U.S. patent application number 17/240944 was filed with the patent office on 2022-03-24 for anti-tigit antibodies and methods of use thereof.
The applicant listed for this patent is Agenus Inc.. Invention is credited to Dhan Sidhartha CHAND, Benjamin Maxime MORIN, Dennis John UNDERWOOD, Nicholas Stuart WILSON.
Application Number | 20220089732 17/240944 |
Document ID | / |
Family ID | 1000005988600 |
Filed Date | 2022-03-24 |
United States Patent
Application |
20220089732 |
Kind Code |
A1 |
CHAND; Dhan Sidhartha ; et
al. |
March 24, 2022 |
ANTI-TIGIT ANTIBODIES AND METHODS OF USE THEREOF
Abstract
The instant disclosure provides antibodies that specifically
bind to T-cell immunoreceptor with Ig and ITIM domains (TIGIT)
(e.g., human TIGIT) and antagonize TIGIT function. Also provided
are pharmaceutical compositions comprising these antibodies,
nucleic acids encoding these antibodies, expression vectors and
host cells for making these antibodies, and methods of treating a
subject using these antibodies.
Inventors: |
CHAND; Dhan Sidhartha;
(Woburn, MA) ; WILSON; Nicholas Stuart; (San
Carlos, CA) ; UNDERWOOD; Dennis John; (Boston,
MA) ; MORIN; Benjamin Maxime; (Somerville,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Agenus Inc. |
Lexington |
MA |
US |
|
|
Family ID: |
1000005988600 |
Appl. No.: |
17/240944 |
Filed: |
April 26, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15968094 |
May 1, 2018 |
11021537 |
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17240944 |
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62492829 |
May 1, 2017 |
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62500345 |
May 2, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/28 20130101;
C07K 2317/734 20130101; C07K 2317/33 20130101; C07K 2317/52
20130101; C07K 16/2818 20130101; A61P 35/04 20180101; G01N 33/505
20130101; C07K 2317/72 20130101; C07K 2317/24 20130101; C07K
2317/34 20130101; C07K 16/2878 20130101; C07K 16/2875 20130101;
C07K 2319/30 20130101; C07K 2317/73 20130101; C07K 2317/92
20130101; C07K 2317/74 20130101; C07K 2317/71 20130101; A61K
2039/57 20130101; C07K 16/30 20130101; C07K 2317/76 20130101; A61K
39/395 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; A61P 35/04 20060101
A61P035/04; C07K 16/30 20060101 C07K016/30; G01N 33/50 20060101
G01N033/50 |
Claims
1. An isolated antibody that specifically binds to human TIGIT, the
antibody comprising a heavy chain variable region comprising
complementarity determining regions (CDRs) CDRH1, CDRH2, and CDRH3,
and a light chain variable region comprising complementarity
determining regions CDRL1, CDRL2, and CDRL3, wherein: (a) CDRH1
comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2;
(b) CDRH2 comprises the amino acid sequence of SEQ ID NO: 3 or SEQ
ID NO: 4; (c) CDRH3 comprises the amino acid sequence of SEQ ID NO:
5; (d) CDRL1 comprises the amino acid sequence of SEQ ID NO: 6; (e)
CDRL2 comprises the amino acid sequence of SEQ ID NO: 7; and/or (f)
CDRL3 comprises the amino acid sequence of SEQ ID NO: 8.
2. The isolated antibody of claim 1, wherein CDRH1, CDRH2, CDRH3,
CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences set forth
in SEQ ID NOs: 1, 3, 5, 6, 7, and 8; or 2, 4, 5, 6, 7, and 8,
respectively.
3. (canceled)
4. The isolated antibody of claim 1, wherein: (a) the heavy chain
variable region comprises an amino acid sequence that is at least
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
the amino acid sequence of SEQ ID NO: 9; optionally wherein the
heavy chain variable region comprises the amino acid sequence of
SEQ ID NO: 9, optionally wherein X in SEQ ID NO: 9 is glutamate (E)
or pyroglutamate (pE); and/or (b) the light chain variable region
comprises an amino acid sequence that is at least 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid
sequence of SEQ ID NO: 10; optionally wherein the light chain
variable region comprises the amino acid of SEQ ID NO: 10,
optionally wherein X in SEQ ID NO: 9 is glutamate (E) or
pyroglutamate (pE).
5-22. (canceled)
23. The isolated antibody of claim 1, wherein: (a) the heavy chain
variable region comprises an amino acid sequence derived from a
human IGHV1-69*01 germline sequence; (b) the heavy chain variable
region comprises an amino acid sequence derived from a human
IGHV1-69*06 germline sequence; (c) the heavy chain variable region
comprises an amino acid sequence derived from a human IGHV1-69*12
germline sequence; (d) the a light chain variable region comprises
an amino acid sequence derived from a human IGLV2-14*01 germline
sequence; (e) the light chain variable region comprises an amino
acid sequence derived from a human IGLV2-23*02 germline sequence;
and/or (f) the light chain variable region comprises an amino acid
sequence derived from a human IGLV2-11*01 germline sequence.
24-28. (canceled)
29. The isolated antibody, of claim 1 wherein: (a) the heavy chain
variable region comprises an amino acid region that is at least
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
the amino acid sequence of SEQ ID NO: 34 or 35, optionally wherein
the heavy chain variable region comprises the amino acid sequence
set forth in SEQ ID NO: 9; and/or (b) the light chain variable
region comprises an amino acid region that is at least 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino
acid sequence of any one of SEQ ID NOs: 37-39 and 60, optionally
wherein the light chain variable region comprises the amino acid
sequence set forth in SEQ ID NO: 10.
30-31. (canceled)
32. An isolated antibody that specifically binds to human TIGIT,
wherein: (a) the antibody binds to the same epitope of human TIGIT
as an antibody comprising a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO: 9 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO: 10; (b) the
antibody binds to an epitope located within a region of human
TIGIT, the amino acid sequence of the region consisting of the
amino acid sequence of any one of SEQ ID NOs: 31-33; (c) the
antibody binds to one or more amino acid residues of human TIGIT
selected from the group consisting of Q35, I47, N49, H90, and T96,
numbered according to the amino acid sequence of SEQ ID NO: 40; (d)
the antibody does not bind to one or more of the amino acid
residues selected from the group consisting of T34, L52, H55, I56,
S57, P58, S59, T98, R100, and F102 of human TIGIT, numbered
according to the amino acid sequence of SEQ ID NO: 40; (e) the
binding of the antibody to a protein comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 36, 44,
45, 46, 48, 51, 52, 53, 57, and 59 is substantially weakened
relative to the binding of the antibody to a protein comprising the
amino acid sequence of SEQ ID NO: 42; and/or (f) the binding of the
antibody to a protein comprising an amino acid sequence selected
from the group consisting of SEQ ID NO: 43, 47, 49, 54, 55, 56, and
58 is not substantially weakened relative to the binding of the
antibody to a protein comprising the amino acid sequence of SEQ ID
NO: 42.
33-106. (canceled)
107. The isolated antibody of claim 1, wherein the antibody further
comprises a heavy chain constant region selected from the group
consisting of human IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4,
IgA.sub.1, and IgA.sub.2, optionally wherein: (a) the antibody
comprises an IgG.sub.1 heavy chain constant region, optionally
wherein (1) the IgG.sub.1 heavy chain constant region comprises the
amino acid sequence of SEQ ID NO: 19, (2) the amino acid sequence
of the IgG.sub.1 heavy chain constant region comprises an N297A
mutation, numbered according to the EU numbering system, (3) the
IgG.sub.1 heavy chain constant region comprises the amino acid
sequence of SEQ ID NO: 20, (4) the amino acid sequence of the
IgG.sub.1 heavy chain constant region comprises L234F, L235F, and
N297A mutations, numbered according to the EU numbering system, (5)
the IgG.sub.1 heavy chain constant region comprises the amino acid
sequence of SEQ ID NO: 21, (6) the amino acid sequence of the
IgG.sub.1 heavy chain constant region comprises S239D and I332E
mutations, numbered according to the EU numbering system, (7) the
IgG.sub.1 heavy chain constant region comprises the amino acid
sequence of SEQ ID NO: 22, (8) the amino acid sequence of the
IgG.sub.1 heavy chain constant region comprises S239D, A330L, and
I332E mutations, numbered according to the EU numbering system, (9)
the IgG.sub.1 heavy chain constant region comprises the amino acid
sequence of SEQ ID NO: 23, (10) the amino acid sequence of the
IgG.sub.1 heavy chain constant region comprises L235V, F243L,
R292P, Y300L, and P396L mutations, numbered according to the EU
numbering system, (11) the IgG.sub.1 heavy chain constant region
comprises the amino acid sequence of SEQ ID NO: 24, (12) the amino
acid sequence of the IgG.sub.1 heavy chain constant region
comprises 5267E and L328F mutations, numbered according to the EU
numbering system, (13) the IgG.sub.1 heavy chain constant region
comprises the amino acid sequence of SEQ ID NO: 25, and/or (14) the
IgG.sub.1 heavy chain constant region is afucosylated; (b) the
antibody comprises an IgG.sub.4 heavy chain constant region,
optionally wherein (1) the amino acid sequence of the IgG.sub.4
heavy chain constant region comprises a S228P mutation, numbered
according to the EU numbering system, and/or (2) the IgG.sub.4
heavy chain constant region comprises the amino acid sequence of
SEQ ID NO: 26; and/or (c) antibody comprises a light chain constant
region comprising the amino acid sequence of SEQ ID NO: 28.
