody Engineering
[0201] Further included are embodiments in which the anti-TIGIT
antibodies and antigen-binding fragments thereof are engineered
antibodies to include modifications to framework residues within
the variable domains of a parental (e.g., mouse or rat) monoclonal
antibody, e.g. to improve the properties of the antibody or
fragment. Typically, such framework modifications are made to
decrease the immunogenicity of the antibody or fragment. This is
usually accomplished by replacing non-CDR residues in the variable
domains (i.e. framework residues) in a parental (e.g. rodent)
antibody or fragment with analogous residues from the immune
repertoire of the species in which the antibody is to be used, e.g.
human residues in the case of human therapeutics. Such an antibody
or fragment is referred to as a "humanized" antibody or fragment.
In some cases it is desirable to increase the affinity, or alter
the specificity of an engineered (e.g. humanized) antibody. One
approach is to "backmutate" one or more framework residues to the
corresponding germline sequence. More specifically, an antibody or
fragment that has undergone somatic mutation can contain framework
residues that differ from the germline sequence from which the
antibody is derived. Such residues can be identified by comparing
the antibody or fragment framework sequences to the germline
sequences from which the antibody or fragment is derived. Another
approach is to revert to the original parental (e.g., rodent)
residue at one or more positions of the engineered (e.g. humanized)
antibody, e.g. to restore binding affinity that may have been lost
in the process of replacing the framework residues. (See, e.g.,
U.S. Pat. No. 5,693,762, U.S. Pat. No. 5,585,089 and U.S. Pat. No.
5,530,101.)
[0202] In certain embodiments, the anti-TIGIT antibodies and
antigen-binding fragments thereof are engineered (e.g. humanized)
to include modifications to in the framework and/or CDRs to improve
their properties. Such engineered changes can be based on molecular
modelling. A molecular model for the variable region for the
parental (non-human) antibody sequence can be constructed to
understand the structural features of the antibody and used to
identify potential regions on the antibody that can interact with
the antigen. Conventional CDRs are based on alignment of
immunoglobulin sequences and identifying variable regions. Kabat et
al., (1991) Sequences of Proteins of Immunological Interest, Kabat,
et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH
Publ. No. 91-3242; Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et
al., (1977) J. Biol. Chem. 252:6609-6616. Chothia and coworkers
carefully examined conformations of the loops in crystal structures
of antibodies and proposed hypervariable loops. Chothia, et al.,
(1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature
342:878-883. There are variations between regions classified as
"CDRs" and "hypervariable loops". Later studies (Raghunathan et al,
(2012) J. Mol Recog. 25, 3, 103-113) analyzed several
antibody-antigen crystal complexes and observed that the antigen
binding regions in antibodies do not necessarily conform strictly
to the "CDR" residues or "hypervarible" loops. The molecular model
for the variable region of the non-human antibody can be used to
guide the selection of regions that can potentially bind to the
antigen. In practice the potential antigen binding regions based on
model differ from the conventional "CDR" s or "hyper variable"
loops. Commercial scientific software such as MOE (Chemical
Computing Group) can be used for molecular modeling. Human
frameworks can be selected based on best matches with the non-human
sequence both in the frameworks and in the CDRs. For FR4 (framework
4) in VH, VJ regions for the human germlines are compared with the
corresponding non-human region. In the case of FR4 (framework 4) in
VL, J-kappa and J-Lambda regions of human germline sequences are
compared with the corresponding non-human region. Once suitable
human frameworks are identified, the CDRs are grafted into the
selected human frameworks. In some cases certain residues in the
VL-VH interface can be retained as in the non-human (parental)
sequence. Molecular models can also be used for identifying
residues that can potentially alter the CDR conformations and hence
binding to antigen. In some cases, these residues are retained as
in the non-human (parental) sequence. Molecular models can also be
used to identify solvent exposed amino acids that can result in
unwanted effects such as glycosylation, deamidation and oxidation.
Developability filters can be introduced early on in the design
stage to eliminate/minimize these potential problems.
[0203] Another type of framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T cell epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in U.S. Pat. No. 7,125,689.
[0204] In particular embodiments, it will be desirable to change
certain amino acids containing exposed side-chains to another amino
acid residue in order to provide for greater chemical stability of
the final antibody, so as to avoid deamidation or isomerization.
The deamidation of asparagine may occur on NG, DG, NG, NS, NA, NT,
QG or QS sequences and result in the creation of an isoaspartic
acid residue that introduces a kink into the polypeptide chain and
decreases its stability (isoaspartic acid effect). Isomerization
can occur at DG, DS, DA or DT sequences. In certain embodiments,
the antibodies of the present disclosure do not contain deamidation
or asparagine isomerism sites.
[0205] For example, an asparagine (Asn) residue may be changed to
Gln or Ala to reduce the potential for formation of isoaspartate at
any Asn-Gly sequences, particularly within a CDR. A similar problem
may occur at a Asp-Gly sequence. Reissner and Aswad (2003) Cell.
Mol. Life Sci. 60:1281. Isoaspartate formation may debilitate or
completely abrogate binding of an antibody to its target antigen.
See, Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734. In one
embodiment, the asparagine is changed to glutamine (Gln). It may
also be desirable to alter an amino acid adjacent to an asparagine
(Asn) or glutamine (Gln) residue to reduce the likelihood of
deamidation, which occurs at greater rates when small amino acids
occur adjacent to asparagine or glutamine. See, Bischoff &
Kolbe (1994) J. Chromatog. 662:261. In addition, any methionine
residues (typically solvent exposed Met) in CDRs may be changed to
Lys, Leu, Ala, or Phe or other amino acids in order to reduce the
possibility that the methionine sulfur would oxidize, which could
reduce antigen-binding affinity and also contribute to molecular
heterogeneity in the final antibody preparation. Id. Additionally,
in order to prevent or minimize potential scissile Asn-Pro peptide
bonds, it may be desirable to alter any Asn-Pro combinations found
in a CDR to Gln-Pro, Ala-Pro, or Asn-Ala. Antibodies with such
substitutions are subsequently screened to ensure that the
substitutions do not decrease the affinity or specificity of the
antibody for TIGIT, or other desired biological activity to
unacceptable levels.
TABLE-US-00002 TABLE 2 Exemplary stabilizing CDR variants CDR
Residue Stabilizing Variant Sequence Asn-Gly Gln-Gly, Ala-Gly, or
Asn-Ala (N-G) (Q-G), (A-G), or (N-A) Asp-Gly Glu-Gly, Ala-Gly or
Asp-Ala (D-G) (E-G), (A-G), or (D-A) Met (typically solvent
exposed) Lys, Leu, Ala, or Phe (M) (K), (L), (A), or (F) Asn Gln or
Ala (N) (Q) or (A) Asn-Pro Gln-Pro, Ala-Pro, or Asn-Ala (N-P)
(Q-P), (A-P), or (N-A)
[0206] In some embodiments of the instant invention, the CDR3 of
SEQ ID NO:3 can be modified at position 110W to reduce or remove
potential oxidation (wherein the numbering is according to Kabat).
Thus, for example SEQ ID NO:3 (MPSFITLASLSTWEGYFDF) can be modified
to any of the following sequences: MPSFITLASLSTFEGYFDF (SEQ ID
NO:79), MPSFITLASLSTYEGYFDF (SEQ ID NO:80), MPSFITLASLSTIEGYFDF
(SEQ ID NO:81), MPSFITLASLSTVEGYFDF (SEQ ID NO:82) or
MPSFITLASLSTLEGYFDF (SEQ ID NO:83). Therefore, in some embodiments
of the instant invention, the anti-TIGIT antibody of the invention
comprises a heavy chain variable region comprising the CDR1 of SEQ
ID NO:1, the CDR2 of SEQ ID NO:2 and the CDR3 of SEQ ID NO: 3, 79,
80, 81, 82 or 83.
[0207] In some embodiments of the instant invention, the CDR2 of
SEQ ID NO:5 can be modified at positions 52N and 53S to reduce or
remove potential deamidation sites (wherein the numbering is
according to Kabat). Thus, for example SEQ ID NO:5 (YANSLQT) can be
modified to any of the following sequences: YASNLQT (SEQ ID NO:65),
YASSLQT (SEQ ID NO:66), YASTLQT (SEQ ID NO:67), YATTLQT (SEQ ID
NO:68), YASYLQT (SEQ ID NO:69), YANQLQT (SEQ ID NO:70), YAGSLQT
(SEQ ID NO:71), YASQLQT (SEQ ID NO:72), YADSLQT (SEQ ID NO:73).
Therefore, in some embodiments of the instant invention, the
anti-TIGIT antibody of the invention comprises a light chain
variable region comprising the CDR1 of SEQ ID NO:4, the CDR2 of SEQ
ID NO:5, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69,
SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:72 or SEQ ID NO:73, and the
CDR3 of SEQ ID NO:6.
[0208] In some embodiments of the instant invention, the CDR3 of
SEQ ID NO:6 can be modified at position 95W to reduce or remove
potential oxidation (wherein the numbering is according to Kabat).
Thus, for example SEQ ID NO:6 (QQYYSGWT) can be modified to any of
the following sequences: QQYYSGFT (SEQ ID NO:74), QQYYSGYT (SEQ ID
NO:75), QQYYSGIT (SEQ ID NO: 76), QQYYSGVT (SEQ ID NO:77), QQYYSGLT
(SEQ ID NO:78). Therefore, in some embodiments of the instant
invention, the anti-TIGIT antibody of the invention comprises a
light chain variable region comprising the CDR1 of SEQ ID NO:4, the
CDR2 of SEQ ID NO:5 and the CDR3 of SEQ ID NO:6, SEQ ID NO:74, SEQ
ID NO:75, SEQ ID NO:76, SEQ ID NO:77 or SEQ ID NO:78.
[0209] In some embodiments of the instant invention, the anti-TIGIT
antibody of the invention comprises a light chain variable region
comprising the CDR1 of SEQ ID NO:4, the CDR2 of SEQ ID NO:5, SEQ ID
NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ
ID NO:72, SEQ ID NO:72 or SEQ ID NO:73, and the CDR3 of SEQ ID
NO:6, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77 or SEQ
ID NO:78.
[0210] In another embodiment of the instant invention, the
anti-TIGIT antibody of the invention comprises a heavy chain FR4
region which comprises the amino acid sequence of any one of SEQ ID
NOs: 7, 9-24 or 38-47, wherein the M at position 122 is substituted
with V, L, A, R, N, P Q, E, G, I, H, K, F, S, T, W, or L to avoid
potential oxidation.
[0211] In another embodiment of the instant invention, the
anti-TIGIT antibody of the invention comprises a heavy chain FR4
region which comprises the amino acid sequence of any one of SEQ ID
NOs: 7, 9-24 or 38-47, wherein the M at position 122 and the V at
position 123 are substituted with T and L respectively to avoid
potential oxidation.
[0212] In some embodiments of the instant invention, the CDR3 of
SEQ ID NO:90 can be modified at position 6 to reduce or remove
potential oxidation. Thus, for example SEQ ID NO:90 (GGPYGWYFDV) be
modified to any of the following sequences: SEQ ID NOs:
154-167.
Antibody Engineering of the Fc Region
[0213] The antibodies (e.g., humanized antibodies) and
antigen-binding fragments thereof disclosed herein (e.g., 14A6,
28H5, 31C6 and humanized versions thereof) can also be engineered
to include modifications within the Fc region, typically to alter
one or more properties of the antibody, such as serum half-life,
complement fixation, Fc receptor binding, and/or effector function
(e.g., antigen-dependent cellular cytotoxicity). Furthermore, the
antibodies and antigen-binding fragments thereof disclosed herein
(e.g., 14A6, 28H5, 31C6 and humanized versions thereof) can be
chemically modified (e.g., one or more chemical moieties can be
attached to the antibody) or be modified to alter its
glycosylation, again to alter one or more properties of the
antibody or fragment. Each of these embodiments is described in
further detail below. The numbering of residues in the Fc region is
that of the EU index of Kabat.
[0214] The antibodies and antigen-binding fragments thereof
disclosed herein (e.g., 14A6, 28H5, 31C6 and humanized versions
thereof) also include antibodies and fragments with modified (or
blocked) Fc regions to provide altered effector functions. See,
e.g., U.S. Pat. No. 5,624,821; WO2003/086310; WO2005/120571;
WO2006/0057702. Such modifications can be used to enhance or
suppress various reactions of the immune system, with possible
beneficial effects in diagnosis and therapy. Alterations of the Fc
region include amino acid changes (substitutions, deletions and
insertions), glycosylation or deglycosylation, and adding multiple
Fc regions. Changes to the Fc can also alter the half-life of
antibodies in therapeutic antibodies, enabling less frequent dosing
and thus increased convenience and decreased use of material. See
Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35.
[0215] In one embodiment, the antibody or antigen-binding fragment
of the invention (e.g., 14A6, 28H5, 31C6 or humanized versions
thereof) is an IgG4 isotype antibody or fragment comprising a
Serine to Proline mutation at a position corresponding to position
228 (S228P; EU index) in the hinge region of the heavy chain
constant region. This mutation has been reported to abolish the
heterogeneity of inter-heavy chain disulfide bridges in the hinge
region (Angal et al. supra; position 241 is based on the Kabat
numbering system).
[0216] In one embodiment of the invention, the hinge region of CH1
is modified such that the number of cysteine residues in the hinge
region is increased or decreased. This approach is described
further in U.S. Pat. No. 5,677,425. The number of cysteine residues
in the hinge region of CH1 is altered, for example, to facilitate
assembly of the light and heavy chains or to increase or decrease
the stability of the antibody.
[0217] In another embodiment, the Fc hinge region of an antibody or
antigen-binding fragment of the invention (e.g., 14A6, 28H5 or 31C6
or humanized versions thereof) is mutated to decrease the
biological half-life of the antibody or fragment. More
specifically, one or more amino acid mutations are introduced into
the CH2-CH3 domain interface region of the Fc-hinge fragment such
that the antibody or fragment has impaired Staphylococcyl protein A
(SpA) binding relative to native Fc-hinge domain SpA binding. This
approach is described in further detail in U.S. Pat. No.
6,165,745.
[0218] In another embodiment, the antibody or antigen-binding
fragment of the invention (e.g., 14A6 or 28H5 or a humanized
version thereof) is modified to increase its biological half-life.
Various approaches are possible. For example, one or more of the
following mutations can be introduced: T252L, T254S, T256F, as
described in U.S. Pat. No. 6,277,375. Alternatively, to increase
the biological half-life, the antibody can be altered within the
CH1 or CL region to contain a salvage receptor binding epitope
taken from two loops of a CH2 domain of an Fc region of an IgG, as
described in U.S. Pat. Nos. 5,869,046 and 6,121,022.
[0219] In yet other embodiments, the Fc region is altered by
replacing at least one amino acid residue with a different amino
acid residue to alter the effector function(s) of the antibody or
antigen-binding fragment. For example, one or more amino acids
selected from amino acid residues 234, 235, 236, 237, 297, 318, 320
and 322 can be replaced with a different amino acid residue such
that the antibody has an altered affinity for an effector ligand
and retains the antigen-binding ability of the parent antibody. The
effector ligand to which affinity is altered can be, for example,
an Fc receptor or the C1 component of complement. This approach is
described in further detail in U.S. Pat. Nos. 5,624,821 and
5,648,260.
[0220] In another example, one or more amino acids selected from
amino acid residues 329, 331 and 322 can be replaced with a
different amino acid residue such that the antibody has altered C1q
binding and/or reduced or abolished complement dependent
cytotoxicity (CDC). This approach is described in further detail in
U.S. Pat. No. 6,194,551.
[0221] In another example, one or more amino acid residues within
amino acid positions 231 and 239 are altered to thereby alter the
ability of the antibody to fix complement. This approach is
described further in PCT Publication WO 94/29351.
[0222] In yet another example, the Fc region is modified to
decrease the ability of the antibody or antigen-binding fragment of
the invention (e.g., 14A6 or 28H5 or a humanized version thereof)
to mediate antibody dependent cellular cytotoxicity (ADCC) and/or
to decrease the affinity of the antibody or fragment for an
Fc.gamma. receptor by modifying one or more amino acids at the
following positions: 238, 239, 243, 248, 249, 252, 254, 255, 256,
258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285,
286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307,
309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334,
335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414,
416, 419, 430, 434, 435, 437, 438 or 439. This approach is
described further in PCT Publication WO 00/42072. Moreover, the
binding sites on human IgG1 for Fc.gamma.R1, Fc.gamma.RII,
Fc.gamma.RIII and FcRn have been mapped and variants with improved
binding have been described (see Shields et al. (2001) J. Biol.
Chem. 276:6591-6604).
[0223] In one embodiment of the invention, the Fc region is
modified to decrease the ability of the antibody of the invention
(e.g., 14A6, 28H5, 31C6 and humanized versions thereof) to mediate
effector function and/or to increase anti-inflammatory properties
by modifying residues 243 and 264. In one embodiment, the Fc region
of the antibody or fragment is modified by changing the residues at
positions 243 and 264 to alanine. In one embodiment, the Fc region
is modified to decrease the ability of the antibody or fragment to
mediate effector function and/or to increase anti-inflammatory
properties by modifying residues 243, 264, 267 and 328.
Effector Function Enhancement
[0224] In some embodiments, the Fc region of an anti-TIGIT antibody
is modified to increase the ability of the antibody or
antigen-binding fragment to mediate effector function and/or to
increase their binding to the Fcgamma receptors (Fc.gamma.Rs).
[0225] The term "Effector Function" as used herein is meant to
refer to one or more of Antibody Dependant Cell mediated Cytotoxic
activity (ADCC), Complement-dependant cytotoxic activity (CDC)
mediated responses, Fc-mediated phagocytosis or antibody dependant
cellular phagocytosis (ADCP) and antibody recycling via the FcRn
receptor.
[0226] The interaction between the constant region of an antigen
binding protein and various Fc receptors (FcR) including FcgammaRI
(CD64), FcgammaRII (CD32) and FcgammaRIII (CD16) is believed to
mediate the effector functions, such as ADCC and CDC, of the
antigen binding protein. The Fc receptor is also important for
antibody cross-linking, which can be important for anti-tumor
immunity.
[0227] Effector function can be measured in a number of ways
including for example via binding of the FcgammaRIII to Natural
Killer cells or via FcgammaRI to monocytes/macrophages to measure
for ADCC effector function. For example an antigen binding protein
of the present invention can be assessed for ADCC effector function
in a Natural Killer cell assay. Examples of such assays can be
found in Shields et al, 2001 J. Biol. Chem., Vol. 276, p 6591-6604;
Chappel et al, 1993 J. Biol. Chem., Vol 268, p 25124-25131; Lazar
et al, 2006 PNAS, 103; 4005-4010.
[0228] The ADCC or CDC properties of antibodies of the present
invention, or their cross-linking properties, may be enhanced in a
number of ways.
[0229] Human IgG1 constant regions containing specific mutations or
altered glycosylation on residue Asn297 have been shown to enhance
binding to Fc receptors. In some cases these mutations have also
been shown to enhance ADCC and CDC (Lazar et al. PNAS 2006, 103;
4005-4010; Shields et al. J Biol Chem 2001, 276; 6591-6604;
Nechansky et al. Mol Immunol, 2007, 44; 1815-1817).
[0230] In one embodiment of the present invention, such mutations
are in one or more of positions selected from 239, 332 and 330
(IgG1), or the equivalent positions in other IgG isotypes. Examples
of suitable mutations are S239D and I332E and A330L. In one
embodiment, the antigen binding protein of the invention herein
described is mutated at positions 239 and 332, for example S239D
and I332E or in a further embodiment it is mutated at three or more
positions selected from 239 and 332 and 330, for example S239D and
I332E and A330L. (EU index numbering).
[0231] In an alternative embodiment of the present invention, there
is provided an antibody comprising a heavy chain constant region
with an altered glycosylation profile such that the antigen binding
protein has enhanced effector function. For example, wherein the
antibody has enhanced ADCC or enhanced CDC or wherein it has both
enhanced ADCC and CDC effector function. Examples of suitable
methodologies to produce antigen binding proteins with an altered
glycosylation profile are described in WO2003011878, WO2006014679
and EP1229125.
[0232] In a further aspect, the present invention provides
"non-fucosylated" or "afucosylated" antibodies. Non-fucosylated
antibodies harbour a tri-mannosyl core structure of complex-type
N-glycans of Fc without fucose residue. These glycoengineered
antibodies that lack core fucose residue from the Fc N-glycans may
exhibit stronger ADCC than fucosylated equivalents due to
enhancement of FcgammaRIIIa binding capacity.
[0233] The present invention also provides a method for the
production of an antibody according to the invention comprising the
steps of: a) culturing a recombinant host cell comprising an
expression vector comprising the isolated nucleic acid as described
herein, wherein the recombinant host cell does not comprise an
alpha-1,6-fucosyltransferase; and b) recovering the antigen binding
protein. The recombinant host cell may be not normally contain a
gene encoding an alpha-1,6-fucosyltransferase (for example yeast
host cells such as Pichia sp.) or may have been genetically
modified to inactive an alpha-1,6-fucosyltransferase. Recombinant
host cells which have been genetically modified to inactivate the
FUT8 gene encoding an alpha-1,6-fucosyltransferase are available.
See, e.g., the POTELLIGENT.TM. technology system available from
BioWa, Inc. (Princeton, N.J.) in which CHOK1SV cells lacking a
functional copy of the FUT8 gene produce monoclonal antibodies
having enhanced antibody dependent cell mediated cytotoxicity
(ADCC) activity that is increased relative to an identical
monoclonal antibody produced in a cell with a functional FUT8 gene.
Aspects of the POTELLIGENT.TM. technology system are described in
U.S. Pat. No. 7,214,775, U.S. Pat. No. 6,946,292, WO0061739 and
WO0231240. Those of ordinary skill in the art will also recognize
other appropriate systems.
[0234] It will be apparent to those skilled in the art that such
modifications may not only be used alone but may be used in
combination with each other in order to further enhance effector
function.
Production of Antibodies with Modified Glycosylation
[0235] In still another embodiment, the antibodies or
antigen-binding fragments of the invention (e.g., 14A6, 28H5, 31C6
and humanized versions thereof) comprise a particular glycosylation
pattern. For example, an afucosylated or an aglycosylated antibody
or fragment can be made (i.e., the antibody lacks fucose or
glycosylation, respectively). The glycosylation pattern of an
antibody or fragment may be altered to, for example, increase the
affinity or avidity of the antibody or fragment for a TIGIT
antigen. Such modifications can be accomplished by, for example,
altering one or more of the glycosylation sites within the antibody
or fragment sequence. For example, one or more amino acid
substitutions can be made that result removal of one or more of the
variable region framework glycosylation sites to thereby eliminate
glycosylation at that site. Such aglycosylation may increase the
affinity or avidity of the antibody or fragment for antigen. See,
e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861.
[0236] Antibodies and antigen-binding fragments disclosed herein
(e.g., 14A6, 28H5, 31C6 and humanized versions thereof) may further
include those produced in lower eukaryote host cells, in particular
fungal host cells such as yeast and filamentous fungi have been
genetically engineered to produce glycoproteins that have
mammalian- or human-like glycosylation patterns (See for example,
Choi et al, (2003) Proc. Natl. Acad. Sci. 100: 5022-5027; Hamilton
et al., (2003) Science 301: 1244-1246; Hamilton et al., (2006)
Science 313: 1441-1443; Nett et al., Yeast 28(3):237-52 (2011);
Hamilton et al., Curr Opin Biotechnol. October; 18(5):387-92
(2007)). A particular advantage of these genetically modified host
cells over currently used mammalian cell lines is the ability to
control the glycosylation profile of glycoproteins that are
produced in the cells such that compositions of glycoproteins can
be produced wherein a particular N-glycan structure predominates
(see, e.g., U.S. Pat. No. 7,029,872 and U.S. Pat. No. 7,449,308).
These genetically modified host cells have been used to produce
antibodies that have predominantly particular N-glycan structures
(See for example, Li et al., (2006) Nat. Biotechnol. 24:
210-215).
[0237] In particular embodiments, the antibodies and
antigen-binding fragments thereof disclosed herein (e.g., 14A6,
28H5, 31C6 and humanized versions thereof) further include those
produced in lower eukaryotic host cells and which comprise
fucosylated and non-fucosylated hybrid and complex N-glycans,
including bisected and multiantennary species, including but not
limited to N-glycans such as GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2;
Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2;
NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2.
[0238] In particular embodiments, the antibodies and
antigen-binding fragments thereof provided herein (e.g., 14A6,
28H5, 31C6 and humanized versions thereof) may comprise antibodies
or fragments having at least one hybrid N-glycan selected from the
group consisting of GlcNAcMan.sub.5GlcNAc.sub.2;
GalGlcNAcMan.sub.5GlcNAc.sub.2; and
NANAGalGlcNAcMan.sub.5GlcNAc.sub.2. In particular aspects, the
hybrid N-glycan is the predominant N-glycan species in the
composition.
[0239] In particular embodiments, the antibodies and
antigen-binding fragments thereof provided herein (e.g., 14A6,
28H5, 31C6 and humanized versions thereof) comprise antibodies and
fragments having at least one complex N-glycan selected from the
group consisting of GlcNAcMan.sub.3GlcNAc.sub.2;
GalGlcNAcMan.sub.3GlcNAc.sub.2; NANAGalGlcNAcMan.sub.3GlcNAc.sub.2;
GlcNAc.sub.2Man.sub.3GlcNAc.sub.2;
GalGlcNAc.sub.2Man.sub.3GlcNAc.sub.2;
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2;
NANAGal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2; and
NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2. In particular
aspects, the complex N-glycan are the predominant N-glycan species
in the composition. In further aspects, the complex N-glycan is a
particular N-glycan species that comprises about 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex
N-glycans in the composition. In one embodiment, the antibody and
antigen binding fragments thereof provided herein comprise complex
N-glycans, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%,
99%, or 100% of the complex N-glycans in comprise the structure
NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2, wherein such
structure is afucosylated. Such structures can be produced, e.g.,
in engineered Pichia pastoris host cells.
[0240] In particular embodiments, the N-glycan is fucosylated. In
general, the fucose is in an .alpha.1,3-linkage with the GlcNAc at
the reducing end of the N-glycan, an .alpha.1,6-linkage with the
GlcNAc at the reducing end of the N-glycan, an .alpha.1,2-linkage
with the Gal at the non-reducing end of the N-glycan, an
.alpha.1,3-linkage with the GlcNac at the non-reducing end of the
N-glycan, or an .alpha.1,4-linkage with a GlcNAc at the
non-reducing end of the N-glycan.
[0241] Therefore, in particular aspects of the above the
glycoprotein compositions, the glycoform is in an
.alpha.1,3-linkage or .alpha.1,6-linkage fucose to produce a
glycoform selected from the group consisting of
Man.sub.5GlcNAc.sub.2(Fuc), GlcNAcMan.sub.5GlcNAc.sub.2(Fuc),
Man.sub.3GlcNAc.sub.2(Fuc), GlcNAcMan.sub.3GlcNAc.sub.2(Fuc),
GlcNAc.sub.2Man.sub.3GlcNAc.sub.2(Fuc),
GalGlcNAc.sub.2Man.sub.3GlcNAc.sub.2(Fuc),
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2(Fuc),
NANAGal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2(Fuc), and
NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2(Fuc); in an
.alpha.1,3-linkage or .alpha.1,4-linkage fucose to produce a
glycoform selected from the group consisting of
GlcNAc(Fuc)Man.sub.5GlcNAc.sub.2, GlcNAc(Fuc)Man.sub.3GlcNAc.sub.2,
GlcNAc.sub.2(Fuc.sub.1-2)Man.sub.3GlcNAc.sub.2,
GalGlcNAc2(Fuc.sub.1-2)Man.sub.3GlcNAc.sub.2,
Gal.sub.2GlcNAc.sub.2(Fuc1-2)Man3GlcNAc2,
NANAGal2GlcNAc2(Fuc.sub.1-2)Man.sub.3GlcNAc.sub.2, and
NANA.sub.2Gal.sub.2GlcNAc.sub.2(Fuc.sub.1-2)Man.sub.3GlcNAc.sub.2;
or in an .alpha.1,2-linkage fucose to produce a glycoform selected
from the group consisting of
Gal(Fuc)GlcNAc.sub.2Man.sub.3GlcNAc.sub.2,
Gal.sub.2(Fuc.sub.1-2)GlcNAc.sub.2Man.sub.3GlcNAc.sub.2,
NANAGal.sub.2(Fuc.sub.1-2)GlcNAc.sub.2Man.sub.3GlcNAc.sub.2, and
NANA.sub.2Gal.sub.2(Fuc.sub.1-2)GlcNAc.sub.2Man.sub.3GlcNAc.sub.2.
[0242] In further aspects, the antibodies (e.g., humanized
antibodies) or antigen-binding fragments thereof comprise high
mannose N-glycans, including but not limited to,
Man.sub.8GlcNAc.sub.2, Man.sub.7GlcNAc.sub.2,
Man.sub.6GlcNAc.sub.2, Man.sub.5GlcNAc.sub.2,
Man.sub.4GlcNAc.sub.2, or N-glycans that consist of the
Man.sub.3GlcNAc.sub.2 N-glycan structure.
[0243] In further aspects of the above, the complex N-glycans
further include fucosylated and non-fucosylated bisected and
multiantennary species.
[0244] As used herein, the terms "N-glycan" and "glycoform" are
used interchangeably and refer to an N-linked oligosaccharide, for
example, one that is attached by an asparagine-N-acetylglucosamine
linkage to an asparagine residue of a polypeptide. N-linked
glycoproteins contain an N-acetylglucosamine residue linked to the
amide nitrogen of an asparagine residue in the protein. The
predominant sugars found on glycoproteins are glucose, galactose,
mannose, fucose, N-acetylgalactosamine (GalNAc),
N-acetylglucosamine (GlcNAc) and sialic acid (e.g.,
N-acetyl-neuraminic acid (NANA)). The processing of the sugar
groups occurs co-translationally in the lumen of the ER and
continues post-translationally in the Golgi apparatus for N-linked
glycoproteins.
[0245] N-glycans have a common pentasaccharide core of
Man.sub.3GlcNAc.sub.2 ("Man" refers to mannose; "Glc" refers to
glucose; and "NAc" refers to N-acetyl; GlcNAc refers to
N-acetylglucosamine). Usually, N-glycan structures are presented
with the non-reducing end to the left and the reducing end to the
right. The reducing end of the N-glycan is the end that is attached
to the Asn residue comprising the glycosylation site on the
protein. N-glycans differ with respect to the number of branches
(antennae) comprising peripheral sugars (e.g., GlcNAc, galactose,
fucose and sialic acid) that are added to the Man.sub.3GlcNAc.sub.2
("Man3") core structure which is also referred to as the
"trimannose core", the "pentasaccharide core" or the "paucimannose
core". N-glycans are classified according to their branched
constituents (e.g., high mannose, complex or hybrid). A "high
mannose" type N-glycan has five or more mannose residues. A
"complex" type N-glycan typically has at least one GlcNAc attached
to the 1,3 mannose arm and at least one GlcNAc attached to the 1,6
mannose arm of a "trimannose" core. Complex N-glycans may also have
galactose ("Gal") or N-acetylgalactosamine ("GalNAc") residues that
are optionally modified with sialic acid or derivatives (e.g.,
"NANA" or "NeuAc", where "Neu" refers to neuraminic acid and "Ac"
refers to acetyl). Complex N-glycans may also have intrachain
substitutions comprising "bisecting" GlcNAc and core fucose
("Fuc"). Complex N-glycans may also have multiple antennae on the
"trimannose core," often referred to as "multiple antennary
glycans." A "hybrid" N-glycan has at least one GlcNAc on the
terminal of the 1,3 mannose arm of the trimannose core and zero or
more mannoses on the 1,6 mannose arm of the trimannose core. The
various N-glycans are also referred to as "glycoforms."
[0246] With respect to complex N-glycans, the terms "G-2", "G-1",
"G0", "G1", "G2", "A1", and "A2" mean the following. "G-2" refers
to an N-glycan structure that can be characterized as
Man.sub.3GlcNAc.sub.2; the term "G-1" refers to an N-glycan
structure that can be characterized as GlcNAcMan.sub.3GlcNAc.sub.2;
the term "G0" refers to an N-glycan structure that can be
characterized as GlcNAc.sub.2Man.sub.3GlcNAc.sub.2; the term "G1"
refers to an N-glycan structure that can be characterized as
GalGlcNAc.sub.2Man.sub.3GlcNAc.sub.2; the term "G2" refers to an
N-glycan structure that can be characterized as
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2; the term "A1" refers to
an N-glycan structure that can be characterized as
NANAGal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2; and, the term "A2"
refers to an N-glycan structure that can be characterized as
NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2. Unless
otherwise indicated, the terms G-2", "G-1", "G0", "G1", "G2", "A1",
and "A2" refer to N-glycan species that lack fucose attached to the
GlcNAc residue at the reducing end of the N-glycan. When the term
includes an "F", the "F" indicates that the N-glycan species
contains a fucose residue on the GlcNAc residue at the reducing end
of the N-glycan. For example, G0F, G1F, G2F, A1F, and A2F all
indicate that the N-glycan further includes a fucose residue
attached to the GlcNAc residue at the reducing end of the N-glycan.
Lower eukaryotes such as yeast and filamentous fungi do not
normally produce N-glycans that produce fucose.
[0247] With respect to multiantennary N-glycans, the term
"multiantennary N-glycan" refers to N-glycans that further comprise
a GlcNAc residue on the mannose residue comprising the non-reducing
end of the 1,6 arm or the 1,3 arm of the N-glycan or a GlcNAc
residue on each of the mannose residues comprising the non-reducing
end of the 1,6 arm and the 1,3 arm of the N-glycan. Thus,
multiantennary N-glycans can be characterized by the formulas
GlcNAc.sub.(2-4)Man3GlcNAc.sub.2,
Gal.sub.(1-4)GlcNAc.sub.(2-4)Man.sub.3GlcNAc.sub.2, or
NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(2-4)Man3GlcNAc.sub.2. The
term "1-4" refers to 1, 2, 3, or 4 residues.
[0248] With respect to bisected N-glycans, the term "bisected
N-glycan" refers to N-glycans in which a GlcNAc residue is linked
to the mannose residue at the reducing end of the N-glycan. A
bisected N-glycan can be characterized by the formula
GlcNAc.sub.3Man.sub.3GlcNAc.sub.2 wherein each mannose residue is
linked at its non-reducing end to a GlcNAc residue. In contrast,
when a multiantennary N-glycan is characterized as
GlcNAc.sub.3Man.sub.3GlcNAc.sub.2, the formula indicates that two
GlcNAc residues are linked to the mannose residue at the
non-reducing end of one of the two arms of the N-glycans and one
GlcNAc residue is linked to the mannose residue at the non-reducing
end of the other arm of the N-glycan.
Antibody Physical Properties
[0249] The antibodies and antigen-binding fragments thereof
disclosed herein (e.g., 14A6, 28H5, 31C6 and humanized versions
thereof) may further contain one or more glycosylation sites in
either the light or heavy chain immunoglobulin variable region.
Such glycosylation sites may result in increased immunogenicity of
the antibody or fragment or an alteration of the pK of the antibody
due to altered antigen-binding (Marshall et al. (1972) Annu Rev
Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94;
Wallick et al (1988) J Exp Med 168:1099-109; Spiro (2002)
Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452-7;
Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation has
been known to occur at motifs containing an N-X-S/T sequence.
[0250] Each antibody or antigen-binding fragment (e.g., 14A6, 28H5,
31C6 or humanized versions thereof) will have a unique isoelectric
point (pI), which generally falls in the pH range between 6 and
9.5. The pI for an IgG1 antibody typically falls within the pH
range of 7-9.5 and the pI for an IgG4 antibody typically falls
within the pH range of 6-8.
[0251] Each antibody or antigen-binding fragment (e.g., 14A6, 28H5,
31C6 or humanized versions thereof) will have a characteristic
melting temperature, with a higher melting temperature indicating
greater overall stability in vivo (Krishnamurthy R and Manning M C
(2002) Curr Pharm Biotechnol 3:361-71). In general, the T.sub.M1
(the temperature of initial unfolding) may be greater than
60.degree. C., greater than 65.degree. C., or greater than
70.degree. C. The melting point of an antibody or fragment can be
measured using differential scanning calorimetry (Chen et al (2003)
Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52)
or circular dichroism (Murray et al. (2002) J. Chromatogr Sci
40:343-9).
[0252] In a further embodiment, antibodies and antigen-binding
fragments thereof (e.g., 14A6, 28H5, 31C6 and humanized versions
thereof) are selected that do not degrade rapidly. Degradation of
an antibody or fragment can be measured using capillary
electrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E
(1995) Anal Chem 67:3626-32).
[0253] In a further embodiment, antibodies (e.g., 14A6, 28H5, 31C6
and humanized versions thereof) and antigen-binding fragments
thereof are selected that have minimal aggregation effects, which
can lead to the triggering of an unwanted immune response and/or
altered or unfavorable pharmacokinetic properties. Generally,
antibodies and fragments are acceptable with aggregation of 25% or
less, 20% or less, 15% or less, 10% or less, or 5% or less.
Aggregation can be measured by several techniques, including
size-exclusion column (SEC), high performance liquid chromatography
(HPLC), and light scattering.
Antibody Conjugates
[0254] The anti-TIGIT antibodies and antigen-binding fragments
thereof disclosed herein (e.g., 14A6, 28H5, 31C6 and humanized
versions thereof) may also be conjugated to a chemical moiety. The
chemical moiety may be, inter alia, a polymer, a radionuclide or a
cytotoxic factor. In particular embodiments, the chemical moiety is
a polymer which increases the half-life of the antibody or fragment
in the body of a subject. Suitable polymers include, but are not
limited to, hydrophilic polymers which include but are not limited
to polyethylene glycol (PEG) (e.g., PEG with a molecular weight of
2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa or 40 kDa), dextran
and monomethoxypolyethylene glycol (mPEG). Lee, et al., (1999)
(Bioconj. Chem. 10:973-981) discloses PEG conjugated single-chain
antibodies. Wen, et al., (2001) (Bioconj. Chem. 12:545-553)
disclose conjugating antibodies with PEG which is attached to a
radiometal chelator (diethylenetriaminpentaacetic acid (DTPA)).
[0255] The antibodies and antigen-binding fragments thereof
disclosed herein (e.g., 14A6, 28H5, 31C6 and humanized versions
thereof) may also be conjugated with labels such as .sup.99Tc,
.sup.90Y, .sup.111In, .sup.32P, .sup.14C, .sup.125I, .sup.3H,
.sup.131I, .sup.11C, .sup.15O, .sup.13N, .sup.18F, .sup.35S,
.sup.51Cr, .sup.57To, .sup.226Ra, .sup.60Co, .sup.59Fe, .sup.57Se,
.sup.152Eu, .sup.67CU, .sup.217Ci, .sup.211At, .sup.212Pb,
.sup.47Sc, .sup.109Pd, .sup.234Th, and .sup.40K, .sup.157Gd,
.sup.55Mn, .sup.52Tr, and .sup.56Fe.
[0256] The antibodies and antigen-binding fragments disclosed
herein (e.g., 14A6, 28H5, 31C6 and humanized versions thereof) may
also be PEGylated, for example to increase its biological (e.g.,
serum) half-life. To PEGylate an antibody or fragment, the antibody
or fragment, typically is reacted with a reactive form of
polyethylene glycol (PEG), such as a reactive ester or aldehyde
derivative of PEG, under conditions in which one or more PEG groups
become attached to the antibody or antibody fragment. In particular
embodiments, the PEGylation is carried out via an acylation
reaction or an alkylation reaction with a reactive PEG molecule (or
an analogous reactive water-soluble polymer). As used herein, the
term "polyethylene glycol" is intended to encompass any of the
forms of PEG that have been used to derivatize other proteins, such
as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or
polyethylene glycol-maleimide. In certain embodiments, the antibody
or fragment to be PEGylated is an aglycosylated antibody or
fragment. Methods for PEGylating proteins are known in the art and
can be applied to the antibodies of the invention. See, e.g., EP 0
154 316 and EP 0 401 384.
[0257] The antibodies and antigen-binding fragments disclosed
herein (e.g., 14A6, 28H5, 31C6 and humanized versions thereof) may
also be conjugated with fluorescent or chemilluminescent labels,
including fluorophores such as rare earth chelates, fluorescein and
its derivatives, rhodamine and its derivatives, isothiocyanate,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde,
fluorescamine, .sup.152Eu, dansyl, umbelliferone, luciferin,
luminal label, isoluminal label, an aromatic acridinium ester
label, an imidazole label, an acridimium salt label, an oxalate
ester label, an aequorin label, 2,3-dihydrophthalazinediones,
biotin/avidin, spin labels and stable free radicals.
[0258] The antibodies and antigen-binding fragments thereof of the
invention (e.g., 14A6, 28H5, 31C6 and humanized versions thereof)
may also be conjugated to a cytotoxic factor such as diptheria
toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain,
abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii
proteins and compounds (e.g., fatty acids), dianthin proteins,
Phytoiacca americana proteins PAPI, PAPII, and PAP-S, momordica
charantia inhibitor, curcin, crotin, saponaria officinalis
inhibitor, mitogellin, restrictocin, phenomycin, and enomycin.
[0259] Any method known in the art for conjugating the antibodies
and antigen-binding fragments thereof of the invention (e.g., 14A6,
28H5, 31C6 and humanized versions thereof) to the various moieties
may be employed, including those methods described by Hunter, et
al., (1962) Nature 144:945; David, et al., (1974) Biochemistry
13:1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren,
J., (1982) Histochem. and Cytochem. 30:407. Methods for conjugating
antibodies and fragments are conventional and very well known in
the art.
Therapeutic Uses of Anti-TIGIT Antibodies
[0260] Further provided are methods for treating subjects,
including human subjects, in need of treatment with the isolated
antibodies or antigen-binding fragments thereof disclosed herein
(e.g., 14A6, 28H5, 31C6 and humanized versions thereof). In one
embodiment of the invention, such subject suffers from an infection
or an infectious disease. In another embodiment of the invention,
such subject suffers from cancer. In one embodiment, the cancer is
a solid tumor which is infiltrated by tumor-infiltrating
lymphocytes which express TIGIT. In one embodiment the cancer is,
e.g., osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer,
leukemia, renal transitional cell cancer, bladder cancer, Wilm's
cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate
cancer, bone cancer, lung cancer (e.g., non-small cell lung
cancer), gastric cancer, colorectal cancer, cervical cancer,
synovial sarcoma, head and neck cancer, squamous cell carcinoma,
multiple myeloma, renal cell cancer, retinoblastoma,
hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor
of the kidney, Ewing's sarcoma, chondrosarcoma, brain cancer,
glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma,
a primitive neuroectodermal tumor, medulloblastoma, astrocytoma,
anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid
plexus papilloma, polycythemia vera, thrombocythemia, idiopathic
myelfibrosis, soft tissue sarcoma, thyroid cancer, endometrial
cancer, carcinoid cancer or liver cancer, breast cancer or gastric
cancer. In an embodiment of the invention, the cancer is metastatic
cancer, e.g., of the varieties described above.
[0261] In an embodiment, the invention provides methods for
treating subjects using an anti-TIGIT antibody or antigen-binding
fragment thereof of the invention (e.g., 14A6, 28H5, 31C6 and
humanized versions thereof), wherein the subject suffers from a
viral infection. In one embodiment, the viral infection is
infection with a virus selected from the group consisting of human
immunodeficiency virus (HIV), hepatitis virus (A, B, or C), herpes
virus (e.g., VZV, HSV-I, HAV-6, HSV-II, and CMV, Epstein Barr
virus), adenovirus, influenza virus, flaviviruses, echovirus,
rhinovirus, coxsackie virus, coronavirus, respiratory syncytial
virus, mumps virus, rotavirus, measles virus, rubella virus,
parvovirus, vaccinia virus, HTLV virus, dengue virus,
papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus
or arboviral encephalitis virus.
[0262] In an embodiment, the invention provides methods for
treating subjects using an anti-TIGIT antibody or antigen-binding
fragment thereof of the invention, wherein the subject suffers from
a bacterial infection. In one embodiment, the bacterial infection
is infection with a bacteria selected from the group consisting of
Chlamydia, rickettsial bacteria, mycobacteria, staphylococci,
streptococci, pneumonococci, meningococci and gonococci,
klebsiella, proteus, serratia, pseudomonas, Legionella,
Corynebacterium diphtherias, Salmonella, bacilli, Vibrio cholerae,
Clostridium tetan, Clostridium botulinum, Bacillus anthricis,
Yersinia pestis, Mycobacterium leprae, Mycobacterium lepromatosis,
and Borriella.
[0263] In an embodiment, the invention provides methods for
treating subjects using an anti-TIGIT antibody or antigen-binding
fragment thereof of the invention, wherein the subject suffers from
a fungal infection. In one embodiment, the fungal infection is
infection with a fungus selected from the group consisting of
Candida (albicans, krusei, glabrata, tropicalis, etc.),
Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.),
Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii,
Blastomyces dermatitidis, Paracoccidioides brasiliensis,
Coccidioides immitis and Histoplasma capsulatum.
[0264] In an embodiment, the invention provides methods for
treating subjects using an anti-TIGIT antibody or antigen-binding
fragment thereof of the invention, wherein the subject suffers from
a parasitic infection. In one embodiment, the parasitic infection
is infection with a parasite selected from the group consisting of
Entamoeba histolytica, Balantidium coli, Naegleria fowleri,
Acanthamoeba, Giardia Zambia, Cryptosporidium, Pneumocystis
carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei,
Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii and
Nippostrongylus brasiliensis.
[0265] In addition, the present invention provides a method for
preventing or inhibiting TIGIT binding to MHC class II, enhancing
antigen-specific T-cell activation or stimulating T-cell production
of interleukin-2 in a subject (e.g., human), for example, wherein
the subject suffers from cancer or infectious disease (e.g., as
discussed herein) comprising administering an effective amount of
anti-TIGIT antibody or antigen-binding fragment thereof (e.g.,
14A6, 28A5, 31C6 and humanized versions thereof), optionally in
association with a further chemotherapeutic agent.
[0266] A "subject" may be a mammal such as a human, dog, cat,
horse, cow, mouse, rat, monkey (e.g., cynomolgous monkey, e.g.,
Macaca fascicularis) or rabbit. In preferred embodiments of the
invention, the subject is a human subject.
[0267] In particular embodiments, the antibodies or antigen-binding
fragments thereof disclosed herein (e.g., 14A6, 28H5, 31C6 and
humanized versions thereof) may be used alone, or in association
with other, further therapeutic agents and/or therapeutic
procedures, for treating or preventing any disease such as cancer,
e.g., as discussed herein, in a subject in need of such treatment
or prevention. Compositions, e.g., pharmaceutical compositions
comprising a pharmaceutically acceptable carrier, comprising such
antibodies and fragments in association with further therapeutic
agents are also part of the present invention.
[0268] In particular embodiments, the antibodies or antigen-binding
fragments thereof disclosed herein (e.g., 14A6, 28H5, 31C6 and
humanized versions thereof) may be used alone, or in association
with tumor vaccines.
[0269] In particular embodiments, the antibodies or antigen-binding
fragments thereof disclosed herein (e.g., 14A6, 28H5, 31C6 and
humanized versions thereof) may be used alone, or in association
with chemotherapeutic agents.
[0270] In particular embodiments, the antibodies or antigen-binding
fragments thereof disclosed herein (e.g., 14A6, 28H5, 31C6 and
humanized versions thereof) may be used alone, or in association
with radiation therapy.
[0271] In particular embodiments, the antibodies or antigen-binding
fragments thereof disclosed herein (e.g., 14A6, 28H5, 31C6 and
humanized versions thereof) may be used alone, or in association
with targeted therapies. Examples of targeted therapies include:
hormone therapies, signal transduction inhibitors (e.g., EGFR
inhibitors, such as cetuximab (Erbitux) and erlotinib (Tarceva));
HER2 inhibitors (e.g., trastuzumab (Herceptin) and pertuzumab
(Perjeta)); BCR-ABL inhibitors (such as imatinib (Gleevec) and
dasatinib (Sprycel)); ALK inhibitors (such as crizotinib (Xalkori)
and ceritinib (Zykadia)); BRAF inhibitors (such as vemurafenib
(Zelboraf) and dabrafenib (Tafinlar)), gene expression modulators,
apoptosis inducers (e.g., bortezomib (Velcade) and carfilzomib
(Kyprolis)), angiogenesis inhibitors (e.g., bevacizumab (Avastin)
and ramucirumab (Cyramza), monoclonal antibodies attached to toxins
(e.g., brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine
(Kadcyla)).
[0272] In particular embodiments, the anti-TIGIT antibodies or
antigen-binding fragments thereof of the invention (e.g., 14A6,
28H5, 31C6 and humanized versions thereof) may be used in
combination with an anti-cancer therapeutic agent or
immunomodulatory drug such as an immunomodulatory receptor
inhibitor, e.g., an antibody or antigen-binding fragment thereof
that specifically binds to the receptor.
[0273] Thus, the present invention includes compositions comprising
an anti-TIGIT antibody or antigen-binding fragment thereof of the
present invention (e.g., 14A6, 28H5, 31C6 and humanized versions
thereof) in association with pembrolizumab; as well as methods for
treating or preventing cancer in a subject comprising administering
an effective amount of the anti-TIGIT antibody or antigen-binding
fragment thereof and pembrolizumab to the subject. Optionally, the
subject is also administered a further therapeutic agent.
[0274] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the present invention (e.g.,
14A6, 28H5, 31C6 and humanized versions thereof) is in association
with an isolated antibody comprising an immunoglobulin heavy chain
comprising the amino acid sequence of SEQ ID NO:33 and an
immunoglobulin light chain comprising the amino acid sequence of
SEQ ID NO:34. SEQ ID NOs: 33 and 34 encode the heavy and light
chain of pembrolizumab.
[0275] In an embodiment of the invention, an anti-TIGIT antibody)
or antigen-binding fragment thereof of the present invention (e.g.,
14A6, 28H5, 31C6 and humanized versions thereof) is in association
with an isolated antibody comprising an immunoglobulin heavy chain
comprising the amino acid sequence of SEQ ID NO:35 and an
immunoglobulin light chain comprising the amino acid sequence of
SEQ ID NO:36. SEQ ID NOs: 35 and 36 encode the heavy and light
chain of nivolumab.
[0276] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 and humanized versions thereof) is in association with
one or more of: anti-PD1 antibody (e.g., pembrolizumab, nivolumab,
pidilizumab (CT-011)), anti-PDL1 antibody, anti-CTLA4 antibody,
anti-CS1 antibody (e.g., elotuzumab), anti-KIR2DL1/2/3 antibody
(e.g., lirilumab), anti-CD137 antibody (e.g., urelumab), anti-GITR
antibody (e.g., TRX518), anti-PD-L1 antibody (e.g., BMS-936559,
MSB0010718C or MPDL3280A), anti-PD-L2 antibody, anti-ILT1 antibody,
anti-ILT2 antibody, anti-ILT3 antibody, anti-ILT4 antibody,
anti-ILT5 antibody, anti-ILT6 antibody, anti-ILT7 antibody,
anti-ILT8 antibody, anti-CD40 antibody, anti-OX40 antibody,
anti-ICOS, anti-SIRP.alpha., anti-KIR2DL1 antibody, anti-KIR2DL2/3
antibody, anti-KIR2DL4 antibody, anti-KIR2DL5A antibody,
anti-KIR2DL5B antibody, anti-KIR3DL1 antibody, anti-KIR3DL2
antibody, anti-KIR3DL3 antibody, anti-NKG2A antibody, anti-NKG2C
antibody, anti-NKG2E antibody, anti-4-1BB antibody (e.g.,
PF-05082566), anti-TSLP antibody, anti-IL-10 antibody, IL-10 or
PEGylated IL-10, or any small organic molecule inhibitor of such
targets.
[0277] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-PD1 antibody.
[0278] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-PDL1 antibody (e.g., BMS-936559, MSB0010718C or
MPDL3280A).
[0279] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-CTLA4 antibody.
[0280] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-CS1 antibody.
[0281] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-KIR2DL1/2/3 antibody.
[0282] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-CD137 (e.g., urelumab) antibody.
[0283] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-GITR (e.g., TRX518) antibody.
[0284] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-PD-L2 antibody.
[0285] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-ITL1 antibody.
[0286] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-ITL2 antibody.
[0287] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-ITL3 antibody.
[0288] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-ITL4 antibody.
[0289] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-ITL5 antibody.
[0290] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-ITL6 antibody.
[0291] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-ITL7 antibody.
[0292] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-ITL8 antibody.
[0293] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-CD40 antibody.
[0294] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-OX40 antibody.
[0295] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-KIR2DL1 antibody.
[0296] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-KIR2DL2/3 antibody.
[0297] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-KIR2DL4 antibody.
[0298] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-KIR2DL5A antibody.
[0299] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-KIR2DL5B antibody.
[0300] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-KIR3DL1 antibody.
[0301] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-KIR3DL2 antibody.
[0302] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-KIR3DL3 antibody.
[0303] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-NKG2A antibody.
[0304] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-NKG2C antibody.
[0305] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-ICOS antibody.
[0306] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-SIRP.alpha. antibody.
[0307] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-4-1BB antibody.
[0308] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-IL-10 antibody.
[0309] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with an
anti-TSLP antibody.
[0310] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with
IL-10 or PEGylated IL-10.
[0311] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with
one or more of an inhibitor (e.g., a small organic molecule or an
antibody or antigen-binding fragment thereof) such as: an MTOR
(mammalian target of rapamycin) inhibitor, a cytotoxic agent, a
platinum agent, an EGFR inhibitor, a VEGF inhibitor, a microtubule
stabilizer, a taxane, a CD20 inhibitor, a CD52 inhibitor, a CD30
inhibitor, a RANK (Receptor activator of nuclear factor kappa-B)
inhibitor, a RANKL (Receptor activator of nuclear factor kappa-B
ligand) inhibitor, an ERK inhibitor, a MAP Kinase inhibitor, an AKT
inhibitor, a MEK inhibitor, a PI3K inhibitor, a HER1 inhibitor, a
HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, a Bcl2
inhibitor, a CD22 inhibitor, a CD79b inhibitor, an ErbB2 inhibitor,
or a farnesyl protein transferase inhibitor.