108-122. (canceled)
123. The isolated antibody of claim 107, wherein the increase of
Fc.gamma.RIIIA and/or Fc.gamma.RIIA activity in a first cytotoxic
cell contacted with the antibody is greater than the increase of
Fc.gamma.RIIIA and/or Fc.gamma.RIIA activity in a second cytotoxic
cell contacted with a reference antibody comprising the same heavy
chain variable region as the antibody, and a heavy chain constant
region comprising the amino acid sequence of SEQ ID NO: 19, wherein
the cytotoxic cell is optionally a natural killer cell.
124-128. (canceled)
129. The isolated antibody of claim 1 comprising: (a) a heavy chain
comprising or consisting of an amino acid sequence selected from
the group consisting of SEQ ID NOs: 11-18; and/or (b) a light chain
comprising or consisting of the amino acid sequence of SEQ ID NO:
27.
130. (canceled)
131. The isolated antibody of claim 1, wherein: (a) the antibody is
a human antibody; (b) the antibody is a bispecific antibody; (c)
the antibody is antagonistic to human TIGIT; (d) the antibody
preferentially kills regulatory T cells over effector T cells in a
population of peripheral blood mononuclear cells (PBMCs) in vitro;
(e) the antibody decreases or inhibits binding of human TIGIT to
PVR or PVRL2 relative to the level of binding in the absence of the
antibody; (f) the antibody induces IL-2 and/or IFN.gamma.
production by PBMCs stimulated with staphylococcal enterotoxin A
(SEA); (g) the antibody is conjugated to a cytotoxic agent,
cytostatic agent, toxin, radionuclide, or detectable label; (h) the
antibody is cross-linked to a second antibody or a fragment
thereof; and/or (i) the antigen-binding fragment specifically binds
to human TIGIT.
132-139. (canceled)
140. A pharmaceutical composition comprising the antibody of claim
1 and a pharmaceutically acceptable carrier or excipient.
141. An isolated polynucleotide encoding a heavy chain and/or light
chain of the antibody of claim 1.
142. A vector comprising the polynucleotide of claim 141.
143. A recombinant host cell comprising the polynucleotide of claim
141.
144. A method of producing an antibody that specifically binds to
human TIGIT, or an antigen-binding fragment thereof, the method
comprising culturing the host cell of claim 143 such that the
polynucleotide is expressed and the antibody, or antigen-binding
fragment, is produced.
145. A method of: (a) increasing T cell activation in response to
an antigen in a subject; (b) decreasing or inhibiting Treg activity
in response to an antigen in a subject; and/or (c) increasing NK
cell activation in response to an antigen in a subject, the method
comprising administering to the subject an effective amount of the
antibody of claim 1.
146-147. (canceled)
148. A method of: (a) treating cancer in a subject; or (b) treating
an infectious disease in a subject, the method comprising
administering to the subject an effective amount of the antibody of
claim 1, optionally wherein the antibody is administered
intravenously, subcutaneously, intratumorally, or is delivered to a
tumor draining lymph node.
149-168. (canceled)
169. A method of: (a) increasing T cell activation in response to
an antigen in a subject; (b) decreasing or inhibiting Treg activity
in response to an antigen in a subject; and/or (c) increasing NK
cell activation in response to an antigen in a subject, the method
comprising administering to the subject an effective amount of the
antibody of claim 32.
170. A method of: (a) treating cancer in a subject; or (b) treating
an infectious disease in a subject, the method comprising
administering to the subject an effective amount of the antibody of
claim 32, optionally wherein the antibody is administered
intravenously, subcutaneously, intratumorally, or is delivered to a
tumor draining lymph node.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Nos. 62/492,829, filed May 1, 2017; and 62/500,345,
filed May 2, 2017, each of which is incorporated by reference
herein in its entirety.
1. FIELD
[0002] The instant disclosure relates to antibodies that
specifically bind to TIGIT (e.g., human TIGIT) and methods of using
the same.
2. BACKGROUND
[0003] The protein T-cell immunoreceptor with Ig and ITIM domains
(TIGIT), also known as VSIG9 or VSTM3, is a type I transmembrane
protein in the immunoglobulin (Ig) superfamily. It has a single Ig
domain, a type I transmembrane domain, a single intracellular
immunoreceptor tyrosine-based inhibitory motif (ITIM) and a single
immunoglobulin tail tyrosine (ITT)-like phosphorylation motif and
is expressed on activated CD4-positive/CD25-positive regulatory T
cells (Tregs), memory CD45RO-positive T cells, and natural killer
(NK) cells, but not naive T cells.
[0004] Poliovirus receptor (PVR, also known as CD155) is highly
expressed on monocytes and dendritic cells, and is capable of
activating effector T cells and NK cells, as well as attenuating
the activity of Tregs, through binding to its two receptors CD226
and CD96. TIGIT binds to PVR and has been shown to antagonize the
interaction of PVR with CD226 and CD96, thereby suppressing T cell-
and NK cell-mediated immune activity.
[0005] Given the apparent role of human TIGIT in modulating immune
responses, therapeutic agents designed to antagonize TIGIT
signaling hold great promise for the treatment of diseases that
involve immune suppression.
3. SUMMARY
[0006] The instant disclosure provides antibodies that specifically
bind to TIGIT (e.g., human TIGIT) and antagonize TIGIT function,
e.g., TIGIT-mediated immune suppression. Also provided are
pharmaceutical compositions comprising these antibodies, nucleic
acids encoding these antibodies, expression vectors and host cells
for making these antibodies, and methods of treating a subject
using these antibodies. The antibodies disclosed herein are
particularly useful for increasing T cell and NK cell activation in
response to an antigen (e.g., a tumor antigen or an infectious
disease antigen) and/or decreasing Treg-mediated immune
suppression, and hence, are useful for treating cancer in a subject
or treating or preventing an infectious disease in a subject.
[0007] Accordingly, in one aspect, the instant disclosure provides
an antibody or isolated antibody comprising a heavy chain variable
region (VH) comprising complementarity determining regions (CDRs)
CDRH1, CDRH2 and CDRH3 and a light chain variable region (VL)
comprising complementarity determining regions CDRL1, CDRL2 and
CDRL3, wherein:
(a) CDRH1 comprises the amino acid sequence of SYGIS (SEQ ID NO: 1)
or GYTFASY (SEQ ID NO: 2); (b) CDRH2 comprises the amino acid
sequence of GITPFFNRVDVAEKFQG (SEQ ID NO: 3) or TPFFNR (SEQ ID NO:
4); (c) CDRH3 comprises the amino acid sequence of CDRH3 comprises
the amino acid sequence of DLRRGGVGDAFDI (SEQ ID NO: 5); (d) CDRL1
comprises the amino acid sequence of CDRL1 comprises the amino acid
sequence of TGTSSDVGSHNYVS (SEQ ID NO: 6); (e) CDRL2 comprises the
amino acid sequence of EVSYRPS (SEQ ID NO: 7); and/or (f) CDRL3
comprises the amino acid sequence of SSYTPSSATV (SEQ ID NO: 8).
[0008] In certain embodiments, the CDRH1, CDRH2, CDRH3, CDRL1,
CDRL2, and CDRL3 comprise the amino acid sequences set forth in SEQ
ID NOs: 1, 3, 5, 6, 7, and 8, respectively. In certain embodiments,
the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino
acid sequences set forth in SEQ ID NOs: 2, 4, 5, 6, 7, and 8,
respectively.
[0009] In certain embodiments, the antibody comprises a heavy chain
variable region comprising an amino acid sequence that is at least
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
the amino acid sequence of SEQ ID NO: 9. In certain embodiments,
the heavy chain variable region comprises the amino acid sequence
of SEQ ID NO: 9. In certain embodiments, the amino acid sequence of
the heavy chain variable region consists of the amino acid sequence
of SEQ ID NO: 9. In certain embodiments, X in SEQ ID NO: 9 is
glutamate (E). In certain embodiments, X in SEQ ID NO: 9 is
pyroglutamate (pE).
[0010] In certain embodiments, the antibody comprises a light chain
variable region comprising an amino acid sequence that is at least
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
the amino acid sequence of SEQ ID NO: 10. In certain embodiments,
the light chain variable region comprises the amino acid sequence
of SEQ ID NO: 10. In certain embodiments, the amino acid sequence
of the light chain variable region consists of the amino acid
sequence of SEQ ID NO: 10. In certain embodiments, X in SEQ ID NO:
10 is glutamine (Q). In certain embodiments, X in SEQ ID NO: 10 is
pyroglutamate (pE).
[0011] In another aspect, the instant disclosure provides an
isolated antibody that specifically binds to human TIGIT, the
antibody comprising a heavy chain variable region comprising the
amino acid sequence of SEQ ID NO: 9. In certain embodiments, the
amino acid sequence of the heavy chain variable region consists of
the amino acid sequence of SEQ ID NO: 9. In certain embodiments, X
in SEQ ID NO: 9 is glutamate (E). In certain embodiments, X in SEQ
ID NO: 9 is pyroglutamate (pE).
[0012] In another aspect, the instant disclosure provides an
isolated antibody that specifically binds to human TIGIT, the
antibody comprising a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 10. In certain embodiments, the
amino acid sequence of the light chain variable region consists of
the amino acid sequence of SEQ ID NO: 10. In certain embodiments, X
in SEQ ID NO: 10 is glutamine (Q). In certain embodiments, X in SEQ
ID NO: 10 is pyroglutamate (pE).
[0013] In another aspect, the instant disclosure provides an
isolated antibody that specifically binds to human TIGIT, the
antibody comprising a heavy chain variable region comprising the
amino acid sequence of SEQ ID NO: 9 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO: 10. In
certain embodiments, the amino acid sequence of the heavy chain
variable region consists of the amino acid sequence of SEQ ID NO: 9
the amino acid sequence of the light chain variable region consists
of the amino acid sequence of SEQ ID NO: 10. In certain
embodiments, X in SEQ ID NO: 9 is glutamate (E). In certain
embodiments, X in SEQ ID NO: 9 is pyroglutamate (pE). In certain
embodiments, X in SEQ ID NO: 10 is glutamine (Q). In certain
embodiments, X in SEQ ID NO: 10 is pyroglutamate (pE).