[0312] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with
any one or more of: 13-cis-retinoic acid,
3-[5-(methylsulfonylpiperadinemethyl)-indolyl]-quinolone,
4-hydroxytamoxifen, 5-deooxyuridine, 5'-deoxy-5-fluorouridine,
5-fluorouracil, 6-mecaptopurine, 7-hydroxystaurosporine, A-443654,
abirateroneacetate, abraxane, ABT-578, acolbifene, ADS-100380,
ALT-110, altretamine, amifostine, aminoglutethimide, amrubicin,
Amsacrine, anagrelide, anastrozole, angiostatin, AP-23573, ARQ-197,
arzoxifene, AS-252424, AS-605240, asparaginase, AT-9263,
atrasentan, axitinib, AZD1152, Bacillus Calmette-Guerin (BCG)
vaccine, batabulin, BC-210, besodutox, bevacizumab, bicalutamide,
Bio111, BIO140, bleomycin, BMS-214662, BMS-247550, BMS-275291,
BMS-310705, bortezimib, buserelin, busulfan, calcitriol,
camptothecin, canertinib, capecitabine, carboplatin, carmustine,
CC8490, Cediranib, CG-1521, CG-781, chlamydocin, chlorambucil,
chlorotoxin, cilengitide, cimitidine, cisplatin, cladribine,
clodronate, COL-3, CP-724714, cyclophosphamide, cyproterone,
cyproteroneacetate, cytarabine, cytosinearabinoside, dacarbazine,
dacinostat, dactinomycin, dalotuzumab, danusertib, dasatanib,
daunorubicin, decatanib, deguelin, denileukin, deoxycoformycin,
depsipeptide, diarylpropionitrile, diethylstilbestrol, diftitox,
docetaxel, dovitinib, doxorubicin, droloxifene, edotecarin,
yttrium-90 labeled-edotreotide, edotreotide, EKB-569, EMD121974,
endostatin, enzalutamide, enzastaurin, epirubicin, epithilone B,
ERA-923, Erbitux, erlotinib, estradiol, estramustine, etoposide,
everolimus, exemestane, ficlatuzumab, finasteride, flavopiridol,
floxuridine, fludarabine, fludrocortisone, fluoxymesterone,
flutamide, FOLFOX regimen, Fulvestrant, galeterone, gefitinib,
gemcitabine, gimatecan, goserelin, goserelin acetate, gossypol,
GSK461364, GSK690693, HMR-3339, hydroxyprogesteronecaproate,
hydroxyurea, IC87114, idarubicin, idoxyfene, ifosfamide, IM862,
imatinib, IMC-1C11, INCB24360, IN01001, interferon, interleukin-12,
ipilimumab, irinotecan, JNJ-16241199, ketoconazole, KRX-0402,
lapatinib, lasofoxifene, letrozole, leucovorin, leuprolide,
leuprolide acetate, levamisole, liposome entrapped paclitaxel,
lomustine, lonafarnib, lucanthone, LY292223, LY292696, LY293646,
LY293684, LY294002, LY317615, marimastat, mechlorethamine,
medroxyprogesteroneacetate, megestrolacetate, melphalan,
mercaptopurine, mesna, methotrexate, mithramycin, mitomycin,
mitotane, mitoxantrone, tozasertib, MLN8054, neovastat, Neratinib,
neuradiab, nilotinib, nilutimide, nolatrexed, NVP-BEZ235,
oblimersen, octreotide, ofatumumab, oregovomab, orteronel,
oxaliplatin, paclitaxel, palbociclib, pamidronate, panitumumab,
pazopanib, PD0325901, PD184352, PEG-interferon, pemetrexed,
pentostatin, perifosine, phenylalaninemustard, PI-103, pictilisib,
PIK-75, pipendoxifene, PKI-166, plicamycin, porfimer, prednisone,
procarbazine, progestins, PX-866, R-763, raloxifene, raltitrexed,
razoxin, ridaforolimus, rituximab, romidepsin, RTA744, rubitecan,
scriptaid, Sdx102, seliciclib, selumetinib, semaxanib, SF1126,
sirolimus, SN36093, sorafenib, spironolactone, squalamine, SR13668,
streptozocin, SU6668, suberoylanalide hydroxamic acid, sunitinib,
synthetic estrogen, talampanel, talimogene laherparepvec,
tamoxifen, temozolomide, temsirolimus, teniposide, tesmilifene,
testosterone, tetrandrine, TGX-221, thalidomide, thioguanine,
thiotepa, ticilimumab, tipifarnib, tivozanib, TKI-258, TLK286,
topotecan, toremifene citrate, trabectedin, trastuzumab, tretinoin,
trichostatin A, triciribinephosphate monohydrate, triptorelin
pamoate, TSE-424, uracil mustard, valproic acid, valrubicin,
vandetanib, vatalanib, VEGF trap, vinblastine, vincristine,
vindesine, vinorelbine, vitaxin, vitespan, vorinostat, VX-745,
wortmannin, Xr311, zanolimumab, ZK186619, ZK-304709, ZM336372,
ZSTK474.
[0313] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is in association with
one or more antiemetics including, but not limited to: casopitant
(GlaxoSmithKline), Netupitant (MGI-Helsinn) and other NK-1 receptor
antagonists, palonosetron (sold as Aloxi by MGI Pharma), aprepitant
(sold as Emend by Merck and Co.; Rahway, N.J.), diphenhydramine
(sold as Benadryl.RTM. by Pfizer; New York, N.Y.), hydroxyzine
(sold as Atarax.RTM. by Pfizer; New York, N.Y.), metoclopramide
(sold as Reglan.RTM. by AH Robins Co; Richmond, Va.), lorazepam
(sold as Ativan.RTM. by Wyeth; Madison, N.J.), alprazolam (sold as
Xanax.RTM. by Pfizer; New York, N.Y.), haloperidol (sold as
Haldol.RTM. by Ortho-McNeil; Raritan, N.J.), droperidol
(Inapsine.RTM.), dronabinol (sold as Marinol.RTM. by Solvay
Pharmaceuticals, Inc.; Marietta, Ga.), dexamethasone (sold as
Decadron.RTM. by Merck and Co.; Rahway, N.J.), methylprednisolone
(sold as Medrol.RTM. by Pfizer; New York, N.Y.), prochlorperazine
(sold as Compazine.RTM. by Glaxosmithkline; Research Triangle Park,
N.C.), granisetron (sold as Kytril.RTM. by Hoffmann-La Roche Inc.;
Nutley, N.J.), ondansetron (sold as Zofran.RTM. by Glaxosmithkline;
Research Triangle Park, N.C.), dolasetron (sold as Anzemet.RTM. by
Sanofi-Aventis; New York, N.Y.), tropisetron (sold as Navoban.RTM.
by Novartis; East Hanover, N.J.).
[0314] Other side effects of cancer treatment include red and white
blood cell deficiency. Accordingly, in an embodiment of the
invention, an anti-TIGIT antibody or antigen-binding fragment
thereof (e.g., 14A6, 28H5, 31C6 or humanized versions thereof) is
in association with an agent which treats or prevents such a
deficiency, such as, e.g., filgrastim, PEG-filgrastim,
erythropoietin, epoetin alfa or darbepoetin alfa.
[0315] In an embodiment of the invention, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) is administered in
association with anti-cancer radiation therapy. For example, in an
embodiment of the invention, the radiation therapy is external beam
therapy (EBT): a method for delivering a beam of high-energy X-rays
to the location of the tumor. The beam is generated outside the
patient (e.g., by a linear accelerator) and is targeted at the
tumor site. These X-rays can destroy the cancer cells and careful
treatment planning allows the surrounding normal tissues to be
spared. No radioactive sources are placed inside the patient's
body. In an embodiment of the invention, the radiation therapy is
proton beam therapy: a type of conformal therapy that bombards the
diseased tissue with protons instead of X-rays. In an embodiment of
the invention, the radiation therapy is conformal external beam
radiation therapy: a procedure that uses advanced technology to
tailor the radiation therapy to an individual's body structures. In
an embodiment of the invention, the radiation therapy is
brachytherapy: the temporary placement of radioactive materials
within the body, usually employed to give an extra dose--or
boost--of radiation to an area.
[0316] In an embodiment of the invention, a surgical procedure
administered in association with an anti-TIGIT antibody or
antigen-binding fragment thereof (e.g., 14A6, 28H5, 31C6 or
humanized versions thereof) is surgical tumorectomy.
[0317] The term "in association with" indicates that the components
administered in a method of the present invention (e.g., an
anti-TIGIT antibody (e.g., humanized antibody) or antigen-binding
fragment thereof (e.g., 14A6, 28H5, 31C6 or humanized versions
thereof) along with pembrolizumab) can be formulated into a single
composition for simultaneous delivery or formulated separately into
two or more compositions (e.g., a kit). Each component can be
administered to a subject at a different time than when the other
component is administered; for example, each administration may be
given non-simultaneously (e.g., separately or sequentially) at
several intervals over a given period of time. Moreover, the
separate components may be administered to a subject by the same or
by a different route.
Experimental and Diagnostic Uses
[0318] The anti-TIGIT antibodies and antigen-binding fragments
thereof disclosed herein (e.g., 14A6, 28H5, 31C6 and humanized
versions thereof) may be used as affinity purification agents. In
this process, the anti-TIGIT antibodies and antigen-binding
fragments thereof are immobilized on a solid phase such a Sephadex,
glass or agarose resin or filter paper, using methods well known in
the art. The immobilized antibody or fragment is contacted with a
sample containing the TIGIT protein (or a fragment thereof) to be
purified, and thereafter the support is washed with a suitable
solvent that will remove substantially all the material in the
sample except the TIGIT protein, which is bound to the immobilized
antibody or fragment. Finally, the support is washed with a solvent
which elutes the bound TIGIT (e.g., protein A). Such immobilized
antibodies and fragments form part of the present invention.
[0319] Further provided are antigens for generating secondary
antibodies which are useful for example for performing Western
blots and other immunoassays discussed herein. In particular,
polypeptides are disclosed which comprise the variable regions
and/or CDR sequences of a therapeutic antibody disclosed herein
(e.g., 14A6, 28H5 or 31C6) and which may be used to generate
anti-idiotypic antibodies for use in specifically detecting the
presence of the antibody, e.g., in a therapeutic context.
[0320] Anti-TIGIT antibodies (e.g., humanized antibodies) and
antigen-binding fragments thereof may also be useful in diagnostic
assays for TIGIT protein, e.g., detecting its expression in
specific cells, tissues, or serum, e.g., tumor cells such as
melanoma cells. Such diagnostic methods may be useful in various
disease diagnoses.
[0321] The present invention includes ELISA assays (enzyme-linked
immunosorbent assay) incorporating the use of an anti-TIGIT
antibody or antigen-binding fragment thereof disclosed herein
(e.g., 14A6 or a humanized version thereof).
[0322] For example, such a method comprises the following
steps:
(a) coat a substrate (e.g., surface of a microtiter plate well,
e.g., a plastic plate) with anti-TIGIT antibody or antigen-binding
fragment thereof; (b) apply a sample to be tested for the presence
of TIGIT to the substrate; (c) wash the plate, so that unbound
material in the sample is removed; (d) apply detectably labeled
antibodies (e.g., enzyme-linked antibodies) which are also specific
to the TIGIT antigen; (e) wash the substrate, so that the unbound,
labeled antibodies are removed; (f) if the labeled antibodies are
enzyme linked, apply a chemical which is converted by the enzyme
into a fluorescent signal; and (g) detect the presence of the
labeled antibody.
[0323] Detection of the label associated with the substrate
indicates the presence of the TIGIT protein.
[0324] In a further embodiment, the labeled antibody or
antigen-binding fragment thereof is labeled with peroxidase which
react with ABTS (e.g.,
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) or
3,3',5,5'-Tetramethylbenzidine to produce a color change which is
detectable. Alternatively, the labeled antibody or fragment is
labeled with a detectable radioisotope (e.g., .sup.3H) which can be
detected by scintillation counter in the presence of a
scintillant.
[0325] An anti-TIGIT antibody or antigen-binding fragment thereof
of the invention (e.g., 14A6, 28H5, 31C6 or humanized versions
thereof) may be used in a Western blot or immune-protein blot
procedure. Such a procedure forms part of the present invention and
includes e.g.:
(1) optionally transferring proteins from a sample to be tested for
the presence of TIGIT (e.g., from a PAGE or SDS-PAGE
electrophoretic separation of the proteins in the sample) onto a
membrane or other solid substrate using a method known in the art
(e.g., semi-dry blotting or tank blotting); contacting the membrane
or other solid substrate to be tested for the presence of bound
TIGIT or a fragment thereof with an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention.
[0326] Such a membrane may take the form of a nitrocellulose or
vinyl-based (e.g., polyvinylidene fluoride (PVDF)) membrane to
which the proteins to be tested for the presence of TIGIT in a
non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or
SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel
electrophoresis) gel have been transferred (e.g., following
electrophoretic separation in the gel). Before contacting the
membrane with the anti-TIGIT antibody or fragment, the membrane is
optionally blocked, e.g., with non-fat dry milk or the like so as
to bind non-specific protein binding sites on the membrane.
(2) washing the membrane one or more times to remove unbound
anti-TIGIT antibody or fragment and other unbound substances; and
(3) detecting the bound anti-TIGIT antibody or fragment.
[0327] Detection of the bound antibody or fragment indicates that
the TIGIT protein is present on the membrane or substrate and in
the sample. Detection of the bound antibody or fragment may be by
binding the antibody or fragment with a secondary antibody (an
anti-immunoglobulin antibody) which is detectably labeled and,
then, detecting the presence of the secondary antibody.
[0328] The anti-TIGIT antibodies and antigen-binding fragments
thereof disclosed herein (e.g., 14A6, 28H5, 31C6 and humanized
versions thereof) may also be used for immunohistochemistry. Such a
method forms part of the present invention and comprises, e.g.,
(1) contacting a cell (e.g., a tumor cell such as a melanoma cell)
to be tested for the presence of TIGIT protein with an anti-TIGIT
antibody or antigen-binding fragment thereof of the invention; and
(2) detecting the antibody or fragment on or in the cell.
[0329] If the antibody or fragment itself is detectably labeled, it
can be detected directly. Alternatively, the antibody or fragment
may be bound by a detectably labeled secondary antibody which is
detected.
[0330] Certain anti-TIGIT antibodies and antigen-binding fragments
thereof disclosed herein (e.g., 14A6, 28H5, 31C6 and humanized
versions thereof) may also be used for in vivo tumor imaging. Such
a method may include injection of a radiolabeled anti-TIGIT
antibody or antigen-binding fragment thereof into the body of a
patient to be tested for the presence of a tumor associated with
TIGIT expression (e.g., which expresses TIGIT, for example, on the
tumor cell surface) followed by nuclear imaging of the body of the
patient to detect the presence of the labeled antibody or fragment
e.g., at loci comprising a high concentration of the antibody or
fragment which are bound to the tumor. The detection of the loci
indicates the presence of the TIGIT.sup.+ tumor and tumor
cells.
[0331] Imaging techniques include SPECT imaging (single photon
emission computed tomography) or PET imaging (positron emission
tomography). Labels include e.g., iodine-123 (.sup.123I) and
technetium-99m (.sup.99mTc), e.g., in conjunction with SPECT
imaging or .sup.11C, .sup.13N, .sup.15O or .sup.18F, e.g., in
conjunction with PET imaging or Indium-111 (See e.g., Gordon et
al., (2005) International Rev. Neurobiol. 67:385-440).
Pharmaceutical Compositions and Administration
[0332] To prepare pharmaceutical or sterile compositions of the
anti-TIGIT antibodies and antigen-binding fragments of the
invention (e.g., 14A6, 28H5, 31C6 and humanized versions thereof),
the antibody or antigen-binding fragment thereof is admixed with a
pharmaceutically acceptable carrier or excipient. See, e.g.,
Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National
Formulary, Mack Publishing Company, Easton, Pa. (1984).
[0333] Formulations of therapeutic and diagnostic agents may be
prepared by mixing with acceptable carriers, excipients, or
stabilizers in the form of, e.g., lyophilized powders, slurries,
aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001)
Goodman and Gilman's The Pharmacological Basis of Therapeutics,
McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science
and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New
York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms:
Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.)
(1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY;
Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:
Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000)
Excipient Toxicity and Safety, Marcel Dekker, Inc., New York,
N.Y.).
[0334] Toxicity and therapeutic efficacy of the antibodies of the
invention, administered alone or in combination with another
therapeutic agent, can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for
determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index (LD.sub.50/ED.sub.50).
The data obtained from these cell culture assays and animal studies
can be used in formulating a range of dosage for use in human. The
dosage of such compounds lies preferably within a range of
circulating concentrations that include the ED.sub.50 with little
or no toxicity. The dosage may vary within this range depending
upon the dosage form employed and the route of administration.
[0335] In a further embodiment, a further therapeutic agent that is
administered to a subject in association with an anti-TIGIT
antibody or antigen-binding fragment thereof of the invention
(e.g., 14A6, 28H5, 31C6) in accordance with the Physicians' Desk
Reference 2003 (Thomson Healthcare; 57th edition (Nov. 1,
2002)).
[0336] The mode of administration can vary. Routes of
administration include oral, rectal, transmucosal, intestinal,
parenteral; intramuscular, subcutaneous, intradermal,
intramedullary, intrathecal, direct intraventricular, intravenous,
intraperitoneal, intranasal, intraocular, inhalation, insufflation,
topical, cutaneous, transdermal, or intra-arterial.
[0337] In particular embodiments, the anti-TIGIT antibodies or
antigen-binding fragments thereof of the invention (e.g., 14A6,
28H5, 31C6 and humanized versions thereof) can be administered by
an invasive route such as by injection. In further embodiments of
the invention, an anti-TIGIT antibody or antigen-binding fragment
thereof, or pharmaceutical composition thereof, is administered
intravenously, subcutaneously, intramuscularly, intraarterially,
intratumorally, or by inhalation, aerosol delivery. Administration
by non-invasive routes (e.g., orally; for example, in a pill,
capsule or tablet) is also within the scope of the present
invention.
[0338] The present invention provides a vessel (e.g., a plastic or
glass vial, e.g., with a cap or a chromatography column, hollow
bore needle or a syringe cylinder) comprising any of the antibodies
or antigen-binding fragments of the invention (e.g., 14A6, 28H5,
31C6 and humanized versions thereof) or a pharmaceutical
composition thereof. The present invention also provides an
injection device comprising any of the antibodies or
antigen-binding fragments of the invention (e.g., 14A6, 28H5, 31C6
and humanized versions thereof) or a pharmaceutical composition
thereof. An injection device is a device that introduces a
substance into the body of a patient via a parenteral route, e.g.,
intramuscular, subcutaneous or intravenous. For example, an
injection device may be a syringe (e.g., pre-filled with the
pharmaceutical composition, such as an auto-injector) which, for
example, includes a cylinder or barrel for holding fluid to be
injected (e.g., antibody or fragment or a pharmaceutical
composition thereof), a needle for piecing skin and/or blood
vessels for injection of the fluid; and a plunger for pushing the
fluid out of the cylinder and through the needle bore. In an
embodiment of the invention, an injection device that comprises an
antibody or antigen-binding fragment thereof of the present
invention or a pharmaceutical composition thereof is an intravenous
(IV) injection device. Such a device includes the antibody or
fragment or a pharmaceutical composition thereof in a cannula or
trocar/needle which may be attached to a tube which may be attached
to a bag or reservoir for holding fluid (e.g., saline; or lactated
ringer solution comprising NaCl, sodium lactate, KCl, CaCl.sub.2
and optionally including glucose) introduced into the body of the
patient through the cannula or trocar/needle. The antibody or
fragment or a pharmaceutical composition thereof may, in an
embodiment of the invention, be introduced into the device once the
trocar and cannula are inserted into the vein of a subject and the
trocar is removed from the inserted cannula. The IV device may, for
example, be inserted into a peripheral vein (e.g., in the hand or
arm); the superior vena cava or inferior vena cava, or within the
right atrium of the heart (e.g., a central IV); or into a
subclavian, internal jugular, or a femoral vein and, for example,
advanced toward the heart until it reaches the superior vena cava
or right atrium (e.g., a central venous line). In an embodiment of
the invention, an injection device is an autoinjector; a jet
injector or an external infusion pump. A jet injector uses a
high-pressure narrow jet of liquid which penetrate the epidermis to
introduce the antibody or fragment or a pharmaceutical composition
thereof to a patient's body. External infusion pumps are medical
devices that deliver the antibody or fragment or a pharmaceutical
composition thereof into a patient's body in controlled amounts.
External infusion pumps may be powered electrically or
mechanically. Different pumps operate in different ways, for
example, a syringe pump holds fluid in the reservoir of a syringe,
and a moveable piston controls fluid delivery, an elastomeric pump
holds fluid in a stretchable balloon reservoir, and pressure from
the elastic walls of the balloon drives fluid delivery. In a
peristaltic pump, a set of rollers pinches down on a length of
flexible tubing, pushing fluid forward. In a multi-channel pump,
fluids can be delivered from multiple reservoirs at multiple
rates.
[0339] The pharmaceutical compositions disclosed herein may also be
administered with a needleless hypodermic injection device; such as
the devices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002;
5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or
4,596,556. Such needleless devices comprising the pharmaceutical
composition are also part of the present invention. The
pharmaceutical compositions disclosed herein may also be
administered by infusion. Examples of well-known implants and
modules for administering the pharmaceutical compositions include
those disclosed in: U.S. Pat. No. 4,487,603, which discloses an
implantable micro-infusion pump for dispensing medication at a
controlled rate;
[0340] U.S. Pat. No. 4,447,233, which discloses a medication
infusion pump for delivering medication at a precise infusion rate;
U.S. Pat. No. 4,447,224, which discloses a variable flow
implantable infusion apparatus for continuous drug delivery; U.S.
Pat. No. 4,439,196, which discloses an osmotic drug delivery system
having multi-chamber compartments. Many other such implants,
delivery systems, and modules are well known to those skilled in
the art and those comprising the pharmaceutical compositions of the
present invention are within the scope of the present
invention.
[0341] Alternately, one may administer the anti-TIGIT antibody or
antigen-binding fragment of the invention (e.g., 14A6, 28H5, 31C6
or humanized versions thereof) in a local rather than systemic
manner, for example, via injection of the antibody or fragment
directly into a tumor, e.g., a TIGIT.sup.+ tumor. Furthermore, one
may administer the antibody or fragment in a targeted drug delivery
system, for example, in a liposome coated with a tissue-specific
antibody, targeting, for example, a tumor e.g., a TIGIT.sup.+
tumor, e.g., characterized by immunopathology. The liposomes will
be targeted to and taken up selectively by the afflicted tissue.
Such methods and liposomes are part of the present invention.
[0342] The administration regimen depends on several factors,
including the serum or tissue turnover rate of the therapeutic
antibody or antigen-binding fragment, the level of symptoms, the
immunogenicity of the therapeutic antibody, and the accessibility
of the target cells in the biological matrix. Preferably, the
administration regimen delivers sufficient therapeutic antibody or
fragment to effect improvement in the target disease state, while
simultaneously minimizing undesired side effects. Accordingly, the
amount of biologic delivered depends in part on the particular
therapeutic antibody and the severity of the condition being
treated. Guidance in selecting appropriate doses of therapeutic
antibodies or fragments is available (see, e.g., Wawrzynczak (1996)
Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK;
Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and
Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.) (1993)
Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases,
Marcel Dekker, New York, N.Y.; Baert, et al. (2003) New Engl. J.
Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med.
341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792;
Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et
al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New
Engl. J. Med. 343:1594-1602).
[0343] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known or suspected in
the art to affect treatment. Generally, the dose begins with an
amount somewhat less than the optimum dose and it is increased by
small increments thereafter until the desired or optimum effect is
achieved relative to any negative side effects. Important
diagnostic measures include those of symptoms of, e.g., the
inflammation or level of inflammatory cytokines produced. In
general, it is desirable that a biologic that will be used is
derived from the same species as the animal targeted for treatment,
thereby minimizing any immune response to the reagent. In the case
of human subjects, for example, humanized and fully human
antibodies are may be desirable.
[0344] Antibodies or antigen-binding fragments thereof disclosed
herein (e.g., 14A6, 28H5, 31C6 and humanized versions thereof) may
be provided by continuous infusion, or by doses administered, e.g.,
daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly,
quarterly, semiannually, annually etc. Doses may be provided, e.g.,
intravenously, subcutaneously, topically, orally, nasally,
rectally, intramuscular, intracerebrally, intraspinally, or by
inhalation. A total weekly dose is generally at least 0.05 .mu.g/kg
body weight, more generally at least 0.2 .mu.g/kg, 0.5 .mu.g/kg, 1
.mu.g/kg, 10 .mu.g/kg, 100 .mu.g/kg, 0.25 mg/kg, 1.0 mg/kg, 2.0
mg/kg, 5.0 mg/ml, 10 mg/kg, 25 mg/kg, 50 mg/kg or more (see, e.g.,
Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al.
(2002) New Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J.
Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al. (20003)
Cancer Immunol. Immunother. 52:151-144). Doses may also be provided
to achieve a pre-determined target concentration of anti-TIGIT
antibody in the subject's serum, such as 0.1, 0.3, 1, 3, 10, 30,
100, 300 .mu.g/ml or more. In other embodiments, An anti-TIGIT
antibody of the present invention is administered, e.g.,
subcutaneously or intravenously, on a weekly, biweekly, "every 4
weeks," monthly, bimonthly, or quarterly basis at 10, 20, 50, 80,
100, 200, 500, 1000 or 2500 mg/subject.
[0345] As used herein, the term "effective amount" refer to an
amount of an anti-TIGIT or antigen-binding fragment thereof of the
invention (e.g., humanized 14A6 or humanized 28H5) that, when
administered alone or in combination with an additional therapeutic
agent to a cell, tissue, or subject, is effective to cause a
measurable improvement in one or more symptoms of disease, for
example cancer or the progression of cancer. An effective dose
further refers to that amount of the antibody or fragment
sufficient to result in at least partial amelioration of symptoms,
e.g., tumor shrinkage or elimination, lack of tumor growth,
increased survival time. When applied to an individual active
ingredient administered alone, an effective dose refers to that
ingredient alone. When applied to a combination, an effective dose
refers to combined amounts of the active ingredients that result in
the therapeutic effect, whether administered in combination,
serially or simultaneously. An effective amount of a therapeutic
will result in an improvement of a diagnostic measure or parameter
by at least 10%; usually by at least 20%; preferably at least about
30%; more preferably at least 40%, and most preferably by at least
50%. An effective amount can also result in an improvement in a
subjective measure in cases where subjective measures are used to
assess disease severity.
Kits
[0346] Further provided are kits comprising one or more components
that include, but are not limited to, an anti-TIGIT antibody or
antigen-binding fragment, as discussed herein (e.g., humanized 14A6
or humanized 28H5 or humanized 31C6) in association with one or
more additional components including, but not limited to a
pharmaceutically acceptable carrier and/or a therapeutic agent, as
discussed herein. The antibody or fragment and/or the therapeutic
agent can be formulated as a pure composition or in combination
with a pharmaceutically acceptable carrier, in a pharmaceutical
composition.
[0347] In one embodiment, the kit includes an anti-TIGIT antibody
or antigen-binding fragment thereof of the invention (e.g.,
humanized 14A6 or humanized 28H5 or humanized 31C6) or a
pharmaceutical composition thereof in one container (e.g., in a
sterile glass or plastic vial) and a pharmaceutical composition
thereof and/or a therapeutic agent in another container (e.g., in a
sterile glass or plastic vial).
[0348] In another embodiment, the kit comprises a combination of
the invention, including an anti-TIGIT antibody or antigen-binding
fragment thereof of the invention (e.g., humanized 14A6 or
humanized 28H5 or humanized 31C6) along with a pharmaceutically
acceptable carrier, optionally in combination with one or more
therapeutic agents formulated together, optionally, in a
pharmaceutical composition, in a single, common container.
[0349] If the kit includes a pharmaceutical composition for
parenteral administration to a subject, the kit can include a
device for performing such administration. For example, the kit can
include one or more hypodermic needles or other injection devices
as discussed above.
[0350] The kit can include a package insert including information
concerning the pharmaceutical compositions and dosage forms in the
kit. Generally, such information aids patients and physicians in
using the enclosed pharmaceutical compositions and dosage forms
effectively and safely. For example, the following information
regarding a combination of the invention may be supplied in the
insert: pharmacokinetics, pharmacodynamics, clinical studies,
efficacy parameters, indications and usage, contraindications,
warnings, precautions, adverse reactions, overdosage, proper dosage
and administration, how supplied, proper storage conditions,
references, manufacturer/distributor information and patent
information.
Detection Kits and Therapeutic Kits
[0351] As a matter of convenience, an anti-TIGIT antibody or
antigen-binding fragment thereof of the invention (e.g., 14A6,
28H5, 31C6 or humanized versions thereof) can be provided in a kit,
i.e., a packaged combination of reagents in predetermined amounts
with instructions for performing the diagnostic or detection assay.
Where the antibody or fragment is labeled with an enzyme, the kit
will include substrates and cofactors required by the enzyme (e.g.,
a substrate precursor which provides the detectable chromophore or
fluorophore). In addition, other additives may be included such as
stabilizers, buffers (e.g., a block buffer or lysis buffer) and the
like. The relative amounts of the various reagents may be varied
widely to provide for concentrations in solution of the reagents
which substantially optimize the sensitivity of the assay.
Particularly, the reagents may be provided as dry powders, usually
lyophilized, including excipients which on dissolution will provide
a reagent solution having the appropriate concentration.
[0352] Also provided are diagnostic or detection reagents and kits
comprising one or more such reagents for use in a variety of
detection assays, including for example, immunoassays such as ELISA
(sandwich-type or competitive format). The kit's components may be
pre-attached to a solid support, or may be applied to the surface
of a solid support when the kit is used. In some embodiments of the
invention, the signal generating means may come pre-associated with
an antibody or fragment of the invention or may require combination
with one or more components, e.g., buffers, antibody-enzyme
conjugates, enzyme substrates, or the like, prior to use. Kits may
also include additional reagents, e.g., blocking reagents for
reducing nonspecific binding to the solid phase surface, washing
reagents, enzyme substrates, and the like. The solid phase surface
may be in the form of a tube, a bead, a microtiter plate, a
microsphere, or other materials suitable for immobilizing proteins,
peptides, or polypeptides. In particular aspects, an enzyme that
catalyzes the formation of a chemilluminescent or chromogenic
product or the reduction of a chemilluminescent or chromogenic
substrate is a component of the signal generating means. Such
enzymes are well known in the art. Kits may comprise any of the
capture agents and detection reagents described herein. Optionally
the kit may also comprise instructions for carrying out the methods
of the invention.
[0353] Also provided is a kit comprising an anti-TIGIT antibody
(e.g., humanized antibody) or antigen-binding fragment thereof
packaged in a container, such as a vial or bottle, and further
comprising a label attached to or packaged with the container, the
label describing the contents of the container and providing
indications and/or instructions regarding use of the contents of
the container to treat one or more disease states as described
herein.
[0354] In one aspect, the kit is for treating cancer and comprises
an anti-TIGIT antibody (e.g., humanized antibody) or
antigen-binding fragment thereof and a further therapeutic agent or
a vaccine. The kit may optionally further include a syringe for
parenteral, e.g., intravenous, administration. In another aspect,
the kit comprises an anti-TIGIT antibody (e.g., humanized antibody)
or antigen-binding fragment thereof and a label attached to or
packaged with the container describing use of the antibody or
fragment with the vaccine or further therapeutic agent. In yet
another aspect, the kit comprises the vaccine or further
therapeutic agent and a label attached to or packaged with the
container describing use of the vaccine or further therapeutic
agent with the anti-TIGIT antibody or fragment. In certain
embodiments, an anti-TIGIT antibody and vaccine or further
therapeutic agent are in separate vials or are combined together in
the same pharmaceutical composition.
[0355] As discussed above in the combination therapy section,
concurrent administration of two therapeutic agents does not
require that the agents be administered at the same time or by the
same route, as long as there is an overlap in the time period
during which the agents are exerting their therapeutic effect.
Simultaneous or sequential administration is contemplated, as is
administration on different days or weeks.
[0356] The therapeutic and detection kits disclosed herein may also
be prepared that comprise at least one of the antibody, peptide,
antigen-binding fragment, or polynucleotide disclosed herein and
instructions for using the composition as a detection reagent or
therapeutic agent. Containers for use in such kits may typically
comprise at least one vial, test tube, flask, bottle, syringe or
other suitable container, into which one or more of the detection
and/or therapeutic composition(s) may be placed, and preferably
suitably aliquoted. Where a second therapeutic agent is also
provided, the kit may also contain a second distinct container into
which this second detection and/or therapeutic composition may be
placed. Alternatively, a plurality of compounds may be prepared in
a single pharmaceutical composition, and may be packaged in a
single container means, such as a vial, flask, syringe, bottle, or
other suitable single container. The kits disclosed herein will
also typically include a means for containing the vial(s) in close
confinement for commercial sale, such as, e.g., injection or
blow-molded plastic containers into which the desired vial(s) are
retained. Where a radiolabel, chromogenic, fluorigenic, or other
type of detectable label or detecting means is included within the
kit, the labeling agent may be provided either in the same
container as the detection or therapeutic composition itself, or
may alternatively be placed in a second distinct container means
into which this second composition may be placed and suitably
aliquoted. Alternatively, the detection reagent and the label may
be prepared in a single container means, and in most cases, the kit
will also typically include a means for containing the vial(s) in
close confinement for commercial sale and/or convenient packaging
and delivery.
[0357] A device or apparatus for carrying out the detection or
monitoring methods described herein is also provided. Such an
apparatus may include a chamber or tube into which sample can be
input, a fluid handling system optionally including valves or pumps
to direct flow of the sample through the device, optionally filters
to separate plasma or serum from blood, mixing chambers for the
addition of capture agents or detection reagents, and optionally a
detection device for detecting the amount of detectable label bound
to the capture agent immunocomplex. The flow of sample may be
passive (e.g., by capillary, hydrostatic, or other forces that do
not require further manipulation of the device once sample is
applied) or active (e.g., by application of force generated via
mechanical pumps, electroosmotic pumps, centrifugal force, or
increased air pressure), or by a combination of active and passive
forces.
[0358] In further embodiments, also provided is a processor, a
computer readable memory, and a routine stored on the computer
readable memory and adapted to be executed on the processor to
perform any of the methods described herein. Examples of suitable
computing systems, environments, and/or configurations include
personal computers, server computers, hand-held or laptop devices,
multiprocessor systems, microprocessor-based systems, set top
boxes, programmable consumer electronics, network PCs,
minicomputers, mainframe computers, distributed computing
environments that include any of the above systems or devices, or
any other systems known in the art.
General Methods
[0359] Standard methods in molecular biology are described
Sambrook, Fritsch and Maniatis (1982 & 1989 2.sup.nd Edition,
2001 3.sup.rd Edition) Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook
and Russell (2001) Molecular Cloning, 3.sup.rd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993)
Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif.).
Standard methods also appear in Ausbel, et al. (2001) Current
Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons,
Inc. New York, N.Y., which describes cloning in bacterial cells and
DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast
(Vol. 2), glycoconjugates and protein expression (Vol. 3), and
bioinformatics (Vol. 4).
[0360] Methods for protein purification including
immunoprecipitation, chromatography, electrophoresis,
centrifugation, and crystallization are described (Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 1, John Wiley and
Sons, Inc., New York). Chemical analysis, chemical modification,
post-translational modification, production of fusion proteins,
glycosylation of proteins are described (see, e.g., Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 2, John Wiley and
Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in
Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, N.Y., pp.
16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life
Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia
Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391).
Production, purification, and fragmentation of polyclonal and
monoclonal antibodies are described (Coligan, et al. (2001) Current
Protcols in Immunology, Vol. 1, John Wiley and Sons, Inc., New
York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane,
supra). Standard techniques for characterizing ligand/receptor
interactions are available (see, e.g., Coligan, et al. (2001)
Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New
York).
[0361] Monoclonal, polyclonal, and humanized antibodies can be
prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal
Antibodies, Oxford Univ. Press, New York, N.Y.; Kontermann and
Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New
York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp.
139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al.
(1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem.
274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684;
Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992)J.
Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).
[0362] An alternative to humanization is to use human antibody
libraries displayed on phage or human antibody libraries in
transgenic mice (Vaughan et al. (1996) Nature Biotechnol.
14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al.
(1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000)
Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; Kay et al. (1996) Phage Display of Peptides and
Proteins: A Laboratory Manual, Academic Press, San Diego, Calif.;
de Bruin et al. (1999) Nature Biotechnol. 17:397-399).
[0363] Single chain antibodies and diabodies are described (see,
e.g., Malecki et al. (2002) Proc. Natl. Acad. Sci. USA 99:213-218;
Conrath et al. (2001) J. Biol. Chem. 276:7346-7350; Desmyter et al.
(2001) J. Biol. Chem. 276:26285-26290; Hudson and Kortt (1999) J.
Immunol. Methods 231:177-189; and U.S. Pat. No. 4,946,778).
Bifunctional antibodies are provided (see, e.g., Mack, et al.
(1995) Proc. Natl. Acad. Sci. USA 92:7021-7025; Carter (2001) J
Immunol. Methods 248:7-15; Volkel, et al. (2001) Protein
Engineering 14:815-823; Segal, et al. (2001) J. Immunol. Methods
248:1-6; Brennan, et al. (1985) Science 229:81-83; Raso, et al.
(1997) J. Biol. Chem. 272:27623; Morrison (1985) Science
229:1202-1207; Traunecker, et al. (1991) EMBO 10:3655-3659; and
U.S. Pat. Nos. 5,932,448, 5,532,210, and 6,129,914).
[0364] Bispecific antibodies are also provided (see, e.g., Azzoni
et al. (1998) J Immunol. 161:3493; Kita et al. (1999) J. Immunol.
162:6901; Merchant et al. (2000) J. Biol. Chem. 74:9115; Pandey et
al. (2000) J. Biol. Chem. 275:38633; Zheng et al. (2001) J. Biol
Chem. 276:12999; Propst et al. (2000) J. Immunol. 165:2214; Long
(1999) Ann. Rev. Immunol. 17:875).
[0365] Purification of antigen is not necessary for the generation
of antibodies. Animals can be immunized with cells bearing the
antigen of interest. Splenocytes can then be isolated from the
immunized animals, and the splenocytes can fused with a myeloma
cell line to produce a hybridoma (see, e.g., Meyaard et al. (1997)
Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242;
Preston et al., supra; Kaithamana et al. (1999) J. Immunol.
163:5157-5164).
[0366] Antibodies can be conjugated, e.g., to small drug molecules,
enzymes, liposomes, polyethylene glycol (PEG). Antibodies are
useful for therapeutic, diagnostic, kit or other purposes, and
include antibodies coupled, e.g., to dyes, radioisotopes, enzymes,
or metals, e.g., colloidal gold (see, e.g., Le Doussal et al.
(1991) J. Immunol. 146:169-175; Gibellini et al. (1998) J. Immunol.
160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811;
Everts et al. (2002) 1 Immunol. 168:883-889).
[0367] Methods for flow cytometry, including fluorescence activated
cell sorting (FACS), are available (see, e.g., Owens, et al. (1994)
Flow Cytometry Principles for Clinical Laboratory Practice, John
Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry,
2.sup.nd ed.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical
Flow Cytometry, John Wiley and Sons, Hoboken, N.J.). Fluorescent
reagents suitable for modifying nucleic acids, including nucleic
acid primers and probes, polypeptides, and antibodies, for use,
e.g., as diagnostic reagents, are available (Molecular Probes
(2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.;
Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).
[0368] Standard methods of histology of the immune system are
described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus:
Histopathology and Pathology, Springer Verlag, New York, N.Y.;
Hiatt, et al. (2000) Color Atlas of Histology, Lippincott,
Williams, and Wilkins, Phila, Pa.; Louis, et al. (2002) Basic
Histology: Text and Atlas, McGraw-Hill, New York, N.Y.).
[0369] Software packages and databases for determining, e.g.,
antigenic fragments, leader sequences, protein folding, functional
domains, glycosylation sites, and sequence alignments, are
available (see, e.g., GenBank, Vector NTI.RTM. Suite (Informax,
Inc, Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San
Diego, Calif.); DeCypher.RTM. (TimeLogic Corp., Crystal Bay, Nev.);
Menne, et al. (2000) Bioinformatics 16: 741-742; Menne, et al.
(2000) Bioinformatics Applications Note 16:741-742; Wren, et al.
(2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne
(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids
Res. 14:4683-4690).
Example 1
Generation of Rat and Mouse Anti-hTIGIT Antibodies
[0370] To generate antibodies to human TIGIT, Lewis rats were
immunized with his-tagged human TIGIT recombinant protein from Sino
Biologicals Cat#10917-H08H) using RIBI adjuvant and footpad
injection on a biweekly schedule. Alternatively, Balb/C mice were
immunized with human Fc-tagged human TIGIT recombinant protein
using RIBI adjuvant and footpad injection on a biweekly schedule.
Immunized animals were bled and serum titers determined for binding
to human TIGIT transfected CHOK1 cells using a cell-based ELISA
(described below). The animals with the highest titers were given a
final boost with recombinant protein and draining popliteal lymph
nodes isolated four days later. Hybridomas were generated by
electrofusion of isolated lymphocytes with the myeloma fusion
partner P3X63-AG8.653 using the Cytopulse Hybrimmune electrofusion
system. Fused cells were plated in 96-well plates in DMEM/F12, 15%
BCS, HAT, IL-6, OPI supplement, and gentamycin.
[0371] Hybridoma supernatants were assayed for binding to human
TIGIT expressing CHOK1 cells and cross-reactivity to rhesus TIGIT
expressing CHO cells using a cell-based ELISA format. Human TIGIT
and rhesus TIGIT expressing CHO-K1 cells were plated in 96-well
tissue-culture plates in 50 .mu.l of DMEM/F12, 10% BCS and
gentamycin (CHO-K1 media). Cells were plated at either
2.times.10.sup.4 cells/well two days prior to the assay or
4.times.10.sup.4 cells/well one day prior to the assay. Media was
removed from the wells prior to the assay and 50 .mu.l of hybridoma
supernatant added. Hybridoma supernatants were incubated for 30-60
minutes at room temperature and washed 3 times with PBS/0.05% Tween
20 using a cell ELISA washing protocol on the Biotek EL405x Select
CW plate washer. Fifty microliters of the detection antibody
(HRP-conjugated goat anti-rat IgG (Southern Biotech cat#3030-05) or
HRP-conjugated goat anti-mouse IgG (Southern Biotech cat#1043-05)),
was added at a 1:2000 dilution in CHO-K1 media and incubated at
room temperature for 30-60 minutes. Assay plates were washed as
above and developed with TMB and stopped with TMB stop solution
(KPL cat#50-85-06) or 0.1N phosphoric acid. The absorbance at 450
nm-620 nm was determined. Positive clones were reactive to both
human TIGIT and rhesus TIGIT transfected CHO-K1 cells, and were
negative for binding to parental CHO-K1 cells. In these assays, if
an antibody showed binding to parental (untransfected) CHO-K1
cells; we discarded that antibody in screening as not specific to
TIGIT.
[0372] Positive hybridomas were subcloned by limiting dilution or
subcloned by plating hybridomas in semi-solid media and clones
picked on the ClonePix.RTM. (Genetix). Two rounds of subcloning
were performed on the parental hybridomas. Final subclones were
grown in small-scale cultures in serum-free hybridoma production
medium and purified to generate purified antibody for further
characterization.
[0373] Using these methods, about 819 hybridomas were
generated.
Example 2
Characterization of Anti-hTIGIT Antibodies
[0374] Supernatants from positive clones were tested for their
ability to block recombinant human CD155-huFc protein binding to
hTIGIT CHOK1 cells in a cell-based ELISA format. Human TIGIT-CHO-K1
cells were plated in 96-well plates as described above. Media was
removed from the plates and 50 .mu.l of hybridoma supernatant was
incubated with the human TIGIT CHO-K1 cells at 4.degree. C. for 30
minutes. Fifty microliters of human CD155-huFc was added to the
plate for a final concentration of 0.5 .mu.g/ml of human CD155-huFc
and incubated for 30 minutes at 4.degree. C. Assay plates were
washed 3 times with PBS/0.05% Tween-20 as above. Binding of human
CD155-huFc to the hTIGIT-CHOK1 cells was detected using an
HRP-conjugate F(ab)'2 goat anti-human IgG secondary antibody
(Jackson 109-036-098) at 1:2000 dilution in CHO-K1 media. Plates
were developed using TMB and stopped using TMB Stop Solution as
described above and the A450-620 nm determined.
[0375] A rat antibody generated according to the above described
method is referred to as 14A6, and was derived from clone
LB155.14A6.G2.A8. This rat antibody (14A6) is of the IgG2/kappa
isotype and comprises the heavy chain variable region of SEQ ID
NO:7 and the light chain variable region of SEQ ID NO:8. Purified
14A6 antibody binds to human TIGIT and rhesus TIGIT as determined
by cell-based ELISA binding to human TIGIT and rhesus TIGIT-CHOK1
cells (FIG. 1) using the methods described above. (An isotype
control antibody did not show any binding (data not shown.)
Purified 14A6 antibody can also block the hTIGIT and hCD155
interaction using a cell-based ELISA blocking assay (FIG. 3) using
the method described above.
[0376] A mouse antibody generated according to the above described
method is referred to as 28H5, and was derived from clone
TC167.28H5.H5. This mouse antibody (28H5) is of the IgG1/kappa
isotype and comprises the heavy chain variable region of SEQ ID
NO:63 and the light chain variable region of SEQ ID NO:64. Purified
28H5 antibody also binds to human TIGIT and rhesus TIGIT as
determined by cell-based ELISA binding to human TIGIT and rhesus
TIGIT-CHOK1 cells (FIG. 2) using the methods described above.
Purified 28H5 antibody also block the hTIGIT and hCD155 interaction
using a cell-based ELISA blocking assay (FIG. 3) using the method
described above.
[0377] Another mouse antibody generated according to the above
described method is referred to as 31C6, and was derived from clone
MEB125.31C6.A1.205. This mouse antibody (31C6) is of the IgG1/kappa
isotype and comprises the heavy chain variable region of SEQ ID
NO:94 and the light chain variable region of SEQ ID NO:95. Purified
31C6 antibody also binds to human TIGIT and rhesus TIGIT as
determined by cell-based ELISA binding to human TIGIT and rhesus
TIGIT-CHOK1 cells (FIG. 4) using the methods described above.
Purified 31C06 antibody also block the hTIGIT and hCD155
interaction using a cell-based ELISA blocking assay (FIG. 4) using
the method described above.
[0378] Affinity Determination for Binding of Parental (Non-Human)
Anti-TIGIT Antibodies to Human TIGIT Recombinant Protein:
[0379] The kinetic binding activity of anti-human TIGIT antibodies
14A6 and 31C6 (made as described in Example 1) and of commercial
antibody MBSA43 was measured by surface plasmon resonance using a
Biacore T200 system (Biacore, GE Healthcare, Piscataway, N.J.).
Approximately 5000 RU of Anti-mouse IgG, GE Healthcare Catalog
Number BR-1008-38, or approximately 13,000 RU of Goat Anti-Rat IgG
Fc gamma, Fragment Specific, Jackson ImmunoResearch Catalog Number
112-006-071, was immobilized via amine coupling chemistry onto a
Series S CM5 sensor chip, catalog number BR-1005-30. Mouse
anti-human TIGIT clones, 31C6 and MBSA43, were each injected over
the immobilized anti-mouse surfaces at 1 ug/mL for a capture level
of 40 RU. Rat anti-human TIGIT clone 14A6 was injected over the
immobilized anti-rat surfaces at 1 ug/mL for a capture level of 40
RU. HBS-EP+ buffer (BR-1006-69) was used as the running buffer with
a flow rate of 30 .mu.L/min. Varying concentrations of human
TIGIT-His protein or TIGIT-Fc protein, ranging from 0.29 nM to 40
nM, at a flow rate of 45 .mu.L/min were injected over the antibody
surfaces. Following each Mouse anti-human TIGIT injection cycle for
clones 31C6 and MBSA43, the Series S CM5 chip surface was
regenerated using one three-minute injection of 10 mM Glycine pH
1.7 at a flow rate of 10 .mu.L/min. Following each Rat anti-human
TIGIT injection cycle, the Series S CM5 chip surface was
regenerated using one 20-second injection of 10 mM Glycine pH 1.5
followed by two 10-second injections of 12.5 mM NaOH at a flow rate
of 60 .mu.L/min.
[0380] Background subtraction binding sensorgrams were used for
analyzing the rate constant of association (k.sub.a) and
dissociation (k.sub.d), and the equilibrium dissociation constant
K.sub.D. The resulting data sets were fitted with a 1:1 Langmuir
Binding Model using the Biacore T200 evaluation software (version
2.0). Table 3 summarizes the affinities for the anti-human TIGIT
antibodies to human TIGIT-His protein and TIGIT-Fc protein.
TABLE-US-00003 TABLE 3 Measurement of Affinity for anti-Human TIGIT
Antibodies to human TIGIT-His protein and TIGIT-Fc protein using
BIAcore. BIAcore KD BIAcore KD (human TIGIT-his) (human TIGIT-Fc)
Clone (pM) (pM) 14A6 (rIgG2a/K) 3.09 3.12 31C6 (mIgG1/K) 34.4 10.9
Comparator MBSA43 36.3 16.5 (mIgG1)
Example 3
In Vitro T-Cell Activity Assay for Antagonistic Anti-hTIGIT
Antibodies
[0381] Of the antibodies tested, approximately 352 monoclonal
antibodies shown to block binding of CD155-Fc to CHO cells
expressing hTIGIT were screened for their capacity enhance T cell
activity in vitro using cell-based functional assays.
[0382] One assay we developed to characterize the functional
consequence of blocking human TIGIT receptor utilized Jurkat cells,
an immortalized line of human T lymphocyte cells (clone, E6-1; ATCC
TIB-152) engineered to over-express human TIGIT (hTIGIT-Jurkat)
which were co-cultured with THP-1 cells, a human monocytic cell
line in the presence or absence of one of the TIGIT ligands, CD155
and CD112. hTIGIT-Jurkat cells co-cultured with THP-1 cells and
stimulated with plate-bound anti-CD3 mAb produce IL-2, but when
TIGIT ligand (CD155 or CD112) is added to the co-culture, IL-2
levels were reduced in a ligand-dependent manner. Treatment with
antibodies that blocked the CD155- or CD112-TIGIT interaction, such
as a commercially available anti-hTIGIT Ab, clone MBSA43
(eBioscience Cat#12-9500-42), rescues IL-2 production in this assay
in a dose-dependent fashion (FIG. 5).
[0383] 96-well flat-bottom plates were coated with mouse anti-human
CD3 antibody (1 ug/ml in PBS; Clone HIT3a; BD Pharmingen
Cat#555336) overnight at 4.degree. C. The next day, hTIGIT-Jurkat
cells (50,000) were plated in the pre-coated plates and
pre-incubated for 30-60 minutes with mAb at varying concentrations.
THP-1 cells (50,000) were added to the culture followed by either
CD155-Fc (ECD of human CD155 fused to human Fc; 1.0 .mu.g/ml) or
CD112-Fc (ECD of human CD112 fused to human Fc; 0.5 .mu.g/ml).
After incubation for 18-24 h at 37.degree. C. and 5.0% CO.sub.2,
IL-2 levels were assessed in culture supernatants by Meso Scale
(Human IL-2 Tissue Culture MESO Kit: Cat#K151AHB-2). Treatment with
MBSA43 (10 .mu.g/ml) rescues IL-2 to a level equal to when
activated hTIGIT Jurkat cells are cultured with THP-1 in the
absence of CD155 or CD112.
[0384] As shown in FIG. 5, a titration of the anti-hTIGIT clone
14A6 from 30 .mu.g/ml down to 0.04 .mu.g/ml gave an EC50 of 0.190
.mu.g/ml as compared to MBSA43 at 0.24 .mu.g/ml using this assay.
Clone 14A6 (30 .mu.g/ml) rescued IL-2 to 82% of MBSA43 (10
.mu.g/ml).
[0385] Also shown in FIG. 5, a titration of the anti-hTIGIT clone
28H5 from 30 .mu.g/ml down to 0.04 .mu.g/ml gave an EC50 of 0.24
.mu.g/ml as compared to MBSA43 at 0.24 .mu.g/ml using this
assay.
[0386] Also shown in FIG. 6, a titration of the anti-hTIGIT clone
31C6 from 30 .mu.g/ml down to 0.04 .mu.g/ml gave an EC50 of 0.14
.mu.g/ml as compared to MBSA43 at 0.24 .mu.g/ml using this assay.
Clone 31C6 (30 .mu.g/ml) rescued IL-2 to 118% of MBSA43 (10
.mu.g/ml).
[0387] Of the 352 monoclonal antibodies tested, approximately 113
were able to enhance T cell activity in vitro using cell-based
functional assays.
Example 4
In Vitro T-Cell Activity Assay for Antagonistic Anti-hTIGIT
Antibodies
[0388] We further analyzed the relative functional antagonism of
TIGIT and rescue of T cell activation and effector function using a
primary human T cell line expressing endogenous TIGIT upon T cell
receptor (TCR) activation. The BC4-clone 49 human T cell line is an
allo-antigen specific human CD4+ T cell clone expressing a TCR
specific for HLA-class II MHC molecules expressed on the EBV
transformed cell line, JY (HLA-DR1,4). The BC4-clone 49 human T
cell line requires re-stimulation with allo antigen every two
weeks, using irradiated (5000 Rads) JY stimulator cells, irradiated
PBMC (4000 Rads) isolated from two buffy coats and PHA-P (2.5
.mu.g/ml; Sigma), as well as 10 ng/ml human IL-2 (R&D)
supplement every 3-4 day following restimulation and expansion.