[0014] In another aspect, the instant disclosure provides an
isolated antibody that specifically binds to human TIGIT, the
antibody comprising a heavy chain variable region having an amino
acid sequence derived from a human IGHV1-69*01 germline sequence.
In another aspect, the instant disclosure provides an isolated
antibody that specifically binds to human TIGIT, the antibody
comprising a heavy chain variable region having an amino acid
sequence derived from a human IGHV1-69*06 germline sequence. In
another aspect, the instant disclosure provides an isolated
antibody that specifically binds to human TIGIT, the antibody
comprising a heavy chain variable region having an amino acid
sequence derived from a human IGHV1-69*12 germline sequence.
[0015] In another aspect, the instant disclosure provides an
isolated antibody that specifically binds to human TIGIT, the
antibody comprising a light chain variable region having an amino
acid sequence derived from a human IGLV2-14*01 germline sequence.
In another aspect, the instant disclosure provides an isolated
antibody that specifically binds to human TIGIT, the antibody
comprising a light chain variable region having an amino acid
sequence derived from a human IGLV2-23*02 germline sequence. In
another aspect, the instant disclosure provides an isolated
antibody that specifically binds to human TIGIT, the antibody
comprising a light chain variable region having an amino acid
sequence derived from a human IGLV2-11*01 germline sequence.
[0016] In another aspect, the instant disclosure provides an
isolated antibody that specifically binds to human TIGIT, the
antibody comprising a heavy chain variable region comprising an
amino acid region that is at least 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ
ID NO: 34 or 35. In another aspect, the instant disclosure provides
an isolated antibody that specifically binds to human TIGIT, the
antibody comprising a light chain variable region comprising an
amino acid region that is at least 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to the amino acid sequence of any
one of SEQ ID NOs: 37-39 and 60.
[0017] In certain embodiments, the antibody binds to the same
epitope of human TIGIT as an antibody comprising a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO: 9
and a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 10.
[0018] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody binds to the same epitope of human TIGIT as an
antibody comprising a heavy chain variable region comprising the
amino acid sequence of SEQ ID NO: 9 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO: 10.
[0019] In certain embodiments, the antibody binds to an epitope
located within a region of human TIGIT, the amino acid sequence of
the region consisting of the amino acid sequence of any one of SEQ
ID NOs: 31-33.
[0020] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody binds to an epitope located within a region of human
TIGIT, wherein the amino acid sequence of the region consists of
the amino acid sequence of any one of SEQ ID NOs: 31-33.
[0021] In certain embodiments, the antibody binds to one or more
amino acid residues of human TIGIT selected from the group
consisting of Q35, I47, N49, H90, and T96, numbered according to
the amino acid sequence of SEQ ID NO: 40. In certain embodiments,
the antibody binds to one or more amino acid residues of human
TIGIT selected from the group consisting of Q35, I47, and T96,
numbered according to the amino acid sequence of SEQ ID NO: 40. In
certain embodiments, the antibody binds to amino acid residue T96
of human TIGIT, numbered according to the amino acid sequence of
SEQ ID NO: 40. In certain embodiments, the binding between the
antibody and a protein comprising the amino acid sequence of SEQ ID
NO: 52 is substantially weakened (e.g., reduced by at least 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%)
relative to the binding of the antibody to a protein comprising the
amino acid sequence of SEQ ID NO: 42. In certain embodiments, the
binding between the antibody and a protein comprising the amino
acid sequence of SEQ ID NO: 53 is substantially weakened (e.g.,
reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42. In
certain embodiments, the antibody binds to amino acid residue Q35
of human TIGIT, numbered according to the amino acid sequence of
SEQ ID NO: 40. In certain embodiments, the binding between the
antibody and a protein comprising the amino acid sequence of SEQ ID
NO: 44 is substantially weakened (e.g., reduced by at least 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%)
relative to the binding of the antibody to a protein comprising the
amino acid sequence of SEQ ID NO: 42. In certain embodiments, the
antibody binds to amino acid residue 147 of human TIGIT, numbered
according to the amino acid sequence of SEQ ID NO: 40. In certain
embodiments, the binding between the antibody and a protein
comprising the amino acid sequence of SEQ ID NO: 45 is
substantially weakened (e.g., reduced by at least 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) relative to
the binding of the antibody to a protein comprising the amino acid
sequence of SEQ ID NO: 42. In certain embodiments, the antibody
binds to amino acid residue N49 of human TIGIT, numbered according
to the amino acid sequence of SEQ ID NO: 40. In certain
embodiments, the binding between the antibody and a protein
comprising the amino acid sequence of SEQ ID NO: 46 is
substantially weakened (e.g., reduced by at least 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) relative to
the binding of the antibody to a protein comprising the amino acid
sequence of SEQ ID NO: 42. In certain embodiments, the binding
between the antibody and a protein comprising the amino acid
sequence of SEQ ID NO: 36 is substantially weakened (e.g., reduced
by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, or 90%) relative to the binding of the antibody to a protein
comprising the amino acid sequence of SEQ ID NO: 42. In certain
embodiments, the antibody binds to amino acid residue H90 of human
TIGIT, numbered according to the amino acid sequence of SEQ ID NO:
40. In certain embodiments, the binding between the antibody and a
protein comprising the amino acid sequence of SEQ ID NO: 51 is
substantially weakened (e.g., reduced by at least 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) relative to
the binding of the antibody to a protein comprising the amino acid
sequence of SEQ ID NO: 42. In certain embodiments, the binding
between the antibody and a protein comprising the amino acid
sequence of SEQ ID NO: 57 is substantially weakened (e.g., reduced
by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, or 90%) relative to the binding of the antibody to a protein
comprising the amino acid sequence of SEQ ID NO: 42. In certain
embodiments, the binding between the antibody and a protein
comprising the amino acid sequence of SEQ ID NO: 59 is
substantially weakened (e.g., reduced by at least 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) relative to
the binding of the antibody to a protein comprising the amino acid
sequence of SEQ ID NO: 42. In certain embodiments, the binding
between the antibody and a protein comprising the amino acid
sequence of SEQ ID NO: 48 is substantially weakened (e.g., reduced
by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, or 90%) relative to the binding of the antibody to a protein
comprising the amino acid sequence of SEQ ID NO: 42.
[0022] In certain embodiments, the antibody does not bind to one or
more of the amino acid residues of human TIGIT selected from the
group consisting of T34, L52, H55, I56, S57, P58, S59, T98, R100,
and F102, numbered according to the amino acid sequence of SEQ ID
NO: 40. In certain embodiments, the antibody does not bind to amino
acid residue T34 of human TIGIT, numbered according to the amino
acid sequence of SEQ ID NO: 40. In certain embodiments, the binding
of the antibody to a protein comprising the amino acid sequence of
SEQ ID NO: 43 is not substantially weakened (e.g., not reduced by
more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, or 90%) relative to the binding of the antibody to a protein
comprising the amino acid sequence of SEQ ID NO: 42. In certain
embodiments, the antibody does not bind to amino acid residue L52
of human TIGIT, numbered according to the amino acid sequence of
SEQ ID NO: 40. In certain embodiments, the binding of the antibody
to a protein comprising the amino acid sequence of SEQ ID NO: 47 is
not substantially weakened (e.g., not reduced by more than 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%)
relative to the binding of the antibody to a protein comprising the
amino acid sequence of SEQ ID NO: 42. In certain embodiments, the
antibody does not bind to amino acid residue H55 of human TIGIT,
numbered according to the amino acid sequence of SEQ ID NO: 40. In
certain embodiments, the binding of the antibody to a protein
comprising the amino acid sequence of SEQ ID NO: 49 is not
substantially weakened (e.g., not reduced by more than 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) relative
to the binding of the antibody to a protein comprising the amino
acid sequence of SEQ ID NO: 42. In certain embodiments, the
antibody does not bind to amino acid residue 156 of human TIGIT,
numbered according to the amino acid sequence of SEQ ID NO: 40. In
certain embodiments, the antibody does not bind to amino acid
residue S57 of human TIGIT, numbered according to the amino acid
sequence of SEQ ID NO: 40. In certain embodiments, the antibody
does not bind to amino acid residue P58 of human TIGIT, numbered
according to the amino acid sequence of SEQ ID NO: 40. In certain
embodiments, the antibody does not bind to amino acid residue S59
of human TIGIT, numbered according to the amino acid sequence of
SEQ ID NO: 40. In certain embodiments, the binding of the antibody
to a protein comprising the amino acid sequence of SEQ ID NO: 58 is
not substantially weakened (e.g., not reduced by more than 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%)
relative to the binding of the antibody to a protein comprising the
amino acid sequence of SEQ ID NO: 42. In certain embodiments, the
antibody does not bind to amino acid residue T98 of human TIGIT,
numbered according to the amino acid sequence of SEQ ID NO: 40. In
certain embodiments, the binding of the antibody to a protein
comprising the amino acid sequence of SEQ ID NO: 54 is not
substantially weakened (e.g., not reduced by more than 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) relative
to the binding of the antibody to a protein comprising the amino
acid sequence of SEQ ID NO: 42. In certain embodiments, the
antibody does not bind to amino acid residue R100 of human TIGIT,
numbered according to the amino acid sequence of SEQ ID NO: 40. In
certain embodiments, the binding of the antibody to a protein
comprising the amino acid sequence of SEQ ID NO: 55 is not
substantially weakened (e.g., not reduced by more than 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) relative
to the binding of the antibody to a protein comprising the amino
acid sequence of SEQ ID NO: 42.
[0023] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody binds to one or more amino acid residues of human
TIGIT selected from the group consisting of Q35, I47, N49, H90, and
T96, numbered according to the amino acid sequence of SEQ ID NO:
40.
[0024] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody binds to one or more amino acid residues of human
TIGIT selected from the group consisting of Q35, I47, and T96,
numbered according to the amino acid sequence of SEQ ID NO: 40.
[0025] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody binds to amino acid residue T96 of human TIGIT,
numbered according to the amino acid sequence of SEQ ID NO: 40.
[0026] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding between the antibody and a protein comprising the amino
acid sequence of SEQ ID NO: 52 is substantially weakened (e.g.,
reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0027] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding between the antibody and a protein comprising the amino
acid sequence of SEQ ID NO: 53 is substantially weakened (e.g.,
reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0028] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody binds to amino acid residue Q35 of human TIGIT,
numbered according to the amino acid sequence of SEQ ID NO: 40.
[0029] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding between the antibody and a protein comprising the amino
acid sequence of SEQ ID NO: 44 is substantially weakened (e.g.,
reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0030] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody binds to amino acid residue 147 of human TIGIT,
numbered according to the amino acid sequence of SEQ ID NO: 40.
[0031] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding between the antibody and a protein comprising the amino
acid sequence of SEQ ID NO: 45 is substantially weakened (e.g.,
reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0032] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody binds to amino acid residue N49 of human TIGIT,
numbered according to the amino acid sequence of SEQ ID NO: 40.
[0033] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding between the antibody and a protein comprising the amino
acid sequence of SEQ ID NO: 46 is substantially weakened (e.g.,
reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0034] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding between the antibody and a protein comprising the amino
acid sequence of SEQ ID NO: 36 is substantially weakened (e.g.,
reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0035] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody binds to amino acid residue H90 of human TIGIT,
numbered according to the amino acid sequence of SEQ ID NO: 40.
[0036] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding between the antibody and a protein comprising the amino
acid sequence of SEQ ID NO: 51 is substantially weakened (e.g.,
reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0037] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding between the antibody and a protein comprising the amino
acid sequence of SEQ ID NO: 57 is substantially weakened (e.g.,
reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0038] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding between the antibody and a protein comprising the amino
acid sequence of SEQ ID NO: 59 is substantially weakened (e.g.,
reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0039] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding between the antibody and a protein comprising the amino
acid sequence of SEQ ID NO: 48 is substantially weakened (e.g.,
reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0040] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody does not bind to one or more of the amino acid
residues selected from the group consisting of T34, L52, H55, I56,
S57, P58, S59, T98, R100, and F102 of human TIGIT, numbered
according to the amino acid sequence of SEQ ID NO: 40.
[0041] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody does not bind to amino acid residue T34 of human
TIGIT, numbered according to the amino acid sequence of SEQ ID NO:
40.
[0042] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding of the antibody to a protein comprising the amino acid
sequence of SEQ ID NO: 43 is not substantially weakened (e.g., not
reduced by more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0043] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody does not bind to amino acid residue L52 of human
TIGIT, numbered according to the amino acid sequence of SEQ ID NO:
40.
[0044] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding of the antibody to a protein comprising the amino acid
sequence of SEQ ID NO: 47 is not substantially weakened (e.g., not
reduced by more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0045] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody does not bind to amino acid residue H55 of human
TIGIT, numbered according to the amino acid sequence of SEQ ID NO:
40.
[0046] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding of the antibody to a protein comprising the amino acid
sequence of SEQ ID NO: 49 is not substantially weakened (e.g., not
reduced by more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0047] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody does not bind to amino acid residue 156 of human
TIGIT, numbered according to the amino acid sequence of SEQ ID NO:
40.
[0048] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody does not bind to amino acid residue S57 of human
TIGIT, numbered according to the amino acid sequence of SEQ ID NO:
40.
[0049] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody does not bind to amino acid residue P58 of human
TIGIT, numbered according to the amino acid sequence of SEQ ID NO:
40.
[0050] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody does not bind to amino acid residue S59 of human
TIGIT, numbered according to the amino acid sequence of SEQ ID NO:
40.
[0051] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding of the antibody to a protein comprising the amino acid
sequence of SEQ ID NO: 58 is not substantially weakened (e.g., not
reduced by more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0052] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody does not bind to amino acid residue T98 of human
TIGIT, numbered according to the amino acid sequence of SEQ ID NO:
40.
[0053] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding of the antibody to a protein comprising the amino acid
sequence of SEQ ID NO: 54 is not substantially weakened (e.g., not
reduced by more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0054] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody does not bind to amino acid residue R100 of human
TIGIT, numbered according to the amino acid sequence of SEQ ID NO:
40.
[0055] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding of the antibody to a protein comprising the amino acid
sequence of SEQ ID NO: 55 is not substantially weakened (e.g., not
reduced by more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0056] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody does not bind to amino acid residue F102 of human
TIGIT, numbered according to the amino acid sequence of SEQ ID NO:
40.
[0057] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the binding of the antibody to a protein comprising the amino acid
sequence of SEQ ID NO: 56 is not substantially weakened (e.g., not
reduced by more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90%) relative to the binding of the antibody to a
protein comprising the amino acid sequence of SEQ ID NO: 42.
[0058] In another aspect the instant disclosure provides, an
isolated antibody that specifically binds to human TIGIT, wherein
the antibody does not bind to any of amino acid residues T34, L52,
H55, I56, S57, P58, S59, T98, R100, and F102 of human TIGIT,
numbered according to the amino acid sequence of SEQ ID NO: 40.
[0059] In certain embodiments, the antibody further comprises a
heavy chain constant region selected from the group consisting of
human IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2.
[0060] In certain embodiments, the antibody comprises an IgG.sub.1
heavy chain constant region.
[0061] In certain embodiments, the antibody comprises a heavy chain
constant region comprising the amino acid sequence of SEQ ID NO:
19. In certain embodiments, the amino acid sequence of the
IgG.sub.1 heavy chain constant region comprises an N297A mutation,
numbered according to the EU numbering system. In certain
embodiments, the antibody comprises a heavy chain constant region
comprising the amino acid sequence of SEQ ID NO: 20.
[0062] In certain embodiments, the amino acid sequence of the
IgG.sub.1 heavy chain constant region comprises L234F, L235F, and
N297A mutations, numbered according to the EU numbering system. In
certain embodiments, the antibody comprises a heavy chain constant
region comprising the amino acid sequence of SEQ ID NO: 21.
[0063] In certain embodiments, the amino acid sequence of the
IgG.sub.1 heavy chain constant region comprises S239D and I332E
mutations, numbered according to the EU numbering system. In
certain embodiments, the antibody comprises a heavy chain constant
region comprising the amino acid sequence of SEQ ID NO: 22.
[0064] In certain embodiments, the amino acid sequence of the
IgG.sub.1 heavy chain constant region comprises S239D, A330L, and
I332E mutations, numbered according to the EU numbering system. In
certain embodiments, the antibody comprises a heavy chain constant
region comprising the amino acid sequence of SEQ ID NO: 23.
[0065] In certain embodiments, the amino acid sequence of the
IgG.sub.1 heavy chain constant region comprises L235V, F243L,
R292P, Y300L, and P396L mutations, numbered according to the EU
numbering system. In certain embodiments, the antibody comprises a
heavy chain constant region comprising the amino acid sequence of
SEQ ID NO: 24.
[0066] In certain embodiments, the IgG.sub.1 heavy chain constant
region is afucosylated.
[0067] In certain embodiments, the amino acid sequence of the
IgG.sub.1 heavy chain constant region comprises S267E and L328F
mutations, numbered according to the EU numbering system. In
certain embodiments, the antibody comprises a heavy chain constant
region comprising the amino acid sequence of SEQ ID NO: 25.
[0068] In certain embodiments, the increase of Fc.gamma.RIIIA
and/or Fc.gamma.RIIA activity in a first cytotoxic cell contacted
with the antibody is greater than the increase of Fc.gamma.RIIIA
and/or
[0069] Fc.gamma.RIIA activity in a second cytotoxic cell contacted
with a reference antibody comprising the same heavy chain variable
region as the antibody, and a heavy chain constant region
comprising the amino acid sequence of SEQ ID NO: 19. In certain
embodiments, the cytotoxic cell is a natural killer cell.
[0070] In certain embodiments, the antibody comprises an IgG.sub.4
heavy chain constant region. In certain embodiments, the amino acid
sequence of the IgG.sub.4 heavy chain constant region comprises an
S228P mutation, numbered according to the EU numbering system.
[0071] In certain embodiments, the antibody comprises a heavy chain
constant region comprising the amino acid sequence of SEQ ID NO:
26. In certain embodiments, the antibody comprises a light chain
constant region comprising the amino acid sequence of SEQ ID NO:
28.
[0072] In another aspect, the instant disclosure provides an
isolated antibody that specifically binds to human TIGIT, the
antibody comprising:
[0073] (a) a heavy chain comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs: 11-18; and/or
[0074] (b) a light chain comprising the amino acid sequence of SEQ
ID NO: 27.
[0075] In certain embodiments, the amino acid sequence of the heavy
chain consists of an amino acid sequence selected from the group
consisting of SEQ ID NOs: 11-18; and/or the amino acid sequence of
the light chain consists of the amino acid sequence of SEQ ID NO:
27.
[0076] In certain embodiments, the antibody is a human antibody. In
certain embodiments, the antibody is a bispecific antibody. In
certain embodiments, the antibody is antagonistic to human
TIGIT.
[0077] In certain embodiments, the antibody preferentially kills
regulatory T cells over effector T cells in a population of
peripheral blood mononuclear cells (PBMCs) in vitro. In certain
embodiments, the antibody decreases or inhibits binding of human
TIGIT to PVR or PVRL2 relative to the level of binding in the
absence of the antibody. In certain embodiments, the antibody
induces IL-2 and/or IFN.gamma. production by PBMCs stimulated with
staphylococcal enterotoxin A (SEA).