Numerous T cell clones (BC1-6, BC4-27, BC4-49) were analyzed by PCR
and then by flow cytometry for relative expression of TIGIT
following restimulation and BC4-clone 49 was selected as it had the
highest expression of TIGIT compared to other clones. The relative
expression and kinetics of TIGIT, CD226, CD96 and CD155 were
monitored following re-stimulation with irradiated (5000 Rads) JY
stimulator cells, irradiated PBMC and PHA-P to assess the best time
to assay antagonist mAbs for relative TIGIT-antagonist activity.
Peak expression of TIGIT was observed at 3-4 days post
restimulation, while expression of CD226 and CD96 decreased over
the same time period. Expression of TIGIT then decreased, while
expression of CD226 and CD96 increased again by day 6
post-restimulation (FIG. 7). Accordingly, candidate TIGIT
antagonist mAbs were assessed at 3-4 days post-restimulation.
Transfectant JY overexpressing human CD155 were generated using
pMX->huTIGIT retroviral vectors as a means to suppress BC4-clone
47 T cell responses in the primary T cell bioassay and to assess
the relative capacity of anti-TIGIT mAbs to antagonize CD155
activation of TIGIT and rescue T cell proliferation and IFN.gamma.
three days later.
[0389] The primary T cell assay for relative anti-hTIGIT antagonist
mAbs was set up as follows. Human CD4.sup.+ T cell line BC4-clone
49 was co-cultured with CD155-Fc blocking mAbs specific for hTIGIT
at various concentrations (33, 11, 3.7, 1.2, 0.4 and 0.1 .mu.g/ml)
for 30 minutes and then plated in round-bottom 96-well plates
(2.times.10.sup.4 c/well). JY-hCD155 transfectants, expressing the
allo-antigen and high levels of hCD155, were irradiated (5000
Rads), washed and then added to the wells containing the anti-TIGIT
mAb treated BC4-clone 49 T cells for a final concentration of
1.times.10.sup.4 (2:1 T cell: target cell ratio) or
5.times.10.sup.3 (1:4 T cell: target cell ratio) in a total volume
of 200 .mu.l/well. Controls included no mAb treatment, co-culture
with isotype mAb treated T cells, T cell only and JY-CD155 cells
only. After three or four days of co-culture, supernatants were
harvested for interferon-gamma (IFN.gamma.) cytokine
quantification, and the T cells were pulsed 6 hours with
tritiated-thymidine to assess relative proliferation.
[0390] As shown in FIGS. 8A and 8B, anti-hTIGIT mAb 14A6, 28H5 and
31C6 treatment of BC4-clone 49 T cells resulted in rescue of
IFN.gamma. and proliferative responses as assessed by increased
responses compared to isotype and untreated T cells. Commercial
antibody MBSA43 and antibodies 37D10 and 25G10 (anti-TIGIT
antibodies produced as described in Example 1) also show activity
in this assay. The increase in IFN.gamma. production after
treatment with 14A6, 28H5 and 31C6 was on about 2 fold on
average.
Example 5
Anti-Tumor Activity of Anti-TIGIT and Anti-PD1 Antibodies in Animal
Tumor Model
[0391] Mice:
[0392] Approximately seven to eight week old female BALB/cAnN mice
with an average body weight of 20 grams were obtained from Taconic
Laboratory (Germantown, N.Y.). Conventional animal chow and water
were provided ad libitum.
[0393] Antibody Reagents:
[0394] A monoclonal antibody of murine isotype IgG1 against murine
PD-1 and isotype controls were obtained from internal sources as
frozen (-80.degree. C.) stocks. The rat anti-mouse TIGIT (GIGD7)
antibody was obtained from eBioscience as a 4.degree. C. stock. The
IgG1 isotype control was a mouse monoclonal antibody specific for
adenoviral hexon 25. The IgG2a isotype control was a rat monoclonal
antibody specific for beta-galactosidase. Murine isotype IgG1 is
the murine counterpart isotype to human isotype IgG4.
[0395] Formulations of Antibody Reagents:
[0396] The formulation buffers were specific for each antibody to
stabilize proteins and prevent precipitation. The formulations were
as follows: mIgG1:75 mM NaCl, 10 mM sodium phosphate, 3% sucrose,
pH7.3; anti-PD-1: 20 mM sodium acetate, 7% sucrose, pH5.5; rat
IgG2a: 20 mM sodium acetate, 9% sucrose pH 5.5; and anti-TIGIT
(GIGD7): PBS pH 7.0.
[0397] Tumor Cell Line Preparation and Implant:
[0398] CT26 colon carcinoma cells were cultured in RPMI medium
supplemented with 10% heat-inactivated fetal bovine serum.
3.times.10.sup.5 log-phase and sub-confluent CT26 cells were
injected subcutaneously (SC) in a 100 .mu.L volume of serum-free
RPMI in the right lower dorsal flank of each mouse. Mice were first
shaved with electronic clippers in the area that would be used for
the implant.
[0399] CT-26 is a murine colorectal adenocarcinoma cell line
syngeneic to the BALB/c mouse strain. CT-26 is a relevant model
system for evaluating the mechanism of action for an anti-PD-1
antibody because of the translatable molecular profile of this
tumor post-anti-PD-1 therapy.
[0400] Tumor Measurements and Body Weights:
[0401] Tumors were measured the day before the first dose and twice
a week thereafter. Tumor length and width were measured using
electronic calipers and tumor volume determined using the formula
Volume (mm.sup.3)=0.5.times.Length.times.Width where length is the
longer dimension. Mice were weighed periodically to monitor general
health but also to estimate actual mg/kg dose delivery per mouse
where needed. Before treatment, mice were weighed and tumors from
individual mice were measured. To prevent bias, any outliers by
weight or tumor volume were removed and the remaining mice
randomized into various treatment groups with equivalent mean tumor
size. When the mean tumor volume in the CT26 tumor-bearing mice
reached .about.100 mm.sup.3, around 7 days post implant, animals
were randomized into treatment groups of 10 mice per group and
dosing was started. Animals were administered at the dosing
concentrations specified below.
[0402] Dosing Solution Preparation, Administration, and
Analyses:
[0403] Frozen stocks of the antibodies to be tested in the animal
model were thawed and transferred to wet ice. To avoid repeated
freeze thaw, each vial of stock was thawed once and aliquots made
in volumes sufficient for one time use. Polypropylene, low adhesion
tubes were used for this purpose. The aliquots were snap frozen in
dry ice and stored at -80.degree. C. Before each dosing, one
aliquot was thawed and diluted to nominal concentration in the
appropriate diluent and dosed immediately. Aliquots of dosing
solutions were snap frozen in dry ice and stored at -80.degree. C.
until analyses. Dosing solutions were assessed using the Meso Scale
Discovery (MSD.RTM., Rockville, Md.) platform which is based on
multi-array technology; a combination of electrochemiluminescence
detection and patterned arrays.
[0404] Dosing Anti-TIGIT/Anti-PD1 Treatment Results:
[0405] CT26 tumor-bearing BALB/cAnN mice were administered rat
anti-mouse TIGIT (GIGD7) at a 20 mg/kg dose, IP, every 4 days for
each of 5 cycles. An anti-mouse PD-1 (described above) was
administered at a 10 mg/kg dose, IP, every 4 days for each of 5
cycles. Post dosing, animals continued to be monitored and tumor
volumes were measured out to 36 days. Treatment was started once
the tumor size averaged 100 mm.sup.3 (75 mm.sup.3-115 mm.sup.3).
Tumors were measured twice weekly. As demonstrated by the results,
which are shown in FIG. 9, the mean anti-tumor response of
combination therapy with the PD-1 antagonist and TIGIT antagonist
was greater than the anti-tumor response observed with either
anti-PD1 single agent (p<0.05) or anti-TIGIT single agent
(p<0.005) treatment. For single agent anti-TIGIT treatment, 21%
tumor growth inhibition (TGI) was observed at day 14. For single
agent anti-PD1 treatment, 52% TGI was observed at day 14.
Combination treatment resulted in 85% TGI at day 14 and
demonstrated 40% complete regressions (CR) such that no measurable
tumor remained in 4 out of 10 mice by day 36. The anti-tumor
efficacy with either antibody delivered as monotherapy was 0-10%
CR.
Example 6
Humanization of Antibodies
[0406] The rat 14A6 and the mouse 31C6 antibody were humanized
using methods described in the specification. From the rat antibody
14A6, the following humanized variable heavy chains were
constructed: SEQ ID NOs: 9-24 and SEQ ID NOs: 37-47; and the
following humanized variable light chains were constructed: SEQ ID
NOs: 25-30 and SEQ ID NOs: 48-52. From mouse antibody 31C6, the
following humanized variable heavy chains were constructed: SEQ ID
NOs: 124-129; and the following humanized variable light chains
were constructed: SEQ ID Nos: 130-133.
Example 7
Effect of Fc Isotype on the Anti-Tumor Activity of Anti-TIGIT
Antibodies in Animal Tumor Model
[0407] Mice:
[0408] Approximately seven to eight week old female BALB/cAnN mice
with an average body weight of 20 grams were obtained from Taconic
Laboratory (Germantown, N.Y.). Conventional animal chow and water
were provided ad libitum.
[0409] Antibody Reagents: [0410] Murine anti-mouse PD1
antibody--IgG1 subtype [0411] Rat anti-mouse TIGIT antibody
(18G10)--rat IgG1 subtype. This antibody is described in the
Figures as 18G10 parental. The 18G10 antibody has the VH sequence
of SEQ ID NO:136 and the VL sequence of SEQ ID NO:137. [0412]
Chimeric rat anti-mouse TIGIT antibody (18G10)--comprising a mouse
Fc region of mIgG1 subtype. This antibody is described in the
Figures as 18G10-G2a. (Murine isotype IgG1 is the murine
counterpart isotype to human isotype IgG4.) [0413] Chimeric rat
anti-mouse 18G10 TIGIT antibody--comprising a mouse Fc region of
mIgG2a subtype. This antibody is described in the Figures as
18G10-G2a. (Murine isotype IgG2a is the murine counterpart isotype
to human isotype IgG1.) [0414] Isotype control murine IgG1 antibody
(mouse IgG1 isotype-matched control monoclonal antibody specific
for adenoviral hexon 25)) [0415] Isotype control rat IgG1 antibody
(rat IgG1 isotype-matched control monoclonocal antibody specific
for human IL-4) [0416] Isotype control murine IgG2a antibody (mouse
IgG2a isotype-matched control monoclonal antibody specific for
adenoviral hexon 25).
[0417] Formulations of Antibody Reagents:
[0418] The formulations were as follows: [0419] mIgG1:75 mM NaCl,
10 mM sodium phosphate, 3% sucrose, pH7.4; [0420] anti-PD-1: 20 mM
sodium acetate, 9% sucrose, pH5.5; mIgG2a: 75 mM NaCl, 10 mM sodium
phosphate, 3% sucrose, pH7.3; [0421] rat IgG1: 20 mM sodium
acetate, 7% sucrose pH 5.5; 18G10: 20 mM NaAc, 100 mM NaCl, 3%
sucrose; 18G10-G1: 20 mM NaAc, 9% sucrose, pH 5.5; 18G10-G2a: 20 mM
NaAc, 9% sucrose, pH 5.5.
[0422] Tumor Cell Line Preparation and Implant:
[0423] CT26 colon carcinoma cells were cultured in RPMI medium
supplemented with 10% heat-inactivated fetal bovine serum.
3.times.10.sup.5 log-phase and sub-confluent CT26 cells were
injected subcutaneously (SC) in a 100 .mu.L volume of serum-free
RPMI in the right lower dorsal flank of each mouse. Mice were first
shaved with electronic clippers in the area that would be used for
the implant.
[0424] CT-26 is a murine colorectal adenocarcinoma cell line
syngeneic to the BALB/c mouse strain. CT-26 is a relevant model
system for evaluating the mechanism of action for an anti-PD-1
antibody because of the translatable molecular profile of this
tumor post-anti-PD-1 therapy.
[0425] Tumor Measurements and Body Weights:
[0426] Tumors were measured the day before the first dose and twice
a week thereafter. Tumor length and width were measured using
electronic calipers and tumor volume determined using the formula
Volume (mm.sup.3)=0.5.times.Length.times.Width where length is the
longer dimension. Mice were weighed periodically to monitor general
health but also to estimate actual mg/kg dose delivery per mouse
where needed. Before treatment, mice were weighed and tumors from
individual mice were measured. To prevent bias, any outliers by
weight or tumor volume were removed and the remaining mice
randomized into various treatment groups with equivalent mean tumor
size.
[0427] Dosing Solution Preparation, Administration, and
Analyses:
[0428] Frozen stocks of the antibodies to be tested in the animal
model were thawed and transferred to wet ice. To avoid repeated
freeze thaw, each vial of stock was thawed once and aliquots made
in volumes sufficient for one time use. Polypropylene, low adhesion
tubes were used for this purpose. The aliquots were snap frozen in
dry ice and stored at -80.degree. C. Before each dosing, one
aliquot was thawed and diluted to nominal concentration in the
appropriate diluent and dosed immediately. Aliquots of dosing
solutions were snap frozen in dry ice and stored at -80.degree. C.
until analyses. Dosing solutions were assessed using the Meso Scale
Discovery (MSD.RTM., Rockville, Md.) platform which is based on
multi-array technology; a combination of electrochemiluminescence
detection and patterned arrays.
[0429] Dosing Anti-TIGIT/Anti-PD1 Treatment Results:
[0430] CT26 tumor-bearing BALB/cAnN mice were randomized into 10
treatment groups when the mean tumor volume of these mice reached a
tumor size average of 100 mm3 (80 mm3-119 mm3): (1) muIgG1 isotype
control+rat IgG1 isotype control; (2) muIgG1 isotype
control+muIgG2a isotype control; (3) muDX400+rat IgG1 isotype
control; (4) muDX400+muIgG2a isotype control; (5) muIgG1 isotype
control+18G10; (6) muIgG1 isotype control+18G10-G1; (7) muIgG1
isotype control+18G10-G2a; (8) muDX400+18G10; (9) muDX400+18G10_G1;
(10) muDX400+18G10_G2a. Animals were administered rat anti-mouse
TIGIT (18G10) or chimeric anti-TIGIT antibodies 18G10-G1 or
18G10-G2a (described above) at a 18 mg/kg dose, IP, every 4 days
for each of 6 cycles. An anti-mouse PD-1 (described above) was
administered at a 10 mg/kg dose, IP, every 4 days for each of 6
cycles. Post dosing, animals continued to be monitored and tumor
volumes were measured out to 76 days for some treatment groups.
Tumors were measured twice weekly. The results are shown in FIGS.
10 and 11. Single agent anti-TIGIT treatment using 18G10-G2a showed
44% tumor growth inhibition (TGI) at day 13; while 18G10 parental
antibody showed 38% and the 18G10-G1 antibody showed 36%. For
single agent anti-PD1 treatment combined with isotype control
rIgG1, 51% TGI was observed at day 13. When anti-PD-1 was combined
with isotype control muIgG2a, 50% TGI and 10% complete regressions
(CR) were observed at day 18, such that 1 complete response out of
10 animals was observed. The combination of anti-PD1 and parental
18G10 showed 80% TGI at day 13 and demonstrated 300% CR by day 39.
The combination of anti-PD1 and 18G10-G1 showed 59% TGI at day 13
and demonstrated 10% CR by day 13. The combination of anti-PD1 and
18G10-G2a showed 88% TGI at day 13 and demonstrated 60% CR at day
63. The combination of anti-PD1 and 18G10-G2a showed greater
anti-tumor activity and more complete regressions compared to
combinations of anti-PD1 with 18G10 parental or 18G10-G1. There was
no significant body weight loss associated with administration of
single agents or combination therapy indicating that treatments
were well tolerated.
Example 8
Effect of Fc Isotype on the Anti-Tumor Activity of Anti-TIGIT
Antibodies in Animal Tumor Model
[0431] The Experiment described in Example 7 was repeated, except
that the rat 18G10 antibody was substituted with rat antibody
11A11.
[0432] Mice:
[0433] Approximately seven to eight week old female BALB/cAnN mice
with an average body weight of 20 grams were obtained from Taconic
Laboratory (Germantown, N.Y.). Conventional animal chow and water
were provided ad libitum.
[0434] Antibody Reagents: [0435] Murine anti-mouse PD1
antibody--IgG1 subtype [0436] Rat anti-mouse TIGIT antibody
(11A11)--rat IgG1 subtype. This antibody is described in the
Figures as 11A11 parental. The 11A11 antibody has the VH sequence
of SEQ ID NO:138 and the VL sequence of SEQ ID NO:139. [0437]
Chimeric rat anti-mouse TIGIT antibody (11A11)--comprising a mouse
Fc region of mIgG1 subtype. This antibody is described in the
Figures as 11A11-G2a. (Murine isotype IgG1 is the murine
counterpart isotype to human isotype IgG4.) [0438] Chimeric rat
anti-mouse 11A11 TIGIT antibody--comprising a mouse Fc region of
mIgG2a subtype. This antibody is described in the Figures as
11A11-G2a. (Murine isotype IgG2a is the murine counterpart isotype
to human isotype IgG1.) [0439] Isotype control murine IgG1 antibody
(mouse IgG1 isotype-matched control monoclonal antibody specific
for adenoviral hexon 25)) [0440] Isotype control rat IgG1 antibody
(rat IgG1 isotype-matched control monoclonocal antibody specific
for human IL-4) [0441] Isotype control murine IgG2a antibody (mouse
IgG2a isotype-matched control monoclonal antibody specific for
adenoviral hexon 25).
[0442] Formulations of Antibody Reagents:
[0443] The formulations were as follows: [0444] mIgG1:75 mM NaCl,
10 mM sodium phosphate, 3% sucrose, pH7.4; [0445] anti-PD-1: 20 mM
sodium acetate, 9% sucrose, pH5.5; mIgG2a: 75 mM NaCl, 10 mM sodium
phosphate, 3% sucrose, pH7.3; [0446] rat IgG1: 20 mM sodium
acetate, 7% sucrose pH 5.5; 11A11: 20 mM NaAc, 100 mM NaCl, 3%
sucrose pH5.5; 11A11-G1: 20 mM NaAc, 9% sucrose, pH 5.5; 11A11-G2a:
20 mM NaAc, 9% sucrose, pH 5.5.
[0447] Tumor Cell Line Preparation and Implant:
[0448] CT26 colon carcinoma cells were cultured in RPMI medium
supplemented with 10% heat-inactivated fetal bovine serum.
3.times.10.sup.5 log-phase and sub-confluent CT26 cells were
injected subcutaneously (SC) in a 100 .mu.L volume of serum-free
RPMI in the right lower dorsal flank of each mouse. Mice were first
shaved with electronic clippers in the area that would be used for
the implant.
[0449] CT-26 is a murine colorectal adenocarcinoma cell line
syngeneic to the BALB/c mouse strain. CT-26 is a relevant model
system for evaluating the mechanism of action for an anti-PD-1
antibody because of the translatable molecular profile of this
tumor post-anti-PD-1 therapy.
[0450] Tumor Measurements and Body Weights:
[0451] Tumors were measured the day before the first dose and twice
a week thereafter. Tumor length and width were measured using
electronic calipers and tumor volume determined using the formula
Volume (mm.sup.3)=0.5.times.Length.times.Width where length is the
longer dimension. Mice were weighed periodically to monitor general
health but also to estimate actual mg/kg dose delivery per mouse
where needed. Before treatment, mice were weighed and tumors from
individual mice were measured. To prevent bias, any outliers by
weight or tumor volume were removed and the remaining mice
randomized into various treatment groups with equivalent mean tumor
size. When the mean tumor volume in the CT26 tumor-bearing mice
reached .about.100 mm.sup.3, around 7 days post implant, animals
were randomized into treatment groups of 10 mice per group and
dosing was started. Animals were administered at the dosing
concentrations specified below.
[0452] Dosing Solution Preparation, Administration, and
Analyses:
[0453] Frozen stocks of the antibodies to be tested in the animal
model were thawed and transferred to wet ice. To avoid repeated
freeze thaw, each vial of stock was thawed once and aliquots made
in volumes sufficient for one time use. Polypropylene, low adhesion
tubes were used for this purpose. The aliquots were snap frozen in
dry ice and stored at -80.degree. C. Before each dosing, one
aliquot was thawed and diluted to nominal concentration in the
appropriate diluent and dosed immediately. Aliquots of dosing
solutions were snap frozen in dry ice and stored at -80.degree. C.
until analyses. Dosing solutions were assessed using the Meso Scale
Discovery (MSD.RTM., Rockville, Md.) platform which is based on
multi-array technology; a combination of electrochemiluminescence
detection and patterned arrays.
[0454] Dosing Anti-TIGIT/Anti-PD1 Treatment Results:
[0455] CT26 tumor-bearing BALB/cAnN mice were randomized into 10
treatment groups when the mean tumor volume of these mice reached a
tumor size average of 100 mm.sup.3 (75 mm.sup.3-115 mm.sup.3): (1)
muIgG1 isotype control+rat IgG1 isotype control; (2) muIgG1 isotype
control+muIgG2a isotype control; (3) muDX400+rat IgG1 isotype
control; (4) muDX400+muIgG2a isotype control; (5) muIgG1 isotype
control+11A11; (6) muIgG1 isotype control+11A11-G1; (7) muIgG1
isotype control+11A11-G2a; (8) muDX400+11A11; (9) muDX400+11A11_G1;
(10) muDX400+11A11_G2a. Animals were administered rat anti-mouse
TIGIT (11A11) or chimeric anti-TIGIT antibodies 11A11-G1 or
11A11-G2a (described above) at a 20 mg/kg dose, IP, every 4 days
for each of 6 cycles. An anti-mouse PD-1 (described above) was
administered at a 10 mg/kg dose, IP, every 4 days for each of 6
cycles. Post dosing, animals continued to be monitored and tumor
volumes were measured out to 54 days for some treatment groups.
Tumors were measured twice weekly. The results are shown in FIGS.
12 and 13. Single agent anti-TIGIT treatment using 11A11-G2a showed
52% tumor growth inhibition (TGI) at day 14; while little to no
activity was observed using the 11A11 parental antibody or the
11A11-G1 antibody. For single agent anti-PD1 treatment, 40-50% TGI
was observed at day 14. When combined with isotype control rat
IgG1, anti-PD-1 showed 10% complete regressions (CR) by day 28,
such that 1 complete response out of 10 animals was observed. The
combination of anti-PD1 and parental 11A11 showed 60% TGI at day 14
and demonstrated 30% CR by day 54. The combination of anti-PD1 and
11A11-G1 showed 56% TGI at day 14 and demonstrated 30% CR by day
25. The combination of anti-PD1 and 11A11-G2a showed 94% TGI at day
14 and demonstrated 70% CR at day 42. The combination of anti-PD1
and 11A11-G2a showed greater anti-tumor activity and more complete
regressions compared to combinations of anti-PD1 with 11A11
parental or 11A11-G1. There was no significant body weight loss
associated with administration of single agents or combination
therapy indicating that treatments were well tolerated.
Example 9
Epitope Mapping of hTIGIT 14A6 Antibody by Hydrogen Deuterium
Exchange Mass Spectrometry
[0456] The contact areas between anti-TIGIT antibody 14A6 and human
TIGIT were determined by use of hydrogen deuterium exchange mass
spectrometry (HDX-MS) analysis. HDX-MS measures the exchange of
deuterium with hydrogen into the amide backbone of the protein. The
exchange rate is influenced by the hydrogen's exposure to solvent.
Comparison of the exchange levels in the antigen when the antibody
is bound can identify regions of the protein where the antibody is
binding.
Materials
[0457] Human TIGIT-His--Comprising the extracellular domain of
hTIGIT (residues 25-150 of SEQ ID NO:31) and a histidine tag (SEQ
ID NO:87). [0458] Rat anti-hTIGIT 14A6 antibody (comprising the
VH/VL sequences of SEQ ID NOs:7/8 (lot#78AGU) and a ratIgG2a Fc
region) (Rat.times.[TIGIT_H] mAb (LB155.14A6.G2.A8) IgG2a/Kappa
(HY)).
Liquid Chromatography-Mass Spectrometry
[0459] The mass spectrometer was a Thermo Scientific
Orbitrap-Velos. For the measurement of deuterium labeled samples,
the mass spectrometer was set to acquire one full scan MS data in
the orbitrap at 60,000 resolving power, a target ion count of 1E6,
a maximum ion injection time of 500 milliseconds and two
microscans. For the acquisition of MS/MS data for peptide
identifications, the mass spectrometer was set to acquire one full
scan spectrum at 60,000 resolving power followed by ten
data-dependent MS/MS spectra in the ion trap.
[0460] The liquid chromatography system was a Waters.RTM.
nanoACQUITY for the analytical column gradient and a Waters.RTM.
515 isocratic pump for the sample digestion and loading. For sample
digestion and loading, the buffer used was 2% acetonitrile and
0.05% trifluoroacetic acid at a flow rate of 80 ul/min. For the
analytical gradient, the buffers were Buffer A) 0.1% formic acid in
water and Buffer B) 0.1% formic acid in acetonitrile.
[0461] The gradient was at 40 ul/min from 2% B to 36% B in 10
minutes, followed by a wash of 80% B for 2 minutes and a
reequilibration at 2% B for 3 minutes. The column was then washed
by cycling the gradient between 2% and 80% B, three times with 1
minute at each step, followed by a final equilibration at 2% B for
5 minutes. The trapping column was a Waters.RTM. Vanguard C18 BEH
1.7 um Guard Column and the analytical column was a Waters.RTM. C18
BEH300, 1.7 um 1.times.50 mm column.
[0462] Sample handling for the deuterium labeling was done by a
Leaptec H/D-X PAL system. The labeling sample tray was set to a
temperature of 25.degree. C., the quenching tray was set to
1.5.degree. C. and the trap and analytical column chamber was set
to 1.5.degree. C. The immobilized pepsin column (Porosyme
Immobilized Pepsin 2.1.times.30 mmm, Life Technologies) was kept
outside the column chamber at room temperature.
Deuterium Labeling
[0463] hTIGIT-His (30 pmol/.mu.l) was mixed with an equal volume of
the antibody (14 pmol/.mu.l) or, in the unbound control, PBS pH
7.6. The antibody bound samples and the unbound control were
incubated at room temperature for 1 hour before beginning the
labeling experiment.
[0464] To deuterium label the samples, 2 .mu.l of sample was mixed
with 25 .mu.l of PBS in dueterium oxide pD 7.6. Labeling time
points were 30, 300, 1500, 4500 or 9000 seconds. After the set
time, 25 .mu.l of the labeling mixture was added to 35 .mu.l of
cold quench buffer (8M Urea, 100 mM TCEP). The quenched sample was
incubated at 1.5.degree. C. for one minute. 55 .mu.l was then
injected into the column cooling chamber where the sample was
passed over the pepsin column and the resulting peptides loaded
onto the trapping column. After three minutes, a valve switch took
the pepsin column out of line and the trap was washed at additional
one minute. After that the trap was switched inline with the
analytical column and the analytical gradient and the mass
spectometer were started.