[0078] In certain embodiments, the antibody is conjugated to a
cytotoxic agent, cytostatic agent, toxin, radionuclide, or
detectable label. In certain embodiments, the antibody is
cross-linked to a second antibody or a fragment thereof.
[0079] In another aspect, the instant disclosure provides an
isolated antigen-binding fragment of the antibody disclosed herein,
wherein the antigen-binding fragment specifically binds to human
TIGIT.
[0080] In another aspect, the instant disclosure provides a
pharmaceutical composition comprising an antibody or
antigen-binding fragment as disclosed herein, and a
pharmaceutically acceptable carrier or excipient.
[0081] In another aspect, the instant disclosure provides an
isolated polynucleotide encoding a heavy chain and/or light chain
of the antibody or antigen-binding fragment as disclosed
herein.
[0082] In another aspect, the instant disclosure provides a vector
comprising a polynucleotide as disclosed herein.
[0083] In another aspect, the instant disclosure provides a
recombinant host cell comprising a polynucleotide or vector as
disclosed herein.
[0084] In another aspect, the instant disclosure provides a method
of producing an antibody that specifically binds to human TIGIT, or
an antigen-binding fragment thereof, the method comprising
culturing a host cell as disclosed herein such that the
polynucleotide is expressed and the antibody, or antigen-binding
fragment, is produced.
[0085] In another aspect, the instant disclosure provides a method
of increasing T cell activation in response to an antigen in a
subject, the method comprising administering to the subject an
effective amount of an antibody, antigen-binding fragment, or
pharmaceutical composition as disclosed herein. In another aspect,
the instant disclosure provides a method of decreasing or
inhibiting Treg activity in response to an antigen in a subject,
the method comprising administering to the subject an effective
amount of an antibody, antigen-binding fragment, or pharmaceutical
composition as disclosed herein. In another aspect, the instant
disclosure provides a method of increasing NK cell activation in
response to an antigen in a subject, the method comprising
administering to the subject an effective amount of an antibody,
antigen-binding fragment, or pharmaceutical composition as
disclosed herein. In another aspect, the instant disclosure
provides a method of treating cancer in a subject, the method
comprising administering to the subject an effective amount of an
antibody, antigen-binding fragment, or pharmaceutical composition
as disclosed herein.
[0086] In certain embodiments, the antibody, antigen-binding
fragment, or pharmaceutical composition is administered
intravenously. In certain embodiments, the antibody,
antigen-binding fragment, or pharmaceutical composition is
administered intravenously at 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3
mg/kg, 6 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, or more, optionally
at an interval of once every three weeks.
[0087] In certain embodiments, the antibody, antigen-binding
fragment, or pharmaceutical composition is administered
intratumorally. In certain embodiments, the antibody,
antigen-binding fragment, or pharmaceutical composition is
administered intratumorally at 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1
mg/kg, 3 mg/kg, 6 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, or more,
optionally at an interval of once every three weeks.
[0088] In certain embodiments, the antibody, antigen-binding
fragment, or pharmaceutical composition is administered
subcutaneously. In certain embodiments, the antibody,
antigen-binding fragment, or pharmaceutical composition is
delivered to a tumor draining lymph node.
[0089] In certain embodiments, a method disclosed herein further
comprises administering an additional therapeutic agent to the
subject. In certain embodiments, the additional therapeutic agent
is administered systemically.
[0090] In certain embodiments, the subject has a solid tumor and
the additional therapeutic agent comprises an anti-PD-1 antibody,
optionally wherein the anti-PD-1 antibody is pembrolizumab or
nivolumab.
[0091] In certain embodiments, the subject has head and neck
squamous cell carcinoma and wherein the additional therapeutic
agent is an anti-EGFR antibody, optionally wherein the anti-EGFR
antibody is cetuximab, optionally wherein the method further
comprises administering a chemotherapeutic agent to the subject,
optionally wherein the chemotherapeutic agent is administered
systemically, and optionally wherein the chemotherapeutic agent is
gemcitabine.
[0092] In certain embodiments, the subject has HER2+ breast cancer
and wherein the additional therapeutic agent is an anti-HER2
antibody, optionally wherein the anti-HER2 antibody is trastuzumab,
optionally wherein the method further comprises administering a
chemotherapeutic agent to the subject, optionally wherein the
chemotherapeutic agent is administered systemically, optionally
wherein the chemotherapeutic agent is gemcitabine.
[0093] In certain embodiments, the additional therapeutic agent is
a chemotherapeutic or a checkpoint targeting agent. In certain
embodiments, the checkpoint targeting agent is selected from the
group consisting of an antagonist anti-PD-1 antibody, an antagonist
anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, an
antagonist anti-CTLA-4 antibody, an antagonist anti-TIM-3 antibody,
an antagonist anti-LAG-3 antibody, an antagonist VISTA antibody, an
antagonist CD96 antibody, an antagonist anti-CEACAM1 antibody, an
agonist anti-CD137 antibody, an agonist anti-GITR antibody, and an
agonist anti-OX40 antibody.
[0094] In certain embodiments, the additional therapeutic agent is
an inhibitor of indoleamine-2,3-dioxygenase (IDO). In certain
embodiments, the inhibitor is selected from the group consisting of
epacadostat, F001287, indoximod, and NLG919.
[0095] In certain embodiments, the additional therapeutic agent is
a vaccine. In certain embodiments, the vaccine comprises a heat
shock protein peptide complex (HSPPC) comprising a heat shock
protein complexed with an antigenic peptide. In certain
embodiments, the heat shock protein is hsc70 and is complexed with
a tumor-associated antigenic peptide. In certain embodiments, the
heat shock protein is gp96 protein and is complexed with a
tumor-associated antigenic peptide, wherein the HSPPC is derived
from a tumor obtained from a subject.
[0096] In another aspect, the instant disclosure provides a method
of treating an infectious disease in a subject, the method
comprising administering to the subject an effective amount of an
antibody, antigen-binding fragment, or pharmaceutical composition
as disclosed herein.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0097] FIGS. 1A-1D are a series of surface plasmon resonance (SPR)
sensorgrams showing the binding of the anti-TIGIT antibody BA002 to
purified TIGIT protein. FIGS. 1A, 1B and 1C show the binding of
human dimeric TIGIT-Fc, cynomolgus dimeric TIGIT-Fc, and human
monomeric TIGIT-His, respectively, to captured BA002. FIG. 1D shows
the binding of BA002 (in Fab format) to captured human dimeric
TIGIT-Fc protein. In each sensorgram, response units (RU) are
plotted against time after protein injection.
[0098] FIGS. 2A-2D are a series of graphs showing the binding of
the anti-TIGIT antibody BA002 or an IgG.sub.1 isotype control
antibody to cells expressing cell surface human TIGIT or cynomolgus
monkey TIGIT. The levels of binding of BA002 or an IgG1 isotype
control antibody to Jurkat cells engineered to express human TIGIT
(FIG. 2A), activated primary CD4+CD25+ T cells (FIG. 2B), activated
primary CD8+CD25+ T cells (FIG. 2C), or CHO cells engineered to
express cynomolgus TIGIT (FIG. 2D), as assessed by median
fluorescence intensity (MFI), are plotted against the
concentrations of BA002 incubated with the cells.
[0099] FIGS. 3A-3B are a series of histograms and graphs showing
that BA002 exhibited no binding to TIGIT-related family members
CD96 and CD226. The levels of binding of BA002 or an IgG1 isotype
control antibody to Jurkat cells engineered to express human TIGIT,
CD96, and CD226 (FIG. 3A) or CD96 and CD226 only (FIG. 3B), as
assessed by median fluorescence intensity (MFI), are plotted
against the concentrations of BA002 incubated with the cells.
[0100] FIGS. 4A-4F are a series of graphs showing that BA002
disrupted binding between TIGIT and its ligand, CD155/PVR, at
levels comparable to or greater than a panel of reference
anti-TIGIT antibodies.
[0101] FIGS. 5A-5F are a series of graphs showing that BA002
disrupted binding between TIGIT and its ligand, CD112/PVRL2, at
levels comparable or greater than a panel of reference anti-TIGIT
antibodies.
[0102] FIG. 6 is a graph showing that BA002 enhanced
interferon-.gamma. (IFN.gamma.) secretion by SEA-stimulated PBMCs
to a greater degree than reference anti-TIGIT antibodies, and that
the combination of BA002 and an anti-PD-1 antibody further enhanced
IFN.gamma. secretion by SEA-stimulated PBMCs beyond that observed
for BA002 alone. The degree of enhancement observed in the
anti-PD-1 combination was also greater for BA002 than for the
reference anti-TIGIT antibodies.
[0103] FIGS. 7A-7B are a series of graphs showing that the
combination of BA002 with an anti-CTLA-4 antibody enhanced
interleukin-2 (IL-2) secretion by SEA-stimulated PBMCs from two
different donors, compared to isotype controls.
[0104] FIGS. 8A-8D are a series of graphs showing that BA006 bound
to cells expressing human TIGIT and cynomolgus monkey TIGIT, and
that BA006 did not bind to the related family members CD96 and
CD226. BA006 bound to activated primary human CD4.sup.+ T cells
(FIG. 8A) and to CHO cells engineered to express cynomolgus TIGIT
(FIG. 8B). BA006 bound to Jurkat cells expressing TIGIT, CD96, and
CD226 (FIG. 8C), but did not bind to Jurkat cells expressing CD96
and CD226 alone (FIG. 8D). In each graph, the median fluorescence
intensity (MFI) is plotted against antibody concentration.
[0105] FIGS. 9A-9D are a series of graphs showing that Fc variants
of BA002 further enhanced PBMC cytokine secretion (FIGS. 9A-9B) and
T cell activation, as measured by upregulation of CD25 (FIGS.
9C-9D). The Fc variants of BA002 also showed further enhancement of
cytokine secretion and T cell activation when combined with an
anti-PD-1 antibody.
[0106] FIGS. 10A-10E are a series of graphs showing that BA002 and
Fc variants thereof enhanced IL-2 secretion in SEA-stimulated PBMCs
from five separate donors in a dose-dependent manner. In one donor,
BA002, the Fc variants BA006 and BA005, and an afucosylated form of
BA002 (BA002_AF) enhanced IL-2 secretion by SEA-stimulated PBMCs
(FIG. 10A). In a second donor, the combination of BA002 or a
variant thereof with an anti-PD-1 antibody also enhanced IL-2
secretion by SEA-stimulated PBMCs (FIG. 10B). BA006 and BA002
enhanced IL-2 secretion by SEA-stimulated PBMCs from a third donor
in the presence of CD155-Fc (FIG. 10C). FIGS. 10D and 10E show
dose-dependent activation by BA006 of PBMCs from two different
donors in the presence of a low concentration (10 ng/mL) of
SEA.
[0107] FIGS. 11A-11B are a series of graphs showing that BA002 and
variants thereof enhanced IFN.gamma. secretion by SEA-stimulated
PBMCs from two different donors, relative to isotype control
antibodies.
[0108] FIGS. 12A-12B are a series of graphs showing the capacity of
various Fc variants of the anti-TIGIT antibody BA002 to signal
through Fc.gamma.RIIA (FIG. 12A) or Fc.gamma.RIIIA (FIG. 12B) when
co-engaged with TIGIT expressing target cells (Jurkat cells
engineered to express human TIGIT). In FIG. 12A, isotype controls
for BA002 (i.e., isotype 002) and each variant (i.e., isotype 003,
isotype 005, isotype 006, and isotype 007) were only tested at the
highest antibody concentration (i.e., 1000 ng/mL). In FIG. 12B, the
isotype controls were only tested at the two highest antibody
concentrations (i.e., 30 and 10 ng/mL).
[0109] FIGS. 13A-13B are two series of graphs showing that BA002
and variants thereof promoted antibody-dependent cell-mediated
cytotoxicity (ADCC) of TIGIT-expressing cells. Percent cell killing
of TIGIT-expressing Jurkat cells at four time points (0 hours, 1
hour, 2 hours, and 3 hours) after incubation with antibodies at
three different concentrations (0.1 .mu.g/mL, 1 .mu.g/mL, and 10
.mu.g/mL) is shown in FIG. 13A. Preferential targeting of
regulatory T cells by BA002 and BA006 for NK cell-mediated ADCC in
a co-culture setting, as compared with activated effector T cells,
is shown in FIG. 13B.
[0110] FIG. 14 is a graph showing that the anti-TIGIT antibodies
BA002 and BA006 enhanced IL-2 secretion by SEA-stimulated PBMCs,
with BA006 exhibiting substantially greater enhancement of IL-2
secretion than reference anti-TIGIT antibodies.
[0111] FIGS. 15A-15I are a series of graphs showing that the
anti-TIGIT antibodies BA002 and BA006 can effectively combine with
an antagonistic anti-PD-1 antibody (FIG. 15A), an antagonistic
anti-PD-L1 antibody (FIGS. 15B and 15C), an agonistic anti-CD137
antibody (FIG. 15D), an antagonistic anti-CTLA-4 antibody (FIG.
15E), an antagonistic anti-LAG-3 antibody (FIGS. 15F and 15G), or
an agonistic anti-OX40 antibody (FIGS. 15H and 15I) to promote IL-2
secretion by SEA-stimulated PBMCs.
[0112] FIGS. 16A-16B are a series of graphs showing production of
IL-2 (FIG. 16A) and IFN.gamma. (FIG. 16B) from cynomolgus PBMCs
after incubation with BA002 or BA006 in the presence or absence of
an anti-PD-1 antibody. The isotype control antibodies for BA002 and
BA006 are "Isotype.G1" and "Isotype.3M," respectively. The isotype
control antibody for the anti-PD-1 antibody is "Isotype.G4."
[0113] FIGS. 17A-17F are a series of graphs and histograms showing
the effect of anti-TIGIT antibodies on MHC class I-mediated memory
T cell recall. FIG. 17A is a graph showing interferon gamma
(IFN.gamma.) production over time by CMV-reactive PBMCs stimulated
with CMV pp65 peptide and BA002 or BA006. FIG. 17B is a set of
representative histograms showing the expression of TIGIT, CD226,
and CD96 on activated CD8 effector memory T cells (grey area; the
black lines with white fills indicate staining of cells with
isotype control antibodies). FIGS. 17C and 17D show the production
of IFN.gamma. and TNF.alpha., respectively, from the stimulated
PBMCs. FIGS. 17E and 17F show the percentage of proliferating
cells, as indicated by Ki67 positive staining, in the CD8 effector
memory T cell population and CD4 effector memory T cell population
from stimulated PBMCs.
[0114] FIGS. 18A-18D are a series of graphs showing the effect of
anti-TIGIT antibodies on MHC class II-mediated memory T cell
recall. FIGS. 18A and 18B are representative graphs from three CMV
seropositive donors showing the levels of TIGIT expression on
subsets of CD4 T cells and CD8 T cells from CMV-reactive PBMCs
stimulated with CMV whole antigen, which were known to be primarily
processed and presented on MHC class II. FIGS. 18C and 18D show the
production of IFN.gamma. by PBMCs from two different donors in the
presence or absence of BA002, BA006, an anti-TIGIT reference
antibody, and/or an anti-PD-1 antibody.
[0115] FIG. 19 is a graph showing the percentage of killing of
NY-ESO-1 expressing tumor cells over time by co-cultured primary
human T cells expressing a NY-ESO-1 TCR in the presence or absence
of BA002 or its isotype control antibody ("IgG1 isotype"), BA006 or
its isotype control antibody ("IgG1-3M isotype"), or an anti-PD-1
antibody or its isotype control antibody ("IgG4 isotype"), either
alone or in combination, as measured by live cell imaging, relative
to the number of the tumor cells at the time of addition of the T
cells.
[0116] FIGS. 20A-20G show the effects of anti-TIGIT antibodies on
NK cell activation. FIG. 20A is a series of graphs showing the
gating parameters for identifying NK cells from the PBMC
population, and a histogram showing the distribution of the CD107a
activation marker. FIGS. 20B-20G are a series of graphs showing the
percentage of cells positive for CD107a (FIGS. 20B and 20E),
IFN.gamma. (FIGS. 20C and 20F), and TNF.alpha. (FIGS. 20D and 20G)
out of all the NK cells in the PBMC population, after incubation of
the PBMC population with the indicated antibodies either alone
(FIGS. 20B-20D) or in a co-culture with K562 cells (FIGS. 20E-20G).
"Ref. 1 IgG1" refers to reference antibody #1 in the IgG1 format,
and "Ref 1-FcE" refers to a variant of reference antibody #1
comprising the S239D/A330L/1332E substitutions in the Fc
region.
[0117] FIG. 21 is a sequence alignment of human TIGIT (SEQ ID NO:
29) and cynomolgus monkey TIGIT (SEQ ID NO: 70). The BA002 epitope
regions identified by hydrogen-deuterium exchange (HDX)-mass
spectrometry are indicated in bold and underlining, with
differences between the human and cynomolgus sequences in these
regions shown without underlining. The signal peptide and
transmembrane domains of TIGIT are indicated with boxes.
[0118] FIGS. 22A-22B are ribbon diagrams showing the structure of
human TIGIT protein with specific amino acid residues highlighted.
FIG. 22A shows the amino acid residues in the BA002 epitope regions
of TIGIT, as identified by HDX, facing the PVR-binding surface of
the protein. FIG. 22B shows Q35, I47, H90, T96, and N49, which may
constitute a conformational epitope bound by BA006.
[0119] FIGS. 23A-23F are a series of graphs showing inhibition of
tumor progression by an anti-TIGIT antibody in a xenograft mouse
model in which the test antibodies were administered at an early
stage of tumor progression. The median tumor volumes are plotted
against time in FIG. 23A, and the tumor volumes of each individual
mouse are plotted against time in FIGS. 23B-23F (n=5 per treatment
group).
[0120] FIGS. 24A-24G are a series of graphs showing inhibition of
tumor progression by various surrogate anti-TIGIT antibodies in
combination with an anti-PD-1 antibody in a xenograft mouse model
in which the test antibodies were administered at an early stage of
tumor progression. The median tumor volumes are plotted against
time in FIGS. 24A and 24B (with different y-axis scales), and the
tumor volumes of each individual mouse are plotted against time in
FIGS. 24C-24G (n=5 per treatment group). Surrogate antibodies
"anti-TIGIT mIgG2a," "anti-TIGIT mIgG2a-N297Q," "anti-TIGIT mIgG1,"
and "anti-TIGIT mIgG2 (Fc enhanced)" differ only in their Fc
regions in accordance with their names.
[0121] FIGS. 25A-25E are a series of graphs showing inhibition of
tumor progression by anti-TIGIT surrogate antibody mIgG2a
("anti-TIGIT mIgG2a") or its isotype control antibody ("Isotype
Control 1"), or surrogate antibody mIgG2a (Fc enhanced)
("anti-TIGIT mIgG2 (Fc enhanced)") or its isotype control antibody
("Isotype Control 2"), in a xenograft mouse model in which the test
antibodies were administered at a late stage of tumor progression.
The mean tumor volumes are plotted against time in FIG. 25A, and
the tumor volumes of each individual mouse are plotted against time
in FIGS. 25B-25E (n=10 per treatment group). The dotted line in
FIGS. 25B-25E represents a standard to euthanize mice having tumor
volumes exceeding 2000 mm.sup.3.