[0465] A fully deuterated sample was generated by incubating 2
.mu.l of hTIGIT with 108 .mu.l of deuterated denaturing buffer (4M
Urea, 100 mM TCEP, 0.01% DDM in 99.5% deueterium oxide). The sample
was incubated at room temperature overnight. 55 .mu.l was then
directly injected into the column chamber and the data acquired as
before.
Data Analysis
[0466] LC-MS/MS data was acquired of an unlabeled sample and
searched before deuterium labeling to verify successful digestion
of the proteins and to generate a list of peptides from pepsin
digestion. Data was database searched using Proteome Discoverer 1.4
and the SEQUEST HT search algorithm (ThermoFisher Scientific). The
protein database used was the human TIGIT-His and anti-hTIGIT
antibody sequences concatenated to the yeast Saccharomycese
cerevisiae uniprot (5/20/13) database.
[0467] MS data from the deuterium labeling experience was processed
by HDExaminer (version 1.3, Sierra Analytics). The mass and
retention time selected by the software for each peptide was
verified manually.
Results
[0468] The human TIGIT peptides protected by bound 14A6 antibody
are illustrated in the heatmap of FIG. 14 and correspond to amino
acid residues 54-57, 68-70 and 76-81 of SEQ ID NO:31. (These same
amino acids are shown as residues 30-33, 44-46 and 52-57 of SEQ ID
NO:87.)
Example 10
Epitope Mapping of hTIGIT 31C6 Antibody by Hydrogen Deuterium
Exchange Mass Spectrometry
[0469] The contact areas between anti-TIGIT antibody 31C6 and human
TIGIT were determined by use of hydrogen deuterium exchange mass
spectrometry (HDX-MS) analysis. HDX-MS measures the exchange of
deuterium with hydrogen into the amide backbone of the protein. One
factor influencing the exchange rate is the hydrogen's exposure to
solvent. Comparison of the exchange levels in the antigen when the
antibody is bound can identify regions of the protein where the
antibody is binding.
Materials
[0470] Human TIGIT-His--Comprising the extracellular domain of
hTIGIT (residues 25-145 of SEQ ID NO:31) and a histidine tag (SEQ
ID NO:87). [0471] Mouse anti-hTIGIT 31C6 antibody (lot#41AHK)
(Mouse.times.[TIGIT_H] mAb (MEB125.31C6.A1.205) IgG1/Kappa
(HY))
Liquid Chromatography-Mass Spectrometry
[0472] The mass spectrometer was a Thermo Scientific
Orbitrap-Elite. For the measurement of deuterium labeled samples,
the mass spectrometer was set to acquire one full scan MS data in
the orbitrap at 120,000 resolving power, a target ion count of 1E6,
a maximum ion injection time of 500 milliseconds and two
microscans. For the acquisition of MS/MS data for peptide
identifications, the mass spectrometer was set to acquire one full
scan spectrum at 120,000 resolving power followed by ten
data-dependent MS/MS spectra in the ion trap.
[0473] The liquid chromatography system was a Waters nanoAcquity
for the analytical column gradient and a Waters 515 isocratic pump
for the sample digestion and loading. For sample digestion and
loading, the buffer used was 2% acetonitrile and 0.05%
trifluoroacetic acid at a flow rate of 80 ul/min. For the
analytical gradient, the buffers were Buffer A) 0.1% formic acid in
water and Buffer B) 0.1% formic acid in acetonitrile.
[0474] The gradient was at 40 ul/min from 2% B to 36% B in 10
minutes, followed by a wash of 80% B for 2 minutes and a
re-equilibration at 2% B for 3 minutes. The column was then washed
by cycling the gradient between 2% and 80% B, three times with 1
minute at each step, followed by a final equilibration at 2% B for
5 minutes. The trapping column was a Waters Vanguard CSH C18 1.7 um
Guard Column and the analytical column was a Waters CSH C18, 1.7 um
1.times.50 mm column.
[0475] Sample handling for the deuterium labeling was done by a
Leaptec H/D-X PAL system. The labeling sample tray was set to a
temperature of 25.degree. C., the quenching tray was set to
1.5.degree. C. and the trap and analytical column chamber was set
to 1.5.degree. C. The immobilized pepsin column (Enzymate BEH
Pepsin, Waters corporation) was kept outside the column chamber at
room temperature.
Deuterium Labeling
[0476] hTIGIT-His (63 pmol/ul) was mixed with an equal volume of
the antibody (32 pmol/ul) or, in the unbound control, PBS pH 7.6.
The antibody bound samples and the unbound control were incubated
at room temperature for 1 hour before beginning the labeling
experiment.
[0477] To deuterium label the samples, 2 .mu.l of sample was mixed
with 25 .mu.l of PBS in deuterium oxide pH 7.6. Labeling time
points were 30, 300, 1500, 4500, 9000 and 13500 seconds. After the
set time, 25 .mu.l of the labeling mixture was added to 35 .mu.l of
cold quench buffer (8M Urea, 100 mM TCEP). The quenched sample was
incubated at 1.5.degree. C. for one minute. 55 .mu.l was then
injected into the column cooling chamber where the sample was
passed over the pepsin column and the resulting peptides loaded
onto the trapping column. After three minutes, a valve switch took
the pepsin column out of line and the trap was washed at additional
one minute. The trap was then switched in-line with the analytical
column and the analytical gradient and the mass spectrometer data
acquisition was started. Each time point was acquired in triplicate
in randomized order.
[0478] A fully deuterated sample was generated by incubating 2
.mu.l of hTIGIT (63 pmol/ul) with 108 .mu.l of deuterated
denaturing buffer (4M Urea, 100 mM TCEP, 0.01% DDM in 99.5%
deuterium oxide). The sample was incubated at room temperature
overnight. 55 .mu.l was then directly injected into the column
chamber and the data acquired as before.
Data Analysis
[0479] LC-MS/MS data was acquired of an unlabeled sample and
database searched to verify successful digestion of the proteins
and to generate a list of peptides from the pepsin digestion.
Database search was done using Proteome Discoverer 1.4 and the
SEQUEST HT search algorithm (ThermoFisher Scientific). The protein
database used was the human TIGIT-His and anti-hTIGIT antibody
sequences concatenated to the yeast Saccharomyces cerevisiae
uniprot (5/20/13) database.
[0480] MS data from the deuterium labeling experience was processed
by HDExaminer (version 1.3, Sierra Analytics). The mass and
retention time selected by the software for each peptide was
verified manually.
Results
[0481] The human TIGIT peptides protected by bound 31C6 antibody
are illustrated in the heatmap of FIG. 15 and correspond to amino
acid residues 53-57, 60-65, 68-70, 72-81, 94-95, 109-119 of SEQ ID
NO:31. (These same amino acids are shown as residues 29-33, 36-41,
44-46, 48-57, 70-71, 85-95 of SEQ ID NO:87.)
Example 11
In Vitro T-Cell Activity Assay for Humanized Anti-hTIGIT
Antibodies
[0482] We further analyzed the activity of various humanized
variants of one of the antibodies of the invention (31C6). In one
assay we characterized the functional consequence of blocking human
TIGIT receptor using hTIGIT-Jurkat cells. In this assay the
hTIGIT-Jurkat were co-cultured with JY cells engineered to express
human CD155 (hCD155-JY). The JY cell line used is an Epstein-Barr
virus (EBV) immortalized B cell lymphoblastoid cell line. As in the
assay described above in Example 3, when the hTIGIT Jurkat cells
are stimulated with plate bound .alpha.-CD3 and co-cultured with
parental JY cells (not expressing human CD155), they produce IL-2.
However, when the hTIGIT-Jurkat are co-cultured with hCD155-JY,
IL-2 levels were reduced in a ligand dependent manner. Treatment
with anti-hTIGIT antibodies rescues IL-2 production in this assay
in a dose-dependent fashion.
[0483] In this assay 96-well flat-bottom plates were coated with
mouse anti-human CD3 antibody (1 .mu.g/ml in PBS; Clone HIT3a; BD
Pharmingen Cat#555336) overnight at 4.degree. C. The next day,
hTIGIT expressing Jurkat cells (50,000) were plated in the
pre-coated plates and pre-incubated for 30-60 minutes with mAb at
varying concentrations. Human CD155 expressing JY cells (50,000)
were added to the culture. After incubation for 18-24 h at
37.degree. C. and 5.0% CO2, IL-2 levels were assessed in culture
supernatants by mesoscale (Human IL-2 Tissue Culture MESO Kit:
Cat#K151AHB-2). The results are shown in FIG. 16. A titration of
the parental mouse anti-hTIGIT clone 31C6 (MEB125.31C6.A1.205
mIgG1) from 30 .mu.g/ml down to 0.04 .mu.g/ml gives an EC.sub.50 of
0.730 .mu.g/ml, and the same titration of the mouse human 31C6
chimera (Mouse human chimera MEB125.31C6.A1.205 IgG1) gives an EC50
of 0.910 .mu.g/ml. (The mouse human 31C6 chimera comprised the
variable regions of the parental 31C6 clone (SEQ ID Nos: 94 and 95)
and a human IgG1 region.) Similarly, a titration of the 31C6
humanized variants from 30 .mu.g/ml down to 0.04 .mu.g/ml gives the
following EC.sub.50s:
TABLE-US-00004 Humanized Variant EC.sub.50 MEB125.31C6.A1.205
VH4/VL1 (An antibody 0.620 .mu.g/ml comprising the VH of SEQ ID NO:
127 and VL of SEQ ID NO: 130, and a human IgG1 Fc region)
MEB125.31C6.A1.205 VH5/VL4 (An antibody 1.2 .mu.g/ml comprising the
VH of SEQ ID NO: 128 and VL of SEQ ID NO: 133, and a human IgG1 Fc
region) MEB125.31C6.A1.205 VH5/VL3 (An antibody 1.2 .mu.g/ml
comprising the VH of SEQ ID NO: 128 and VL of SEQ ID NO: 132, and a
human IgG1 Fc region)
[0484] We also used a primary cell-based assay to demonstrate that
the humanized anti-hTIGIT antibodies had activity in primary cells.
Several lots of human peripheral blood mononuclear cells (PBMCs)
were screened for TIGIT expression after stimulation (mixed
lymphocyte reaction stimulation and .alpha.-CD3 stimulation). PBMCs
were then chosen for primary cell-based assays based on their TIGIT
expression after stimulation. HuCD155-Fc was coated onto tissue
culture plates and PBMCs were stimulated with anti-CD3.
[0485] In this assay 96-well high binding plates (Corning 3361)
were coated with human CD155-Fc (in-house generated, 1 ug/ml in
PBS) overnight at 4.degree. C. The next day, 50,000 human PBMC
(Precision Bioservice Cat#83000C-1.0, lot#12920 in RPMI+10% human
serum Bio-world cat#30611043-1, lot#V15022401, RBCs were removed by
BD Pharmlyse BD cat#555899) were plated in the pre-coated plates
and pre-incubated for 30-60 minutes with anti-TIGIT mAbs at varying
concentrations. Anti-CD3 antibody (eBioscience 16-0037-85) at final
concentration 1 ug/ml was then added. After incubation for 48 h at
37.degree. C. and 5.0% CO2, Proinflammatory cytokines (IFN.gamma.,
IL1.beta., IL6 and TNF.alpha.) were assessed in culture
supernatants by mesoscale assay (Human Proinflammaatory-4 I tissue
culture MESO Kit: Cat#K15009B-4). As shown in FIG. 17, humanized
variants of 31C6 were able to stimulate IL-6, TNF.alpha. and
IFN.gamma. in a dose dependent manner similar to the mouse human
31C6 chimeric antibody.
The labels in FIG. 17 correspond to the following antibodies:
[0486] Mouse human chimera MEB125.31C6.A1.205 IgG1 corresponds to
an antibody comprising the variable regions of the parental 31C6
clone (SEQ ID Nos: 94 and 95) and a human IgG1 region.) [0487]
MEB125.31C6.A1.205 VH4/VL1 corresponds to an antibody comprising
the VH of SEQ ID NO: 127 and VL of SEQ ID NO:130, and a human IgG1
Fc region) [0488] MEB125.31C6.A1.205 VH5/VL4 corresponds to an
antibody comprising the VH of SEQ ID NO: 128 and VL of SEQ ID
NO:133, and a human IgG1 Fc region) [0489] MEB125.31C6.A1.205
VH5/VL3 corresponds to an antibody comprising the VH of SEQ ID NO:
128 and VL of SEQ ID NO:133, and a human IgG1 Fc region) All
references cited herein are incorporated by reference to the same
extent as if each individual publication, database entry (e.g.
Genbank sequences or GeneID entries), patent application, or
patent, was specifically and individually indicated to be
incorporated by reference. This statement of incorporation by
reference is intended by Applicants, pursuant to 37 C.F.R.
.sctn.1.57(b)(1), to relate to each and every individual
publication, database entry (e.g. Genbank sequences or GeneID
entries), patent application, or patent, each of which is clearly
identified in compliance with 37 C.F.R. .sctn.1.57(b)(2), even if
such citation is not immediately adjacent to a dedicated statement
of incorporation by reference. The inclusion of dedicated
statements of incorporation by reference, if any, within the
specification does not in any way weaken this general statement of
incorporation by reference. Citation of the references herein is
not intended as an admission that the reference is pertinent prior
art, nor does it constitute any admission as to the contents or
date of these publications or documents.
[0490] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0491] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. Various modifications of the invention in addition to
those shown and described herein will become apparent to those
skilled in the art from the foregoing description and fall within
the scope of the appended claims.
TABLE-US-00005 TABLE 4 Sequence Information SEQ ID Description NO:
SEQUENCE 14A6 H-CDR1 1 SDYWG 14A6 H-CDR2 2 FITYSGSTSYNPSLKS 14A6
H-CDR3 3 MPSFITLASLSTWEGYFDF 14A6 L-CDR1 4 KASQSIHKNLA 14A6 L-CDR2
5 YANSLQT 14A6 L-CDR3 6 QQYYSGWT 14A6 PARENTAL 7
EVQLQESGPGLVKPSQSLSLTCSVTGSSIASDYWGWIRKFPGNKMEWMGFI VH
TYSGSTSYNPSLKSRISITRDTSKNQFFLQLHSVTTDDTATYSCARMPSFI
TLASLSTWEGYFDFWGPGTMVTVSS 14A6 PARENTAL 8
DIQMTQSPSLLSASVGDRVTLNCKASQSIHKNLAWYQQKLGEAPKFLIYYA VL
NSLQTGIPSRFSGSGSGTDFTLTISGLQPEDVATYFCQQYYSGWTFGGGTK VELK Hu14A6VH.1
9 EVQLQESGPGLVKPSETLSLTCTVSGGSISSDYWGWIRQPPGKGLEWIGFI
TYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.1a 10
EVQLQESGPGLVKPSETLSLTCTVSGGSISSDYWGWIRQPPGKGLEWIGFI
TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.1b 11
EVQLQESGPGLVKPSETLSLTCTVSGGSISSDYWGWIRQPPGKGLEWIGFI
TYSGSTSYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.1c 12
EVQLQESGPGLVKPSETLSLTCTVSGSSISSDYWGWIRQPPGKGLEWMGFI
TYSGSTSYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.1d 13
EVQLQESGPGLVKPSETLSLTCTVSGGSISSDYWGWIRQPPGKGLEWIGFI
TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLHSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.1e 14
EVQLQESGPGLVKPSETLSLTCTVSGGSISSDYWGWIRQPPGKGLEWIGFI
TYSGSTSYNPSLKSRITISRDTSKNQFSLKLHSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.1f 15
EVQLQESGPGLVKPSETLSLTCTVSGSSISSDYWGWIRQPPGKGLEWIGFI
TYSGSTSYNPSLKSRITISRDTSKNQFSLKLHSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.1g 16
EVQLQESGPGLVKPSETLSLTCTVSGSSISSDYWGWIRQPPGKGLEWMGFI
TYSGSTSYNPSLKSRITISVDTSKNQFSLKLHSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.2 17
EVQLQESGPGLVKPSETLSLTCAVSGYSISSDYWGWIRQPPGKGLEWIGFI
TYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.2a 18
EVQLQESGPGLVKPSETLSLTCAVSGYSISSDYWGWIRQPPGKGLEWIGFI
TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.2b 19
EVQLQESGPGLVKPSETLSLTCAVSGYSISSDYWGWIRQPPGKGLEWIGFI
TYSGSTSYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.2c 20
EVQLQESGPGLVKPSETLSLTCAVSGSSISSDYWGWIRQPPGKGLEWMGFI
TYSGSTSYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.2d 21
EVQLQESGPGLVKPSETLSLTCAVSGYSISSDYWGWIRQPPGKGLEWIGFI
TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLHSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.2e 22
EVQLQESGPGLVKPSETLSLTCAVSGYSISSDYWGWIRQPPGKGLEWIGFI
TYSGSTSYNPSLKSRITISRDTSKNQFSLKLHSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.2f 23
EVQLQESGPGLVKPSETLSLTCAVSGSSISSDYWGWIRQPPGKGLEWIGFI
TYSGSTSYNPSLKSRITISRDTSKNQFSLKLHSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6VH.2e 24
EVQLQESGPGLVKPSETLSLTCAVSGSSISSDYWGWIRQPPGKGLEWMGFI
TYSGSTSYNPSLKSRITISRDTSKNQFSLKLHSVTAADTAVYYCARMPSFI
TLASLSTWEGYFDFWGQGTMVTVSS Hu14A6Vk.1 25
DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKLLIYYA
NSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTK VEIK
Hu14A6Vk.1a 26 DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKFLIYYA
NSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTK VEIK
Hu14A6Vk.1b 27 DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKFLIYYA
NSLQTGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTK VEIK Hu14A6Vk.2
28 DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKVPKLLIYYA
NSLQTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYYSGWTFGGGTK VEIK
Hu14A6Vk.2a 29 DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKVPKFLIYYA
NSLQTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYYSGWTFGGGTK VEIK
Hu14A6Vk.2b 30 DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKVPKFLIYYA
NSLQTGIPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYYSGWTFGGGTK VEIK Human
TIGIT 31 mrwcllliwa gglrqaplas gmmtgtiett gnisaekggs iilqchlsst
taqvtqvnwe qqdqllaicn adlgwhisps fkdrvapgpg lgltlqsltv ndtgeyfciy
htypdgtytg riflevless vaehgarfqi pllgamaatl vvictavivv valtrkkkal
rihsvegdlr rksagqeews psapsppgsc vqaeaapagl cgeqrgedca elhdyfnvls
yrslgncsff tetg Cyno/Rhesus 32 mrwclfliwa gglrqaplas gmmtgtiett
gnisakkggs TIGIT vilqchlsst maqvtqvnwe qhdhsllair naelgwhiyp
afkdrvapgp glgltlqslt mndtgeyfct yhtypdgtyr griflevles svaehsarfq
ipllgamamm lvviciaviv vvvlarkkks lrihsvesgl grkstgqeeq ipsapsppgs
cvqaeaapag lcgeqqgddc aelhdyfnvl syrslgscsf ftetg Pembrolizumab 33
QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGI Heavy chain
NPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYR
FDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN
VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Pembrolizumab 34
EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLL Light chain
IYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTF
GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC
Nivolumab 35 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVI
heavy chain WYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDY
WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN
TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS
RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Nivolumab 36
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDA light chain
SNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGT
KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC
16AHA_tigit_ 37 EVQLQESGPGLVKPSETLSLTCTVSGSSIASDYWGWIRQPPGKGLEWIGFI
14a6_humanized_ TYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMPSFI
VH1 TLASLSTWEGYFDFWGQGTMVTVSSAS LB155.14A6.G2. A8_VH1 18AHA_tigit_
38 EVQLQESGPGLVKPSETLSLICTVSGSSIASDYWGWIRQPPGKGLEWIGFI
14a6_humanized_ TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI
VH2 TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH2 20AHA_tigit_ 39
EVQLQESGPGLVKPSETLSLICTVSGSSIASDYWGWIRKPPGKGLEWIGFI 14a6_humanized_
TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH3
TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH3 21AHA_tigit_ 40
EVQLQESGPGLVKPSETLSLICTVSGSSIASDYWGWIRQPPGKKLEWIGFI 14a6_humanized_
TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH4
TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH4 19AHA_tigit_ 41
EVQLQESGPGLVKPSETLSLICTVSGSSIASDYWGWIRQPPGKGMEWIGFI 14a6_humanized_
TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH5
TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH5 22AHA_tigit_ 42
EVQLQESGPGLVKPSETLSLICTVSGSSIASDYWGWIRKPPGKKMEWIGFI 14a6_humanized_
TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH6
TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2 .A8_VH6 23AHA_tigit_ 43
EVQLQESGPGLVKPSETLSLICTVSGSSIASDYWGWIRQFPGKGLEWIGFI 14a6_humanized_
TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTADDTAVYYCARMPSFI VH7
TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH7 24AHA_tigit_ 44
EVQLQESGPGLVKPSETLSLICTVSGSSIASDYWGWIRKPPGKKMEWIGFI 14a6_humanized_
TYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH8
TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH8 25AHA_tigit_ 45
EVQLQESGPGLVKPSETLSLICSVIGSSIASDYWGWIRQPPGKGLEWIGFI 14a6_humanized_
TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH9
TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH9 26AHA_tigit_ 46
EVQLQQSGAGLLKPSETLSLICSVIGSSIASDYWGWIRQPPGKGLEWIGFI 14a6_humanized_
TYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH10
TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH10 27AHA_tigit_ 47
EVQLQESGPGLVKPPGTLSLTCSVTGSSIASDYWGWVRQPPGKGLEWIGFI 14a6_humanized_
TYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH11
TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH11 09AHA_tigit_ 48
DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKLLIYYA 14a6_humanized_
NSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTK VL1 VEIK
LB155.14A6.G2. A8_VL1 11AHA_tigit_ 49
DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKFLIYYA 14a6_humanized_
NSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTK VL2 VEIK
LB155.14A6.G2. A8_VL2 12AHA_tigit_ 50
DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKLLIYYA 14a6_humanized_
NSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYYSGWTFGGGTK VL3 VEIK
LB155.14A6.G2. A8_VL3 13AHA_tigit_ 51
DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKFLIYYA 14a6_humanized_
NSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYYSGWTFGGGTK VL4 VEIK
LB155.14A6.G2. A8_VL4
15AHA_tigit_ 52 DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKLLIYYA
14a6_humanized_ NSLQTGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTK
VL5 VEIK LB155.14A6.G2. A8_VL5 Leader 53 MEWSWVFLFFLSVTTGVHS
sequence heavy chains Leader 54 MSVPTQVLGLLLLWLTDARC sequence light
chains Heavy chain 55
TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF constant
PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP domain-
PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF IgG4
NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK S228P
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM
HEALHNHYTQKSLSLSLGK Kappa light 56
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ chain
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR constant GEC
domain 28H5 H-CDR1 57 GYSITSDYAWN 28H5 H-CDR2 58 YISNSGSASYNPSLKS
28H5 H-CDR3 59 LIYYDYGGAMNF 28H5 L-CDR1 60 KASQGVSTTVA 28H5 L-CDR2
61 SASYRYT 28H5 L-CDR3 62 QHYYSTPWT 28H5 PARENTAL 63
DVQLQESGPGLVKPSQSLSLICTVTGYSITSDYAWNWIRQFPGNKLEWMGY VH
ISNSGSASYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCATL IYY
DYGGAMNFWGQGTSVTVSS 28H5 PARENTAL 64
DIVMTQSHKFMSTSVGDRVSITCKASQGVSTIVAWYQQKPGQSPKLLIYSA VL
SYRYTGVPDRFTGSGSGTDFTFTISSVQSEDLAVYYCQHYYSTPWTFGGGT KLEIK 14H6
L-CDR2 65 YASNLQT variant 14H6 L-CDR2 65 YASSLQT variant 14H6
L-CDR2 67 YASTLQT variant 14H6 L-CDR2 68 YATTLQT variant 14H6
L-CDR2 69 YASYLQT variant 14H6 L-CDR2 70 YANQLQT variant 14H6
L-CDR2 71 YAGSLQT variant 14H6 L-CDR2 72 YASQLQT variant 14H6
L-CDR2 73 YADSLQT variant 14H6 L-CDR3 74 QQYYSGFT variant 14H6
L-CDR3 75 QQYYSGYT variant 14H6 L-CDR3 76 QQYYSGIT variant 14H6
L-CDR3 77 QQYYSGVT variant 14H6 L-CDR3 78 QQYYSGLT variant 14H6
H-CDR3 79 MPSFITLASLSTFEGYFDF variant 14H6 H-CDR3 80
MPSFITLASLSTYEGYFDF variant 14H6 H-CDR3 81 MPSFITLASLSTIEGYFDF
variant 14H6 H-CDR3 82 MPSFITLASLSTVEGYFDF variant 14H6 H-CDR3 83
MPSFITLASLSTLEGYFDF variant Nucleic acid 84
GATGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCAC
encoding 28H5
CTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGATCCGACAGTTTC
PARENTAL VH
CAGGAAACAAACTGGAGTGGATGGGCTACATAAGCAACAGTGGTAGCGCTAGCTACAACCCA
TCTCTCAAAAGTCGCATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTT
GAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAACCCTGATCTACTATGATT
ACGGGGGGGCTATGAACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA Nucleic acid
85 GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCAT
encoding 28H5
CACCTGCAAGGCCAGTCAGGGTGTGAGTACTACTGTGGCCTGGTATCAACAGAAACCAGGAC
PARENTAL VL
AATCTCCTAAACTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTC
ACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGTCTGAAGACCT
GGCAGTTTATTACTGTCAGCATTATTATAGTACTCCGTGGACGTTCGGTGGAGGCACCAAGC
TGGAAATCAAA Heavy chain 86
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
constant
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF
domain-
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV IgG1
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK hTIGIT-HIS 87 gtiett gnisaekggs iilqchlsst
taqvtqvnwe qqdqllaicn adlgwhisps fkdrvapgpg lgltlqsltv ndtgeyfciy
htypdgtytg riflevless vaehgarfqi pllga hhhhhhhhhggq 31C6 H-CDR1 88
SYVMH 31C6 H-CDR2 89 YIDPYNDGAKYNEKFKG 31C6 H-CDR3 90 GGPYGWYFDV
31C6 L-CDR1 91 RASEHIYSYLS 31C6 L-CDR2 92 NAKTLAE 31C6 L-CDR3 93
QHHFGSPLT 31C6 PARENTAL 94
EVQLQQSGPELVKPGSSVKMSCKASGYTFSSYVMHWVKQKPGQGLEWIGYIDPYNDGAKYNE VH
(with CDRs
KFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGPYGWYFDVWGAGTTVTVSS
underlined) 31C6 PARENTAL 95
DIQMTQSPASLSASVGETVTITCRASEHIYSYLSWYQQKQGKSPQLLVYNAKTLAEGVPSRF VL
(with CDRs SGSGSGTQFSLKINSLQPEDFGTYYCQHHFGSPLTFGAGTTLELK
underlined) 31C6 H-CDR2 96 YIDPYNrGAKYNEKFG VARIANT (D56R) 31C6
H-CDR2 97 YIDPYNlGAKYNEKG VARIANT F (D56L) 31C6 H-CDR2 98
YIDPYNkGAKYNEKFG VARIANT (D56K) 31C6 H-CDR2 99 YIDPYNfGAKYNEKFG
VARIANT (D56F) 31C6 H-CDR2 100 YIDPYNsGAKYNEKFG VARIANT (D56S) 31C6
H-CDR2 101 YIDPYNyGAKYNEKFG VARIANT (D56Y) 31C6 H-CDR2 102
YIDPYNvGAKYNEKFG VARIANT (D56V) 31C6 H-CDR2 103 YIDPYNDrAKYNEKFKG
VARIANT (G57R) 31C6 H-CDR2 104 YIDPYNDnAKYNEKFKG VARIANT (G57N)
31C6 H-CDR2 105 YIDPYNDqAKYNEKFKG VARIANT (G57Q) 31C6 H-CDR2 106
YIDPYNDeAKYNEKFKG VARIANT (G57E) 31C6 H-CDR2 107 YIDPYND1AKYNEKFKG
VARIANT (G57L) 31C6 H-CDR2 108 YIDPYNDkAKYNEKFKG VARIANT (G57K)
31C6 H-CDR2 109 YIDPYNDsAKYNEKFKG VARIANT (G57S) 31C6 H-CDR2 110
YIDPYNDyAKYNEKFKG VARIANT (G57Y) 31C6 H-CDR2 111 YIDPYNDvAKYNEKFKG
VARIANT (G57V) 31C6 L-CDR2 112 AAKTLAE variant (N50A) 31C6 L-CDR2
113 YAKTLAE variant (N50Y) 31C6 L-CDR2 114 WAKTLAE variant (N50W)
31C6 L-CDR2 115 SAKTLAE variant (N50S) 31C6 L-CDR2 116 TAKTLAE
variant (N50T) 31C6 L-CDR2 117 IAKTLAE variant (N50I) 31C6 L-CDR2
118 VAKTLAE variant (N50V)
31C6 L-CDR2 119 NNKTLAE variant (A51N) 31C6 L-CDR2 120 NIKTLAE
variant (A51I) 31C6 L-CDR2 121 NLLTLAE variant (A51L) 31C6 L-CDR2
122 NTKTLAE variant (A51T) 31C6 L-CDR2 123 NVKTLAE variant (A51V)
31C6_HUMZ_VH1 124
EVQLVQSGAEVKKPGASVKVSCKASGYTFSSYVMHWVRQAPGQRLEWIGYIDPYNDGAKYSQ
(with CDRs
KFQGRVTLTRDTSASTAYMELSSLRSEDTAVYYCARGGPYGWYFDVWGQGTTVTVSS
underlined) 31C6_HUMZ_VH2 125
EVQLVQSGAEVKKPGASVKVSCKASGYTFSSYVMHWVRQAPGQRLEWIGYIDPYNDGAKYSQ
(with CDRs
KFQGRVTLTSDKSASTAYMELSSLRSEDTAVYYCARGGPYGWYFDVWGQGTTVTVSS
underlined) 31C6_HUMZ_VH3 126
EVQLVQSGAEVKKPGASVKVSCKASGYTFSSYVMHWVRQAPGQGLEWIGYIDPYNDGAKYAQ
(with CDRs
KFQGRVTLTRDTSTSTVYMELSSLRSEDTAVYYCARGGPYGWYFDVWGQGTTVTVSS
underlined) 31C6_HUMZ_VH4 127
EVQLVQSGAEVKKPGASVKVSCKASGYTFSSYVMHWVRQAPGQGLEWIGYIDPYNDGAKYAQ
(with CDRs
KFQGRVTLTSDKSTSTVYMELSSLRSEDTAVYYCARGGPYGWYFDVWGQGTTVTVSS
underlined) 31C6_HUMZ_VH5 128
EVQLVQSGAEVKKPGSSVKVSCKASGYTFSSYVMHWVRQAPGQGLEWIGYIDPYNDGAKYAQ
(with CDRs
KFQGRVTLTSDKSTSTAYMELSSLRSEDTAVYYCARGGPYGWYFDVWGQGTTVTVSS
underlined) 31C6_HUMZ_VH6 129
EVQLVQSGAEVKKPGASVKVSCKASGYTFSSYVMHWVRQAPGQGLEWIGYIDPYNDGAKYAQ
(with CDRs
KFQGRVTLTSDKSISTAYMELSRLRSDDTVVYYCARGGPYGWYFDVWGQGTTVTVSS
underlined) 31C6 Humz L1 130
DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKAPKLLIYNAKTLAE (with CDRs
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHFGSPLTFGQGTRLEIK underlined)
31C6 Humz L2 131
DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKAPKLLIYNAKTLAE (with CDRs
GVPSRFSGSGSGTQFTLTISSLQPEDFATYYCQHHFGSPLTFGQGTRLEIK underlined)
31C6 Humz L3 132
DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKVPKLLIYNAKTLAE (with CDRs
GVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHHFGSPLTFGQGTRLEIK underlined)
31C6 Humz L4 133
DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKVPKLLIYNAKTLAE (with CDRs
GVPSRFSGSGSGTQFTLTISSLQPEDVATYYCQHHFGSPLTFGQGTRLEIK underlined)
31C6 H- 134 YIDPYNDGAKYAQKFQG CDR2 variant 31C6 H- 135
YIDPYNDGAKYSQKFQG CDR2 variant 18G10-VH 136
QVQLMESGPGLVQPSQTLSLTCTVSGFPLTSYTVHWVRQPPGKGLEWIGAIWSSGSTDYNSA
sequence LKSRLNINRDSSKSQVFLKMNSLQTEDTAIYFCTKSGWAFFDYWGQGVMVTVSS
18G10-VL 137
DIQMTQSPSLLSASVGDRVTLNCIASQNIYKSLAWYQLKLGEAPKLLIYNANSLQAGIPSRF
sequence SGSGSGTDFALTISGLQPEDVATYFCQQYSGGYTFGAGTKLELK 11A11-VH 138
EVQLVESGGDLVQPGRSLKISCVASGFTFSDYYMAWVRLAPQKGLEWVASISYEGSRTHYGD
sequence SVRGRFIISRDNPKNILYLQMNSLGSEDTATYFCARHTGTLDWLVYWGQGTLVIVSS
11A11-VL 139
NIVMAQSPKSMSISAGDRVTMNCKASQNVDNNIAWYQQKPGQSPKLLIFYASNRYSGVPDRF
sequence TGGGYGTDFTLTIKSVQAEDAAFYYCQRIYNFPTFGSGTKLEIK 14A6 H-CDR3
140 MPSFITLASLSTXEGYFDF CONSENSUS X = W, F, Y, I, V, L 14A6 L-CDR2
141 YAX.sub.1X.sub.2LQT CONSENSUS X.sub.1 = N, S, T, G, D X.sub.2 =
S, N, S, T, Y, 4 14A6 L-CDR3 142 QQYYSGXT CONSENSUS X = W, F, Y, I,
V, L 14A6 VH 143
EVQLQX.sub.1SGX.sub.2GLX.sub.3KPX.sub.4X.sub.5X.sub.6LSLTCX.sub.7VX.sub.8-
GX.sub.30SIX.sub.31SDYWGWX.sub.9RX.sub.10X.sub.11PGX.sub.12X.sub.13X.sub.1-
4EW PARENTAL
X.sub.15GFITYSGSTSYNPSLKSRX.sub.16X.sub.17IX.sub.18X.sub.19DTSKN-
QFX.sub.20LX.sub.21LX.sub.22SVTX.sub.23X.sub.24DTAX.sub.25Y
CONSENSUS
X.sub.26CARMPSFITLASLSTX.sub.27EGYFDFWGX.sub.32GTX.sub.28X.sub.-
29TVSS X.sub.1 = E or Q X.sub.2 = P or A X.sub.3 = V or L X.sub.4 =
S or P X.sub.5 = Q or E or G X.sub.6 = S or T X.sub.7 = S or T or A
X.sub.8 = T or S X.sub.9 = I or V X.sub.10 = K or Q X.sub.11 = F or
P X.sub.12 = N or K X.sub.13 = K or G X.sub.14 = M or L X.sub.15 =
M or I X.sub.16 = I or V X.sub.17 = S or T X.sub.18 = T or S
X.sub.19 = R or V X.sub.20 = F or S X.sub.21 = Q or K X.sub.22 = H
or S X.sub.23 = T or A X.sub.24 = D or A X.sub.25 = T or V X.sub.26
= S or Y, X.sub.27 = W, F, Y, I, V or L X.sub.28 = M, V, L, A, R,
N, P, Q, E, G, I, H, K, F, S, T, W or Y X.sub.29 = V, T or L
X.sub.30 = S or G or Y X.sub.31 = A or S X.sub.32 = P or Q 14A6 VH
144
EVQLQX.sub.1SGX.sub.2GLX.sub.3KPX.sub.4X.sub.5TLSLTCX.sub.6VX.sub.7GX.sub-
.8SIX.sub.9SDYWGWX.sub.10RX.sub.11X.sub.12PGKX.sub.13X.sub.14EWX.sub.15
HUMANIZED
GFITYSGSTSYNPSLKSRX.sub.16TISX.sub.17DTSKNQFSLKLX.sub.18SVTAX.s-
ub.19DTAVYYCARMPSFITLA CONSENSUS
SLSTX.sub.20EGYFDFWGQGTX.sub.21X.sub.22TVSS X.sub.1 = E or Q
X.sub.2 = P or A X.sub.3 = V or L X.sub.4 = S or P X.sub.5 = E or G
X.sub.6 = T or A or S X.sub.7 = S or T X.sub.8 = G or S or Y
X.sub.9 = S or A X.sub.10 = I or V X.sub.11 = Q or K X.sub.12 = P
or F X.sub.13 = G or K X.sub.14 = L or M X.sub.15 = I or M X.sub.16
= V or I X.sub.17 = V or R X.sub.18 = S or H X.sub.19 = A or D
X.sub.20 = W, F, Y, I, V, L X.sub.21 = M, V, L, A, R, N, P, Q, E,
G, I, H, K, F, S, T, W or Y X.sub.22 = V, T or L 14A6 VL 145
DIQMTQSPSX.sub.1LSASVGDRVTX.sub.2X.sub.3CKASQSIHKNLAWYQQKX.sub.4GX.sub.5X-
.sub.15PKX.sub.6LIYYAX.sub.7X.sub.8LQT PARENTAL
GX.sub.9PSRFSGSGSGTDFTLTISX.sub.10LQPEDX.sub.11ATYX.sub.12CQQYYS-
GX.sub.13TFGGGTKVEX.sub.14K CONSENSUS X.sub.1 = L or S X.sub.2 = L
or I X.sub.3 = N or T X.sub.4 = L or P X.sub.5 = E or K X.sub.6 = F
or L X.sub.7 = N, S, T, G or D X.sub.8 = S, N, T, Y or Q X.sub.9 =
I or V X.sub.10 = G or S X.sub.11 = V or F X.sub.12 = F or Y
X.sub.13 = W, F, Y, I, V or L X.sub.14 = L or I X.sub.15 = A or V
14A6 VL 146
DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKX.sub.6PKX.sub.1LIYYAX.sub.2X.s-
ub.3LQTGX.sub.4P HUMANIZED
SRFSGSGSGTDFTLTISSLQPEDX.sub.7ATYYCQQYYSGX.sub.5TFGGGTKVEIK
CONSENSUS X.sub.1 = L or F X.sub.2 = N, S, T, G or D X.sub.3 = S,
N, T, Y or Q X.sub.4 = V or I X.sub.5 = W, F, Y, I, V or L X.sub.6
= A or V X.sub.7 = F or V 31C6 H-CDR2 147
YIDPYNX.sub.1X.sub.2AKYX.sub.3X.sub.4KFX.sub.5G CONSENSUS X.sub.1 =
D, R, L, K, F, 5, Y or V X.sub.2 = G, R, N, Q, E, L K, S, Y or V
X.sub.3 = N, A or S X.sub.4 = E or Q X.sub.5 = K or Q 31C6 L-CDR2
148 X.sub.1X.sub.2KTLAE CONSENSUS X.sub.1 = N, A, V, W, S, T, R, H
G, I or V X.sub.2 = A, N, I, L, T or V 31C6 VH 149
EVQLX.sub.1QSGX.sub.2EX.sub.3X.sub.4KPGX.sub.5SVKX.sub.6SCKASGYTFSSYVMHWV-
X.sub.7QX.sub.8PGQX.sub.9LEWIGYIDPYN PARENTAL
X.sub.10X.sub.11AKYX.sub.12X.sub.13KFX.sub.14GX.sub.15X.sub.16TL-
TX.sub.17DX.sub.18SX.sub.19STX.sub.20YMELSX.sub.21LX.sub.22SX.sub.23DX.sub-
.24X.sub.25VYYC CONSENSUS ARGGPYGX.sub.26YFDVWGX.sub.27GTTVTVSS
X.sub.1 = Q or V X.sub.2 = P or A X.sub.3 = V or L X.sub.4 = V or K
X.sub.5 = S or A X.sub.6 = M or V X.sub.7 = K or R X.sub.8 = K or A
X.sub.9 = G or R X.sub.10 = D, R, L, K, F, S, Y or V X.sub.11 = G,
R, N, Q, E, L K, S, Y or V X.sub.12 = N, A or S X.sub.13 = E or Q
X.sub.14 = K or Q X.sub.15 = R or K X.sub.16 = A or V X.sub.17 = S
or R X.sub.18 = K or T X.sub.19 = S, I , A or T X.sub.20 = A or V
X.sub.21 = R or S X.sub.22 = T or R X.sub.23 = D or E X.sub.24 = S
or T X.sub.25 = A or V X.sub.26 = W, A, D, E, F, G, I, K, N, Q, R,
S, T, V or Y X.sub.27 = A or Q 31C6 VH 150
EVQLVQSGAEVKKPGX.sub.1SVKVSCKASGYTFSSYVMHWVRQAPGQX.sub.2LEWIG
HUMANIZED
YIDPYNX.sub.3X.sub.4AKYX.sub.5X.sub.5KFX.sub.7GRVTLTX.sub.8DX.s-
ub.9SX.sub.10STX.sub.11YMELSX.sub.12LRSX.sub.13DT
CONSENSUS X.sub.14VYYCARGGPYGX.sub.15YFDVWGQGTTVTVSS X.sub.1 = A or
S X.sub.2 = R or G X.sub.3 = D, R, L, K, F, S, Y or V X.sub.4 = G,
R, N, Q, E, L K, S, Y or V X.sub.5 = N, A or S X.sub.6 = E or Q
X.sub.7 = K or Q X.sub.8 = R or S X.sub.9 = T or K X.sub.10 = A, T
or I X.sub.11 = A or V X.sub.12 = S or R X.sub.13 = E or D X.sub.14
= A or V X.sub.15 = W, A, D, E, F, G, I, K, N, Q, R, S, T, V or Y
31C6 VL 151
DIQMTQSPX.sub.1SLSASVGX.sub.2X.sub.3VTITCRASEHIYSYLSWYQQKX.sub.4GKX.sub.5-
PX.sub.6LLX.sub.7YX.sub.8X.sub.9KTLAE PARENTAL
GVPSRFSGSGSGTX.sub.10FX.sub.11LX.sub.12IX.sub.13SLQPEDX.sub.14X.-
sub.15TYYCQHHFGSPLTFGX.sub.16GTX.sub.17LEX.sub.18 CONSENSUS K
X.sub.1 = A or S X.sub.2 = E or D X.sub.3 = T or R X.sub.4 = Q or P
X.sub.5 = A or V X.sub.6 = Q or K X.sub.7 = V or I X.sub.8 = N, A,
Y, W, S, T, I or V X.sub.9 = A, N, I, L, T or V X.sub.10 = Q or D
X.sub.11 = S or T X.sub.12 = K or T X.sub.13 = N or S X.sub.14 = F
or V X.sub.15 = G or A X.sub.16 = A or Q X.sub.17 = T or R X.sub.18
= L or I 31C6 L-VL 152
DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKX.sub.1PKLLIY HUMANIZED
X.sub.2X.sub.3KTLAEGVPSRFSGSGSGTX.sub.4FTLTISSLQPEDX.sub.5ATYYC-
QHHFGSPLTFGQGTR CONSENSUS LEIK X.sub.1 = A or V X.sub.2 = N, A, W,
W, S, T, I or V X.sub.3 = A, N, I, L, T or V X.sub.4 = D or Q
X.sub.5 = F or V 31C6 H-CDR3 153 GGPYGXYFDV CONSENSUS X.sub.15 = W,
A, D, E, F, G, I, K, N, Q, R, S, T, V or Y 31C6 H-CDR3 154
GGPYGAYFDV VARIANT 31C6 H-CDR3 155 GGPYGDYFDV VARIANT 31C6 H-CDR3
156 GGPYGEYFDV VARIANT 31C6 H-CDR3 157 GGPYGFYFDV VARIANT 31C6
H-CDR3 158 GGPYGGYFDV VARIANT 31C6 H-CDR3 159 GGPYGIYFDV VARIANT
31C6 H-CDR3 160 GGPYGKYFDV VARIANT 31C6 H-CDR3 161 GGPYGNYFDV
VARIANT 31C6 H-CDR3 162 GGPYGQYFDV VARIANT 163 GGPYGRYFDV 31C6
H-CDR3 164 GGPYGSYFDV VARIANT 165 GGPYGTYFDV 31C6 H-CDR3 166
GGPYGVYFDV VARIANT 167 GGPYGYYFDV
Sequence CWU 1
1
16715PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 1Ser Asp Tyr Trp Gly 1 5 216PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
2Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys Ser 1
5 10 15 319PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 3Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser
Thr Trp Glu Gly Tyr 1 5 10 15 Phe Asp Phe 411PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
4Lys Ala Ser Gln Ser Ile His Lys Asn Leu Ala 1 5 10 57PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
5Tyr Ala Asn Ser Leu Gln Thr 1 5 68PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
6Gln Gln Tyr Tyr Ser Gly Trp Thr 1 5 7127PRTRattus norvegicus 7Glu
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10
15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Ser Ser Ile Ala Ser Asp
20 25 30 Tyr Trp Gly Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu
Trp Met 35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn
Pro Ser Leu Lys 50 55 60 Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser
Lys Asn Gln Phe Phe Leu 65 70 75 80 Gln Leu His Ser Val Thr Thr Asp
Asp Thr Ala Thr Tyr Ser Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile
Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly 100 105 110 Tyr Phe Asp Phe
Trp Gly Pro Gly Thr Met Val Thr Val Ser Ser 115 120 125
8106PRTRattus norvegicus 8Asp Ile Gln Met Thr Gln Ser Pro Ser Leu
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Leu Asn Cys Lys
Ala Ser Gln Ser Ile His Lys Asn 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Leu Gly Glu Ala Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Ala Asn
Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80
Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Trp Thr 85
90 95 Phe Gly Gly Gly Thr Lys Val Glu Leu Lys 100 105
9127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Humanized antibody chain polypeptide 9Glu Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asp 20 25 30 Tyr
Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys
50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser
Leu Ser Thr Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly
Thr Met Val Thr Val Ser Ser 115 120 125 10127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Humanized
antibody chain polypeptide 10Glu Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Gly Ser Ile Ser Ser Asp 20 25 30 Tyr Trp Gly Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile
Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser
Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70
75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr
Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val
Thr Val Ser Ser 115 120 125 11127PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Humanized antibody chain
polypeptide 11Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly
Ser Ile Ser Ser Asp 20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Thr Tyr Ser Gly
Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Thr Ile
Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly 100 105
110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115
120 125 12127PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Humanized antibody chain polypeptide 12Glu Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ser Ser Asp 20
25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Met 35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro
Ser Leu Lys 50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys
Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr
Leu Ala Ser Leu Ser Thr Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp
Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125
13127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Humanized antibody chain polypeptide 13Glu Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asp 20 25 30
Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu
Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln
Phe Ser Leu 65 70 75 80 Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala
Ser Leu Ser Thr Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120 125 14127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Humanized
antibody chain polypeptide 14Glu Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Gly Ser Ile Ser Ser Asp 20 25 30 Tyr Trp Gly Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile
Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser
Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70
75 80 Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr
Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val
Thr Val Ser Ser 115 120 125 15127PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Humanized antibody chain
polypeptide 15Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser
Ser Ile Ser Ser Asp 20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Thr Tyr Ser Gly
Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Thr Ile
Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu His
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly 100 105
110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115
120 125 16127PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Humanized antibody chain polypeptide 16Glu Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ser Ser Asp 20
25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Met 35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro
Ser Leu Lys 50 55 60 Ser Arg Ile Thr Ile Ser Val Asp Thr Ser Lys
Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu His Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr
Leu Ala Ser Leu Ser Thr Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp
Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125
17127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Humanized antibody chain polypeptide 17Glu Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu
Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Asp 20 25 30
Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu
Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala
Ser Leu Ser Thr Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120 125 18127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Humanized
antibody chain polypeptide 18Glu Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala
Val Ser Gly Tyr Ser Ile Ser Ser Asp 20 25 30 Tyr Trp Gly Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile
Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser
Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70
75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr
Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val
Thr Val Ser Ser 115 120 125 19127PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Humanized antibody chain
polypeptide 19Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr
Ser Ile Ser Ser Asp 20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Thr Tyr Ser Gly
Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Thr Ile
Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly 100 105
110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115
120 125 20127PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Humanized antibody chain polypeptide 20Glu Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Ser Ser Ile Ser Ser Asp 20
25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Met 35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro
Ser Leu Lys 50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys
Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr
Leu Ala Ser Leu Ser Thr Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp
Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125
21127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Humanized antibody chain polypeptide 21Glu Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu
Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Asp 20 25 30
Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu
Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln
Phe Ser Leu 65 70 75 80 Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala
Ser Leu Ser Thr Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120 125 22127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Humanized
antibody chain polypeptide 22Glu Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala
Val Ser Gly Tyr Ser Ile Ser Ser Asp 20 25 30 Tyr Trp Gly Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile
Thr Tyr Ser Gly
Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Thr Ile
Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu His
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly 100 105
110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115
120 125 23127PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Humanized antibody chain polypeptide 23Glu Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Ser Ser Ile Ser Ser Asp 20
25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Ile 35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro
Ser Leu Lys 50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys
Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu His Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr
Leu Ala Ser Leu Ser Thr Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp
Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125
24127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Humanized antibody chain polypeptide 24Glu Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu
Ser Leu Thr Cys Ala Val Ser Gly Ser Ser Ile Ser Ser Asp 20 25 30
Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met 35
40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu
Lys 50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln
Phe Ser Leu 65 70 75 80 Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala
Ser Leu Ser Thr Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120 125 25106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Humanized
antibody chain polypeptide 25Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Gln Ser Ile His Lys Asn 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala
Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp
Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
26106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Humanized antibody chain polypeptide 26Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile 35
40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Tyr Ser Gly Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 105 27106PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Humanized antibody chain polypeptide 27Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu
Ile 35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Tyr Tyr Ser Gly Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105 28106PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Humanized antibody chain polypeptide
28Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys
Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys
Leu Leu Ile 35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr
Cys Gln Gln Tyr Tyr Ser Gly Trp Thr 85 90 95 Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 29106PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Humanized antibody chain
polypeptide 29Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln
Ser Ile His Lys Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Val Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Ala Asn Ser Leu Gln
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val
Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr 85 90 95 Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 30106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Humanized
antibody chain polypeptide 30Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Gln Ser Ile His Lys Asn 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Val Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Ala
Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp
Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
31244PRTHomo sapiens 31Met Arg Trp Cys Leu Leu Leu Ile Trp Ala Gln
Gly Leu Arg Gln Ala 1 5 10 15 Pro Leu Ala Ser Gly Met Met Thr Gly
Thr Ile Glu Thr Thr Gly Asn 20 25 30 Ile Ser Ala Glu Lys Gly Gly
Ser Ile Ile Leu Gln Cys His Leu Ser 35 40 45 Ser Thr Thr Ala Gln
Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln 50 55 60 Leu Leu Ala
Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser 65 70 75 80 Phe
Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln 85 90
95 Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr
100 105 110 Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val
Leu Glu 115 120 125 Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile
Pro Leu Leu Gly 130 135 140 Ala Met Ala Ala Thr Leu Val Val Ile Cys
Thr Ala Val Ile Val Val 145 150 155 160 Val Ala Leu Thr Arg Lys Lys
Lys Ala Leu Arg Ile His Ser Val Glu 165 170 175 Gly Asp Leu Arg Arg
Lys Ser Ala Gly Gln Glu Glu Trp Ser Pro Ser 180 185 190 Ala Pro Ser
Pro Pro Gly Ser Cys Val Gln Ala Glu Ala Ala Pro Ala 195 200 205 Gly
Leu Cys Gly Glu Gln Arg Gly Glu Asp Cys Ala Glu Leu His Asp 210 215
220 Tyr Phe Asn Val Leu Ser Tyr Arg Ser Leu Gly Asn Cys Ser Phe Phe
225 230 235 240 Thr Glu Thr Gly 32245PRTMacaca fascicularis 32Met
Arg Trp Cys Leu Phe Leu Ile Trp Ala Gln Gly Leu Arg Gln Ala 1 5 10
15 Pro Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn
20 25 30 Ile Ser Ala Lys Lys Gly Gly Ser Val Ile Leu Gln Cys His
Leu Ser 35 40 45 Ser Thr Met Ala Gln Val Thr Gln Val Asn Trp Glu
Gln His Asp His 50 55 60 Ser Leu Leu Ala Ile Arg Asn Ala Glu Leu
Gly Trp His Ile Tyr Pro 65 70 75 80 Ala Phe Lys Asp Arg Val Ala Pro
Gly Pro Gly Leu Gly Leu Thr Leu 85 90 95 Gln Ser Leu Thr Met Asn
Asp Thr Gly Glu Tyr Phe Cys Thr Tyr His 100 105 110 Thr Tyr Pro Asp
Gly Thr Tyr Arg Gly Arg Ile Phe Leu Glu Val Leu 115 120 125 Glu Ser
Ser Val Ala Glu His Ser Ala Arg Phe Gln Ile Pro Leu Leu 130 135 140
Gly Ala Met Ala Met Met Leu Val Val Ile Cys Ile Ala Val Ile Val 145
150 155 160 Val Val Val Leu Ala Arg Lys Lys Lys Ser Leu Arg Ile His
Ser Val 165 170 175 Glu Ser Gly Leu Gln Arg Lys Ser Thr Gly Gln Glu
Glu Gln Ile Pro 180 185 190 Ser Ala Pro Ser Pro Pro Gly Ser Cys Val
Gln Ala Glu Ala Ala Pro 195 200 205 Ala Gly Leu Cys Gly Glu Gln Gln
Gly Asp Asp Cys Ala Glu Leu His 210 215 220 Asp Tyr Phe Asn Val Leu
Ser Tyr Arg Ser Leu Gly Ser Cys Ser Phe 225 230 235 240 Phe Thr Glu
Thr Gly 245 33447PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Antibody chain polypeptide 33Gln Val Gln Leu Val
Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr
Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe
50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr
Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly
Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295
300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420
425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440 445 34218PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Antibody chain polypeptide 34Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly
Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40
45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala
50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln His Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170
175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
35440PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Antibody chain polypeptide 35Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5
10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn
Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135
140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr
Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260
265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385
390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Lys 435 440
36214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Antibody chain polypeptide 36Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr
Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp
Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205 Phe Asn Arg Gly Glu Cys 210 37129PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Humanized
antibody chain polypeptide 37Glu Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Ser Ser Ile Ala Ser Asp 20 25 30 Tyr Trp Gly Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile
Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70
75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr
Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val
Thr Val Ser Ser Ala 115 120 125 Ser 38127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Humanized
antibody chain polypeptide 38Glu Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Ser Ser Ile Ala Ser Asp 20 25 30 Tyr Trp Gly Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile
Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser
Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70
75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr
Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val
Thr Val Ser Ser 115 120 125 39127PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Humanized antibody chain
polypeptide 39Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser
Ser Ile Ala Ser Asp 20 25 30 Tyr Trp Gly Trp Ile Arg Lys Pro Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Thr Tyr Ser Gly
Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile
Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly 100 105
110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115
120 125 40127PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Humanized antibody chain polypeptide 40Glu Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp 20
25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Lys Leu Glu Trp
Ile 35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro
Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys
Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr
Leu Ala Ser Leu Ser Thr Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp
Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125
41127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Humanized antibody chain polypeptide 41Glu Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp 20 25 30
Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Met Glu Trp Ile 35
40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu
Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln
Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala
Ser Leu Ser Thr Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120 125 42127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Humanized
antibody chain polypeptide 42Glu Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Ser Ser Ile Ala Ser Asp 20 25 30 Tyr Trp Gly Trp Ile
Arg Lys Pro Pro Gly Lys Lys Met Glu Trp Ile 35 40 45 Gly Phe Ile
Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser
Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70
75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr
Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val
Thr Val Ser Ser 115 120 125 43127PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Humanized antibody chain
polypeptide 43Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser
Ser Ile Ala Ser Asp 20 25 30 Tyr Trp Gly Trp Ile Arg Gln Phe Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Thr Tyr Ser Gly
Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile
Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser
Ser Val Thr Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly 100 105
110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115
120 125 44127PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Humanized antibody chain polypeptide 44Glu Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp 20
25 30 Tyr Trp Gly Trp Ile Arg Lys Pro Pro Gly Lys Lys Met Glu Trp
Ile 35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro
Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys
Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr
Leu Ala Ser Leu Ser Thr Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp
Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125
45127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Humanized antibody chain polypeptide 45Glu Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu
Ser Leu Thr Cys Ser Val Thr Gly Ser Ser Ile Ala Ser Asp 20 25 30
Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu
Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln
Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala
Ser Leu Ser Thr Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Gln
Gly Thr Met Val Thr Val Ser Ser 115 120 125 46127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Humanized
antibody chain polypeptide 46Glu Val Gln Leu Gln Gln Ser Gly Ala
Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser
Val Thr Gly Ser Ser Ile Ala Ser Asp 20 25 30 Tyr Trp Gly Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile
Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70
75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr
Trp Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val
Thr Val Ser Ser 115 120 125 47127PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Humanized antibody chain
polypeptide 47Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Pro Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Ser
Ser Ile Ala Ser Asp 20 25 30 Tyr Trp Gly Trp Val Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Thr Tyr Ser Gly
Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile
Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly 100 105