[0122] FIGS. 26A-26B are a series of graphs showing inhibition of
tumor progression by anti-TIGIT surrogate antibody mIgG2a
("anti-TIGIT mIgG2a") or its isotype control antibody ("Isotype
Control 1"), or surrogate antibody mIgG2a (Fc enhanced)
("anti-TIGIT mIgG2 (Fc enhanced)") or its isotype control antibody
("Isotype Control 2"), in combination with another checkpoint
modulating antibody in a xenograft mouse model in which the test
antibodies were administered at a late stage of tumor progression.
The mean tumor volumes of mice treated with an anti-TIGIT antibody
and an anti-PD-1 antibody (FIG. 26A) or an anti-CTLA-4 antibody
(FIG. 26B) are plotted against time (n=10 per treatment group for
each figure).
[0123] FIGS. 27A-27F are a series of graphs showing a study design
and comparisons of the amounts of T cell subsets in tumors and
tumor-draining lymph nodes (TDLNs) in a mouse xenograft model after
administration of anti-TIGIT surrogate antibody mIgG2a ("anti-TIGIT
mIgG2a") or its isotype control antibody ("Isotype Control 1"), or
surrogate antibody mIgG2a (Fc enhanced) ("anti-TIGIT mIgG2 (Fc
enhanced)") or its isotype control antibody ("Isotype Control 2").
An agonistic anti-GITR antibody ("DTA-1 (mIgG2a)") was used as a
positive control for regulatory T cell depletion. FIG. 27A
illustrates the study design. The relative changes in the amounts
of intratumoral FoxP3.sup.+ regulatory T cells (Tregs) (FIG. 27B),
intratumoral CD4.sup.+ non-Tregs (FIG. 27C), FoxP3.sup.+ Tregs in
tumor-draining lymph nodes (TDLNs) (FIG. 27D), and intratumoral
CD8.sup.+ T cells (FIG. 27E) are plotted against time post
injection of anti-TIGIT antibody. The ratios of intratumoral
CD8.sup.+ T cells to intratumoral Tregs are shown in FIG. 27F (n=4
per treatment group and time point).
[0124] FIGS. 28A-28C are a series of graphs showing the involvement
of Fc.gamma.RIV in anti-TIGIT antibody-mediated T cell activation.
FIG. 28A shows the results of cell-based luciferase reporter assays
that examined the effect of various concentrations of anti-TIGIT
surrogate antibody mIgG2a ("anti-TIGIT mIgG2a") or its isotype
control antibody ("Isotype Control 1"), or surrogate antibody
mIgG2a (Fc enhanced) ("anti-TIGIT mIgG2 (Fc enhanced)") or its
isotype control antibody ("Isotype Control 2") on effector T cell
activation in a co-culture of Fc.gamma.RIV-expressing effector T
cells and murine TIGIT-expressing CHO cells. The relative
luciferase activity (RLU) is plotted against antibody
concentration. FIGS. 28B and 28C show the results of a murine in
vivo immune activation assay that examined the effect of anti-TIGIT
mIgG2a or an mIgG2 anti-CTLA-4 antibody ("anti-CLTA-4 mIgG2a") on
CD4.sup.+ (FIG. 28B) and CD8.sup.+ (FIG. 28C) T cell proliferation
in response to SEB superantigen in the presence or absence of an
anti-Fc.gamma.RIV antibody (n=4 mice per group, data representative
of at least two independent experiments). Proliferation was
determined by assaying the percentage of Ki67.sup.+ T cells using
flow cytometry.
5. DETAILED DESCRIPTION
[0125] The instant disclosure provides antibodies that specifically
bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and
antagonize TIGIT function, e.g., TIGIT-mediated immune suppression.
Also provided are pharmaceutical compositions comprising these
antibodies, nucleic acids encoding these antibodies, expression
vectors and host cells for making these antibodies, and methods of
treating a subject using these antibodies. The antibodies disclosed
herein are particularly useful for increasing T cell and NK cell
activation in response to an antigen (e.g., a tumor antigen or an
infectious disease antigen), and hence, are useful for treating
cancer in a subject or treating or preventing an infectious disease
in a subject. All instances of "isolated antibodies" described
herein are additionally contemplated as antibodies that may be, but
need not be, isolated. All instances of "isolated polynucleotides"
described herein are additionally contemplated as polynucleotides
that may be, but need not be, isolated. All instances of
"antibodies" described herein are additionally contemplated as
antibodies that may be, but need not be, isolated. All instances of
"polynucleotides" described herein are additionally contemplated as
polynucleotides that may be, but need not be, isolated.
5.1 Definitions
[0126] As used herein, the terms "about" and "approximately," when
used to modify a numeric value or numeric range, indicate that
deviations of 5% to 10% above (e.g., up to 5% to 10% above) and 5%
to 10% below (e.g., up to 5% to 10% below) the value or range
remain within the intended meaning of the recited value or
range.
[0127] As used herein, the term "TIGIT" refers to T-cell
immunoreceptor with Ig and ITIM domains (also known as VSIG9 or
VSTM3) that in humans is encoded by the TIGIT gene. As used herein,
the term "human TIGIT" refers to a TIGIT protein encoded by a
wild-type human TIGIT gene (e.g., GenBank.TM. accession number
NM_173799.3) or an extracellular domain of such a protein. An
exemplary amino acid sequence of an immature human TIGIT protein is
provided as SEQ ID NO: 29. An exemplary amino acid sequence of a
mature human TIGIT protein is provided as SEQ ID NO: 40. Exemplary
amino acid sequences of an extracellular domain of a mature human
TIGIT protein are provided as SEQ ID NOs: 30, 41, and 42.
[0128] As used herein, the terms "antibody" and "antibodies"
include full length antibodies, antigen-binding fragments of full
length antibodies, and molecules comprising antibody CDRs, VH
regions, and/or VL regions. Examples of antibodies include, without
limitation, monoclonal antibodies, recombinantly produced
antibodies, monospecific antibodies, multispecific antibodies
(including bispecific antibodies), human antibodies, humanized
antibodies, chimeric antibodies, immunoglobulins, synthetic
antibodies, tetrameric antibodies comprising two heavy chain and
two light chain molecules, an antibody light chain monomer, an
antibody heavy chain monomer, an antibody light chain dimer, an
antibody heavy chain dimer, an antibody light chain-antibody heavy
chain pair, intrabodies, heteroconjugate antibodies, antibody-drug
conjugates, single domain antibodies, monovalent antibodies, single
chain antibodies or single-chain Fvs (scFv), camelized antibodies,
affybodies, Fab fragments, F(ab').sub.2 fragments, disulfide-linked
Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g.,
anti-anti-Id antibodies), and antigen-binding fragments of any of
the above. In certain embodiments, antibodies described herein
refer to polyclonal antibody populations. Antibodies can be of any
type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g.,
IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1 or
IgA.sub.2), or any subclass (e.g., IgG.sub.2a or IgG.sub.2b) of
immunoglobulin molecule. In certain embodiments, antibodies
described herein are IgG antibodies, or a class (e.g., human
IgG.sub.1 or IgG.sub.4) or subclass thereof. In a specific
embodiment, the antibody is a humanized monoclonal antibody. In
another specific embodiment, the antibody is a human monoclonal
antibody.
[0129] As used herein, the terms "VH region" and "VL region" refer,
respectively, to single antibody heavy and light chain variable
regions, comprising FR (Framework Regions) 1, 2, 3 and 4 and CDR
(Complementarity Determining Regions) 1, 2 and 3 (see Kabat et al.,
(1991) Sequences of Proteins of Immunological Interest (NIH
Publication No. 91-3242, Bethesda), which is herein incorporated by
reference in its entirety).
[0130] As used herein, the term "CDR" or "complementarity
determining region" means the noncontiguous antigen combining sites
found within the variable region of both heavy and light chain
polypeptides. These particular regions have been described by Kabat
et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al.,
Sequences of protein of immunological interest. (1991), by Chothia
et al., J. Mol. Biol. 196:901-917 (1987), and by MacCallum et al.,
J. Mol. Biol. 262:732-745 (1996), all of which are herein
incorporated by reference in their entireties, where the
definitions include overlapping or subsets of amino acid residues
when compared against each other. In certain embodiments, the term
"CDR" is a CDR as defined by MacCallum et al., J. Mol. Biol.
262:732-745 (1996) and Martin A. "Protein Sequence and Structure
Analysis of Antibody Variable Domains," in Antibody Engineering,
Kontermann and Dubel, eds., Chapter 31, pp. 422-439,
Springer-Verlag, Berlin (2001). In certain embodiments, the term
"CDR" is a CDR as defined by Kabat et al., J. Biol. Chem. 252,
6609-6616 (1977) and Kabat et al., Sequences of protein of
immunological interest. (1991). In certain embodiments, heavy chain
CDRs and light chain CDRs of an antibody are defined using
different conventions. In certain embodiments, heavy chain CDRs
and/or light chain CDRs are defined by performing structural
analysis of an antibody and identifying residues in the variable
region(s) predicted to make contact with an epitope region of a
target molecule (e.g., human and/or cynomolgus TIGIT). CDRH1, CDRH2
and CDRH3 denote the heavy chain CDRs, and CDRL1, CDRL2 and CDRL3
denote the light chain CDRs.
[0131] As used herein, the term "framework (FR) amino acid
residues" refers to those amino acids in the framework region of an
immunoglobulin chain. The term "framework region" or "FR region" as
used herein, includes the amino acid residues that are part of the
variable region, but are not part of the CDRs (e.g., using the
Kabat or MacCallum definition of CDRs).