110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115
120 125 48106PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Humanized antibody chain polypeptide 48Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg
Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr
Ser Gly Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 49106PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Humanized antibody chain polypeptide 49Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu
Ile 35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Tyr Tyr Ser Gly Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105 50106PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Humanized antibody chain polypeptide
50Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys
Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe
Cys Gln Gln Tyr Tyr Ser Gly Trp Thr 85 90 95 Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 51106PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Humanized antibody chain
polypeptide 51Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln
Ser Ile His Lys Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Ala Asn Ser Leu Gln
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Trp Thr 85 90 95 Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 52106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Humanized
antibody chain polypeptide 52Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Gln Ser Ile His Lys Asn 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala
Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp
Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
5319PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Leader sequence heavy chains peptide 53Met Glu Trp Ser
Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His
Ser 5420PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Leader sequence light chains peptide 54Met Ser Val Pro
Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala
Arg Cys 20 55325PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Heavy chain constant domain-IgG4 S228P
polypeptide 55Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr 1 5 10 15 Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro 20 25 30 Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val 35 40 45 His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser 50 55 60 Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr 65 70 75 80 Cys Asn Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 85 90 95 Glu
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 100 105
110 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
115 120 125 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val 130 135 140 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val 145 150 155 160 Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser 165 170 175 Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu 180 185 190 Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 195 200 205 Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 210 215 220 Gln
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 225 230
235 240 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala 245 250 255 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr 260 265 270 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Arg Leu 275 280 285 Thr Val Asp Lys Ser Arg Trp Gln Glu
Gly Asn Val Phe Ser Cys Ser 290 295 300 Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser 305 310 315 320 Leu Ser Leu Gly
Lys 325 56105PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Kappa light chain constant domain polypeptide
56Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 1
5 10 15 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro 20 25 30 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly 35 40 45 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr 50 55 60 Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His 65 70 75 80 Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val 85 90 95 Thr Lys Ser Phe Asn
Arg Gly Glu Cys 100 105 5711PRTArtificial SequenceDescription of
Artificial Sequence Synthetic CDR peptide 57Gly Tyr Ser Ile Thr Ser
Asp Tyr Ala Trp Asn 1 5 10 5816PRTArtificial SequenceDescription of
Artificial Sequence Synthetic CDR peptide 58Tyr Ile Ser Asn Ser Gly
Ser Ala Ser Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 5912PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
59Leu Ile Tyr Tyr Asp Tyr Gly Gly Ala Met Asn Phe 1 5 10
6011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 60Lys Ala Ser Gln Gly Val Ser Thr Thr Val Ala
1 5 10 617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 61Ser Ala Ser Tyr Arg Tyr Thr 1 5
629PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 62Gln His Tyr Tyr Ser Thr Pro Trp Thr 1 5
63121PRTMus musculus 63Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr
Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg
Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser
Asn Ser Gly Ser Ala Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg
Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu
Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90
95 Ala Thr Leu Ile Tyr Tyr Asp Tyr Gly Gly Ala Met Asn Phe Trp Gly
100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 64107PRTMus
musculus 64Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser
Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Gly
Val Ser Thr Thr 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile Ser Ser Val Gln Ser 65 70 75 80 Glu Asp Leu Ala
Val Tyr Tyr Cys Gln His Tyr Tyr Ser Thr Pro Trp 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 657PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
65Tyr Ala Ser Asn Leu Gln Thr 1 5 667PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
66Tyr Ala Ser Ser Leu Gln Thr 1 5 677PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
67Tyr Ala Ser Thr Leu Gln Thr 1 5 687PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
68Tyr Ala Thr Thr Leu Gln Thr 1 5 697PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
69Tyr Ala Ser Tyr Leu Gln Thr 1 5 707PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
70Tyr Ala Asn Gln Leu Gln Thr 1 5 717PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
71Tyr Ala Gly Ser Leu Gln Thr 1 5 727PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
72Tyr Ala Ser Gln Leu Gln Thr 1 5 737PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
73Tyr Ala Asp Ser Leu Gln Thr 1 5 748PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
74Gln Gln Tyr Tyr Ser Gly Phe Thr 1 5 758PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
75Gln Gln Tyr Tyr Ser Gly Tyr Thr 1 5 768PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
76Gln Gln Tyr Tyr Ser Gly Ile Thr 1 5 778PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
77Gln Gln Tyr Tyr Ser Gly Val Thr 1 5 788PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
78Gln Gln Tyr Tyr Ser Gly Leu Thr 1 5 7919PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
79Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Phe Glu Gly Tyr 1
5 10 15 Phe Asp Phe 8019PRTArtificial SequenceDescription of
Artificial Sequence Synthetic CDR peptide 80Met Pro Ser Phe Ile Thr
Leu Ala Ser Leu Ser Thr Tyr Glu Gly Tyr 1 5 10 15 Phe Asp Phe
8119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 81Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser
Thr Ile Glu Gly Tyr 1 5 10 15 Phe Asp Phe 8219PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
82Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Val Glu Gly Tyr 1
5 10 15 Phe Asp Phe 8319PRTArtificial SequenceDescription of
Artificial Sequence Synthetic CDR peptide 83Met Pro Ser Phe Ile Thr
Leu Ala Ser Leu Ser Thr Leu Glu Gly Tyr 1 5 10 15 Phe Asp Phe
84363DNAMus musculus 84gatgtgcagc ttcaggagtc gggacctggc ctggtgaaac
cttctcagtc tctgtccctc 60acctgcactg tcactggcta ctcaatcacc agtgattatg
cctggaactg gatccgacag 120tttccaggaa acaaactgga gtggatgggc
tacataagca acagtggtag cgctagctac 180aacccatctc tcaaaagtcg
catctctatc actcgagaca catccaagaa ccagttcttc 240ctgcagttga
attctgtgac tactgaggac acagccacat attactgtgc aaccctgatc
300tactatgatt acgggggggc tatgaacttc tggggtcaag gaacctcagt
caccgtctcc 360tca 36385321DNAMus musculus 85gacattgtga tgacccagtc
tcacaaattc atgtccacat cagtaggaga cagggtcagc 60atcacctgca aggccagtca
gggtgtgagt actactgtgg cctggtatca acagaaacca 120ggacaatctc
ctaaactact gatttactcg gcatcctacc ggtacactgg agtccctgat
180cgcttcactg gcagtggatc tgggacggat ttcactttca ccatcagcag
tgtgcagtct 240gaagacctgg cagtttatta ctgtcagcat tattatagta
ctccgtggac gttcggtgga 300ggcaccaagc tggaaatcaa a 32186330PRTHomo
sapiens 86Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115
120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235
240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr 305
310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
87133PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Fusion protein polypeptide 87Gly Thr Ile Glu Thr Thr Gly
Asn Ile Ser Ala Glu Lys Gly Gly Ser 1 5 10 15 Ile Ile Leu Gln Cys
His Leu Ser Ser Thr Thr Ala Gln Val Thr Gln 20 25 30 Val Asn Trp
Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys Asn Ala Asp 35 40 45 Leu
Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val Ala Pro Gly 50 55
60 Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn Asp Thr Gly
65 70 75 80 Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr Tyr
Thr Gly 85 90 95 Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala
Glu His Gly Ala 100 105 110 Arg Phe Gln Ile Pro Leu Leu Gly Ala His
His His His His His His 115 120 125 His His Gly Gly Gln 130
885PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 88Ser Tyr Val Met His 1 5 8917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
89Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Asn Glu Lys Phe Lys 1
5 10 15 Gly 9010PRTArtificial SequenceDescription of Artificial
Sequence Synthetic CDR peptide 90Gly Gly Pro Tyr Gly Trp Tyr Phe
Asp Val 1 5 10 9111PRTArtificial SequenceDescription of Artificial
Sequence Synthetic CDR peptide 91Arg Ala Ser Glu His Ile Tyr Ser
Tyr Leu Ser 1 5 10 927PRTArtificial SequenceDescription of
Artificial Sequence Synthetic CDR peptide 92Asn Ala Lys Thr Leu Ala
Glu 1 5 939PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 93Gln His His Phe Gly Ser Pro Leu Thr 1 5
94119PRTMus musculus 94Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln
Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro
Tyr Asn Asp Gly Ala Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys
Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Pro Tyr Gly Trp Tyr Phe Asp Val Trp Gly Ala Gly
100 105 110 Thr Thr Val Thr Val Ser Ser 115 95107PRTMus musculus
95Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu His Ile Tyr Ser
Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln
Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu
Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr
Cys Gln His His Phe Gly Ser Pro Leu 85 90 95 Thr Phe Gly Ala Gly
Thr Thr Leu Glu Leu Lys 100 105 9616PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
96Tyr Ile Asp Pro Tyr Asn Arg Gly Ala Lys Tyr Asn Glu Lys Phe Gly 1
5 10 15 9716PRTArtificial SequenceDescription of Artificial
Sequence Synthetic CDR peptide 97Tyr Ile Asp Pro Tyr Asn Leu Gly
Ala Lys Tyr Asn Glu Lys Gly Phe 1 5 10 15 9816PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
98Tyr Ile Asp Pro Tyr Asn Lys Gly Ala Lys Tyr Asn Glu Lys Phe Gly 1
5 10 15 9916PRTArtificial SequenceDescription of Artificial
Sequence Synthetic CDR peptide 99Tyr Ile Asp Pro Tyr Asn Phe Gly
Ala Lys Tyr Asn Glu Lys Phe Gly 1 5 10 15 10016PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
100Tyr Ile Asp Pro Tyr Asn Ser Gly Ala Lys Tyr Asn Glu Lys Phe Gly
1 5 10 15 10116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic CDR peptide 101Tyr Ile Asp Pro Tyr Asn Tyr Gly
Ala Lys Tyr Asn Glu Lys Phe Gly 1 5 10 15 10216PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
102Tyr Ile Asp Pro Tyr Asn Val Gly Ala Lys Tyr Asn Glu Lys Phe Gly
1 5 10 15 10317PRTArtificial SequenceDescription of Artificial
Sequence Synthetic CDR peptide 103Tyr Ile Asp Pro Tyr Asn Asp Arg
Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly 10417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
104Tyr Ile Asp Pro Tyr Asn Asp Asn Ala Lys Tyr Asn Glu Lys Phe Lys
1 5 10 15 Gly 10517PRTArtificial SequenceDescription of Artificial
Sequence Synthetic CDR peptide 105Tyr Ile Asp Pro Tyr Asn Asp Gln
Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly 10617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
106Tyr Ile Asp Pro Tyr Asn Asp Glu Ala Lys Tyr Asn Glu Lys Phe Lys
1 5 10 15 Gly 10717PRTArtificial SequenceDescription of Artificial
Sequence Synthetic CDR peptide 107Tyr Ile Asp Pro Tyr Asn Asp Leu
Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly 10817PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
108Tyr Ile Asp Pro Tyr Asn Asp Lys Ala Lys Tyr Asn Glu Lys Phe Lys
1 5 10 15 Gly 10917PRTArtificial SequenceDescription of Artificial
Sequence Synthetic CDR peptide 109Tyr Ile Asp Pro Tyr Asn Asp Ser
Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly 11017PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
110Tyr Ile Asp Pro Tyr Asn Asp Tyr Ala Lys Tyr Asn Glu Lys Phe Lys
1 5 10 15 Gly 11117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic CDR peptide 111Tyr Ile Asp Pro Tyr Asn Asp Val
Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly 1127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
112Ala Ala Lys Thr Leu Ala Glu 1 5 1137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
113Tyr Ala Lys Thr Leu Ala Glu 1 5 1147PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
114Trp Ala Lys Thr Leu Ala Glu 1 5 1157PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
115Ser Ala Lys Thr Leu Ala Glu 1 5 1167PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
116Thr Ala Lys Thr Leu Ala Glu 1 5 1177PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
117Ile Ala Lys Thr Leu Ala Glu 1 5 1187PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
118Val Ala Lys Thr Leu Ala Glu 1 5 1197PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
119Asn Asn Lys Thr Leu Ala Glu 1 5 1207PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
120Asn Ile Lys Thr Leu Ala Glu 1 5 1217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
121Asn Leu Leu Thr Leu Ala Glu 1 5 1227PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
122Asn Thr Lys Thr Leu Ala Glu 1 5 1237PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
123Asn Val Lys Thr Leu Ala Glu 1 5 124119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Humanized
antibody chain polypeptide 124Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Val Met His Trp Val
Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Tyr Ile
Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ser Gln Lys Phe 50 55 60 Gln
Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Trp Tyr Phe Asp Val Trp
Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115
125119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Humanized antibody chain polypeptide 125Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30
Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35
40 45 Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ser Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Ala Ser
Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Trp Tyr
Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser
115 126119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Humanized antibody chain polypeptide 126Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30
Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Trp Tyr
Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser
115 127119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Humanized antibody chain polypeptide 127Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30
Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser
Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Trp Tyr
Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser
115 128119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Humanized antibody chain polypeptide 128Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30
Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser
Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Trp Tyr
Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser
115 129119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Humanized antibody chain polypeptide 129Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30
Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Ile Ser
Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr
Val Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Trp Tyr
Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser
115 130107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Humanized antibody chain polypeptide 130Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Glu His Ile Tyr Ser Tyr 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His
Phe Gly Ser Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu
Ile Lys 100 105 131107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Humanized antibody chain polypeptide
131Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu His Ile Tyr
Ser Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln His His Phe Gly Ser Pro Leu 85 90 95 Thr Phe Gly Gln
Gly Thr Arg Leu Glu Ile Lys 100 105 132107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Humanized
antibody chain polypeptide 132Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Glu His Ile Tyr Ser Tyr 20 25
30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln His
His Phe Gly Ser Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu
Glu Ile Lys 100 105 133107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Humanized antibody chain polypeptide
133Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu His Ile Tyr
Ser Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro
Lys Leu Leu Ile 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr
Tyr Cys Gln His His Phe Gly Ser Pro Leu 85 90 95 Thr Phe Gly Gln
Gly Thr Arg Leu Glu Ile Lys 100 105 13417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
134Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys Phe Gln
1 5 10 15 Gly 13517PRTArtificial SequenceDescription of Artificial
Sequence Synthetic CDR peptide 135Tyr Ile Asp Pro Tyr Asn Asp Gly
Ala Lys Tyr Ser Gln Lys Phe Gln 1 5 10 15 Gly 136116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic VH sequence
polypeptide 136Gln Val Gln Leu Met Glu Ser Gly Pro Gly Leu Val Gln
Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe
Pro Leu Thr Ser Tyr 20 25 30 Thr Val His Trp Val Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Trp Ser Ser Gly
Ser Thr Asp Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Asn Ile
Asn Arg Asp Ser Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn
Ser Leu Gln Thr Glu Asp Thr Ala Ile Tyr Phe Cys Thr 85 90 95 Lys
Ser Gly Trp Ala Phe Phe Asp Tyr Trp Gly Gln Gly Val Met Val 100 105
110 Thr Val Ser Ser 115 137106PRTArtificial SequenceDescription of
Artificial Sequence Synthetic VL sequence polypeptide 137Asp Ile
Gln Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Leu Asn Cys Ile Ala Ser Gln Asn Ile Tyr Lys Ser 20
25 30 Leu Ala Trp Tyr Gln Leu Lys Leu Gly Glu Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Asn Ala Asn Ser Leu Gln Ala Gly Ile Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ala Leu Thr Ile
Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Gln
Gln Tyr Ser Gly Gly Tyr Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu
Glu Leu Lys 100 105 138119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic VH sequence polypeptide 138Glu Val
Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Arg 1 5 10 15
Ser Leu Lys Ile Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20
25 30 Tyr Met Ala Trp Val Arg Leu Ala Pro Gln Lys Gly Leu Glu Trp
Val 35 40 45 Ala Ser Ile Ser Tyr Glu Gly Ser Arg Thr His Tyr Gly
Asp Ser Val 50 55 60 Arg Gly Arg Phe Ile Ile Ser Arg Asp Asn Pro
Lys Asn Ile Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Gly Ser Glu
Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg His Thr Gly Thr Leu
Asp Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Ile Val
Ser Ser 115 139106PRTArtificial SequenceDescription of Artificial
Sequence Synthetic VL sequence polypeptide 139Asn Ile Val Met Ala
Gln Ser Pro Lys Ser Met Ser Ile Ser Ala Gly 1 5 10 15 Asp Arg Val
Thr Met Asn Cys Lys Ala Ser Gln Asn Val Asp Asn Asn 20 25 30 Ile
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40
45 Phe Tyr Ala Ser Asn Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60 Gly Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Lys Ser Val
Gln Ala 65 70 75 80 Glu Asp Ala Ala Phe Tyr Tyr Cys Gln Arg Ile Tyr
Asn Phe Pro Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 14019PRTArtificial SequenceDescription of Artificial
Sequence Synthetic CDR peptide 140Met Pro Ser Phe Ile Thr Leu Ala
Ser Leu Ser Thr Xaa Glu Gly Tyr 1 5 10 15 Phe Asp Phe
1417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 141Tyr Ala Xaa Xaa Leu Gln Thr 1 5
1428PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 142Gln Gln Tyr Tyr Ser Gly Xaa Thr 1 5
143127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic consensus polypeptide 143Glu Val Gln Leu Gln Xaa Ser Gly
Xaa Gly Leu Xaa Lys Pro Xaa Xaa 1 5 10 15 Xaa Leu Ser Leu Thr Cys
Xaa Val Xaa Gly Xaa Ser Ile Xaa Ser Asp 20 25 30 Tyr Trp Gly Trp
Xaa Arg Xaa Xaa Pro Gly Xaa Xaa Xaa Glu Trp Xaa 35 40 45 Gly Phe
Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60
Ser Arg Xaa Xaa Ile Xaa Xaa Asp Thr Ser Lys Asn Gln Phe Xaa Leu 65
70 75 80 Xaa Leu Xaa Ser Val Thr Xaa Xaa Asp Thr Ala Xaa Tyr Xaa
Cys Ala 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser
Thr Xaa Glu Gly 100 105 110 Tyr Phe Asp Phe Trp Gly Xaa Gly Thr Xaa
Xaa Thr Val Ser Ser 115 120 125 144127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
polypeptide 144Glu Val Gln Leu Gln Xaa Ser Gly Xaa Gly Leu Xaa Lys
Pro Xaa Xaa 1 5 10 15 Thr Leu Ser Leu Thr Cys Xaa Val Xaa Gly Xaa
Ser Ile Xaa Ser Asp 20 25 30 Tyr Trp Gly Trp Xaa Arg Xaa Xaa Pro
Gly Lys Xaa Xaa Glu Trp Xaa 35 40 45 Gly Phe Ile Thr Tyr Ser Gly
Ser Thr Ser Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Xaa Thr Ile
Ser Xaa Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Xaa
Ser Val Thr Ala Xaa Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Xaa Glu Gly 100 105
110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Xaa Xaa Thr Val Ser Ser 115
120 125 145106PRTArtificial SequenceDescription of Artificial
Sequence Synthetic consensus polypeptide 145Asp Ile Gln Met Thr Gln
Ser Pro Ser Xaa Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Xaa Xaa Cys Lys Ala Ser Gln Ser Ile His Lys Asn 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Xaa Gly Xaa Xaa Pro Lys Xaa Leu Ile 35 40 45
Tyr Tyr Ala Xaa Xaa Leu Gln Thr Gly Xaa Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Xaa Leu Gln
Pro 65 70 75 80 Glu Asp Xaa Ala Thr Tyr Xaa Cys Gln Gln Tyr Tyr Ser
Gly Xaa Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Xaa Lys 100
105 146106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic consensus polypeptide 146Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Lys Ala Ser Gln Ser Ile His Lys Asn 20 25 30 Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Xaa Pro Lys Xaa Leu Ile 35 40 45 Tyr Tyr
Ala Xaa Xaa Leu Gln Thr Gly Xaa Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Xaa Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly
Xaa Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
14717PRTArtificial SequenceDescription of Artificial Sequence
Synthetic consensus peptide 147Tyr Ile Asp Pro Tyr Asn Xaa Xaa Ala
Lys Tyr Xaa Xaa Lys Phe Xaa 1 5 10 15 Gly 1487PRTArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
peptide 148Xaa Xaa Lys Thr Leu Ala Glu 1 5 149119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
polypeptide 149Glu Val Gln Leu Xaa Gln Ser Gly Xaa Glu Xaa Xaa Lys
Pro Gly Xaa 1 5 10 15 Ser Val Lys Xaa Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Ser Ser Tyr 20 25 30 Val Met His Trp Val Xaa Gln Xaa Pro
Gly Gln Xaa Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn
Xaa Xaa Ala Lys Tyr Xaa Xaa Lys Phe 50 55 60 Xaa Gly Xaa Xaa Thr
Leu Thr Xaa Asp Xaa Ser Xaa Ser Thr Xaa Tyr 65 70 75 80 Met Glu Leu
Ser Xaa Leu Xaa Ser Xaa Asp Xaa Xaa Val Tyr Tyr Cys 85 90 95 Ala
Arg Gly Gly Pro Tyr Gly Xaa Tyr Phe Asp Val Trp Gly Xaa Gly 100 105
110 Thr Thr Val Thr Val Ser Ser 115 150119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
polypeptide 150Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Xaa 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Ser Ser Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro
Gly Gln Xaa Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn
Xaa Xaa Ala Lys Tyr Xaa Xaa Lys Phe 50 55 60 Xaa Gly Arg Val Thr
Leu Thr Xaa Asp Xaa Ser Xaa Ser Thr Xaa Tyr 65 70 75 80 Met Glu Leu
Ser Xaa Leu Arg Ser Xaa Asp Thr Xaa Val Tyr Tyr Cys 85 90 95 Ala
Arg Gly Gly Pro Tyr Gly Xaa Tyr Phe Asp Val Trp Gly Gln Gly 100 105
110 Thr Thr Val Thr Val Ser Ser 115 151107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
polypeptide 151Asp Ile Gln Met Thr Gln Ser Pro Xaa Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Xaa Xaa Val Thr Ile Thr Cys Arg Ala Ser Glu
His Ile Tyr Ser Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Xaa Gly
Lys Xaa Pro Xaa Leu Leu Xaa 35 40 45 Tyr Xaa Xaa Lys Thr Leu Ala
Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Xaa Phe Xaa Leu Xaa Ile Xaa Ser Leu Gln Pro 65 70 75 80 Glu Asp Xaa
Xaa Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu 85 90 95 Thr
Phe Gly Xaa Gly Thr Xaa Leu Glu Xaa Lys 100 105 152107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
polypeptide 152Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu
His Ile Tyr Ser Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly
Lys Xaa Pro Lys Leu Leu Ile 35 40 45 Tyr Xaa Xaa Lys Thr Leu Ala
Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Xaa Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Xaa
Ala Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu 85 90 95 Thr
Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 15310PRTArtificial
SequenceDescription of Artificial Sequence Synthetic CDR peptide
153Gly Gly Pro Tyr Gly Xaa Tyr Phe Asp Val 1 5 10
15410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 154Gly Gly Pro Tyr Gly Ala Tyr Phe Asp Val 1
5 10 15510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 155Gly Gly Pro Tyr Gly Asp Tyr Phe Asp Val 1
5 10 15610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 156Gly Gly Pro Tyr Gly Glu Tyr Phe Asp Val 1
5 10 15710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 157Gly Gly Pro Tyr Gly Phe Tyr Phe Asp Val 1
5 10 15810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 158Gly Gly Pro Tyr Gly Gly Tyr Phe Asp Val 1
5 10 15910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 159Gly Gly Pro Tyr Gly Ile Tyr Phe Asp Val 1
5 10 16010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 160Gly Gly Pro Tyr Gly Lys Tyr Phe Asp Val 1
5 10 16110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 161Gly Gly Pro Tyr Gly Asn Tyr Phe Asp Val 1
5 10 16210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 162Gly Gly Pro Tyr Gly Gln Tyr Phe Asp Val 1
5 10 16310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 163Gly Gly Pro Tyr Gly Arg Tyr Phe Asp Val 1
5 10 16410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 164Gly Gly Pro Tyr Gly Ser Tyr Phe Asp Val 1
5 10 16510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 165Gly Gly Pro Tyr Gly Thr Tyr Phe Asp Val 1
5 10 16610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 166Gly Gly Pro Tyr Gly Val Tyr Phe Asp Val 1
5 10 16710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CDR peptide 167Gly Gly Pro Tyr Gly Tyr Tyr Phe Asp Val 1
5 10
* * * * *