[0132] As used herein, the terms "variable region" and "variable
domain" are used interchangeably and are common in the art. The
variable region typically refers to a portion of an antibody,
generally, a portion of a light or heavy chain, typically about the
amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in
the mature heavy chain and about 90 to 115 amino acids in the
mature light chain, which differ extensively in sequence among
antibodies and are used in the binding and specificity of a
particular antibody for its particular antigen. The variability in
sequence is concentrated in those regions called complementarity
determining regions (CDRs) while the more highly conserved regions
in the variable domain are called framework regions (FR). Without
wishing to be bound by any particular mechanism or theory, it is
believed that the CDRs of the light and heavy chains are primarily
responsible for the interaction and specificity of the antibody
with antigen. In certain embodiments, the variable region is a
human variable region. In certain embodiments, the variable region
comprises rodent or murine CDRs and human framework regions (FRs).
In particular embodiments, the variable region is a primate (e.g.,
non-human primate) variable region. In certain embodiments, the
variable region comprises rodent or murine CDRs and primate (e.g.,
non-human primate) framework regions (FRs).
[0133] The terms "VL" and "VL domain" are used interchangeably to
refer to the light chain variable region of an antibody.
[0134] The terms "VH" and "VH domain" are used interchangeably to
refer to the heavy chain variable region of an antibody.
[0135] As used herein, the terms "constant region" and "constant
domain" are interchangeable and are common in the art. The constant
region is an antibody portion, e.g., a carboxyl terminal portion of
a light and/or heavy chain which is not directly involved in
binding of an antibody to antigen but which can exhibit various
effector functions, such as interaction with an Fc receptor (e.g.,
Fc gamma receptor). The constant region of an immunoglobulin
molecule generally has a more conserved amino acid sequence
relative to an immunoglobulin variable domain.
[0136] As used herein, the term "heavy chain" when used in
reference to an antibody can refer to any distinct type, e.g.,
alpha (.alpha.), delta (.delta.), epsilon (.epsilon.), gamma
(.gamma.), and mu (.mu.), based on the amino acid sequence of the
constant domain, which give rise to IgA, IgD, IgE, IgG, and IgM
classes of antibodies, respectively, including subclasses of IgG,
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, and IgG.sub.4.
[0137] As used herein, the term "light chain" when used in
reference to an antibody can refer to any distinct type, e.g.,
kappa (.kappa.) or lambda (.lamda.) based on the amino acid
sequence of the constant domains. Light chain amino acid sequences
are well known in the art. In specific embodiments, the light chain
is a human light chain.
[0138] As used herein, the term "EU numbering system" refers to the
EU numbering convention for the constant regions of an antibody, as
described in Edelman, G. M. et al., Proc. Natl. Acad. USA, 63,
78-85 (1969) and Kabat et al, Sequences of Proteins of
Immunological Interest, U.S. Dept. Health and Human Services, 5th
edition, 1991, each of which is herein incorporated by reference in
its entirety.
[0139] "Binding affinity" generally refers to the strength of the
sum total of non-covalent interactions between a single binding
site of a molecule (e.g., an antibody) and its binding partner
(e.g., an antigen). Unless indicated otherwise, as used herein,
"binding affinity" refers to intrinsic binding affinity which
reflects a 1:1 interaction between members of a binding pair (e.g.,
antibody and antigen). The affinity of a molecule X for its partner
Y can generally be represented by the dissociation constant
(K.sub.D). Affinity can be measured and/or expressed in a number of
ways known in the art, including, but not limited to, equilibrium
dissociation constant (K.sub.D), and equilibrium association
constant (K.sub.A). The K.sub.D is calculated from the quotient of
k.sub.off/k.sub.on, whereas K.sub.A is calculated from the quotient
of k.sub.on/k.sub.off. k.sub.on refers to the association rate
constant of, e.g., an antibody to an antigen, and k.sub.off refers
to the dissociation rate constant of, e.g., an antibody to an
antigen. The k.sub.on and k.sub.off can be determined by techniques
known to one of ordinary skill in the art, such as BIAcore.RTM. or
KinExA. As used herein, a "lower affinity" refers to a larger
K.sub.D.
[0140] As used herein, the terms "specifically binds,"
"specifically recognizes," "immunospecifically binds," and
"immunospecifically recognizes" are analogous terms in the context
of antibodies and refer to molecules that bind to an antigen (e.g.,
epitope or immune complex) as such binding is understood by one
skilled in the art. For example, a molecule that specifically binds
to an antigen can bind to other peptides or polypeptides, generally
with lower affinity as determined by, e.g., immunoassays,
BIAcore.RTM., KinExA 3000 instrument (Sapidyne Instruments, Boise,
Id.), or other assays known in the art. In a specific embodiment,
molecules that specifically bind to an antigen bind to the antigen
with a K.sub.A that is at least 2 logs (e.g., factors of 10), 2.5
logs, 3 logs, 4 logs or greater than the K.sub.A when the molecules
bind non-specifically to another antigen.
[0141] In another specific embodiment, molecules that specifically
bind to an antigen do not cross react with other proteins under
similar binding conditions. In another specific embodiment,
molecules that specifically bind to TIGIT do not cross react with
other non-TIGIT proteins. In a specific embodiment, provided herein
is an antibody that binds to TIGIT (e.g., human TIGIT) with higher
affinity than to another unrelated antigen. In certain embodiments,
provided herein is an antibody that binds to TIGIT (e.g., human
TIGIT) with a 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95% or higher affinity than to another,
unrelated antigen as measured by, e.g., a radioimmunoassay, surface
plasmon resonance, or kinetic exclusion assay. In a specific
embodiment, the extent of binding of an anti-TIGIT antibody
described herein to an unrelated, non-TIGIT protein is less than
10%, 15%, or 20% of the binding of the antibody to TIGIT protein as
measured by, e.g., a radioimmunoassay.
[0142] As used herein, an "epitope" is a term in the art and refers
to a localized region of an antigen to which an antibody can
specifically bind. An epitope can be, for example, contiguous amino
acids of a polypeptide (linear or contiguous epitope) or an epitope
can, for example, come together from two or more non-contiguous
regions of a polypeptide or polypeptides (conformational,
non-linear, discontinuous, or non-contiguous epitope). In certain
embodiments, the epitope to which an antibody binds can be
determined by, e.g., NMR spectroscopy, X-ray diffraction
crystallography studies, ELISA assays, hydrogen/deuterium exchange
coupled with mass spectrometry (e.g., liquid chromatography
electrospray mass spectrometry), array-based oligo-peptide scanning
assays (e.g., constraining peptides using CLIPS (Chemical Linkage
of Peptides onto Scaffolds) to map discontinuous or conformational
epitopes), and/or mutagenesis mapping (e.g., site-directed
mutagenesis mapping). For X-ray crystallography, crystallization
may be accomplished using any of the known methods in the art
(e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr
50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23;
Chayen N E (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol
Chem 251: 6300-6303, each of which is herein incorporated by
reference in its entirety). Antibody:antigen crystals may be
studied using well known X-ray diffraction techniques and may be
refined using computer software such as X-PLOR (Yale University,
1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth
Enzymol (1985) volumes 114 & 115, eds Wyckoff H W et al., U.S.
2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D
Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol
276A: 361-423, ed Carter C W; Roversi P et al., (2000) Acta
Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323), each of which
is herein incorporated by reference in its entirety. Mutagenesis
mapping studies may be accomplished using any method known to one
of skill in the art. See, e.g., Champe M et al., (1995) J Biol Chem
270: 1388-1394 and Cunningham B C & Wells J A (1989) Science
244: 1081-1085, each of which is herein incorporated by reference
in its entirety, for a description of mutagenesis techniques,
including alanine scanning mutagenesis techniques. CLIPS (Chemical
Linkage of Peptides onto Scaffolds) is a technology to present one
or more peptides in a structurally constrained configuration to
behave as functional mimics of complex protein domains. See, e.g.,
U.S. Publication Nos. US 2008/0139407 A1 and US 2007/099240 A1, and
U.S. Pat. No. 7,972,993, each of which is herein incorporated by
reference in its entirety. In a specific embodiment, the epitope of
an antibody is determined using alanine scanning mutagenesis
studies. In a specific embodiment, the epitope of an antibody is
determined using hydrogen/deuterium exchange coupled with mass
spectrometry. In a specific embodiment, the epitope of an antibody
is determined using CLIPS Epitope Mapping Technology from Pepscan
Therapeutics. In a specific embodiment, the epitope of an antibody
is determined by protein mutagenesis, e.g., by generating switch
mutants of an antigen with portions of its ortholog from another
species and then testing the switch mutants for loss of antibody
binding (e.g., by a FACS-based cell binding assay, as described
herein).
[0143] As used herein, the term "an epitope located within" a
region of human TIGIT refers to an epitope comprising one or more
of the amino acid residues of the specified region. In certain
embodiments, the epitope comprises each one of the amino acid
residues located within the specified region. In certain
embodiments, the epitope consists of each one of the amino acid
residues located within the specified region. In certain
embodiments, one or more additional amino acid residues of human
TIGIT outside the specified region bind to an antibody together
with an epitope located within the specified region.
[0144] As used herein, the binding between a test antibody and a
first antigen is "substantially weakened" relative to the binding
between the test antibody and a second antigen if the binding
between the test antibody and the first antigen is reduced by at
least 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to the binding
between the test antibody and the second antigen, e.g., in a given
experiment, or using mean values from multiple experiments, as
assessed by, e.g., a binding assay disclosed herein.
[0145] As used herein, the terms "T cell receptor" and "TCR" are
used interchangeably and refer to full length heterodimeric
.alpha..beta. or .gamma..delta. TCRs, antigen-binding fragments of
full length TCRs, and molecules comprising TCR CDRs or variable
regions. Examples of TCRs include, but are not limited to, full
length TCRs, antigen-binding fragments of full length TCRs, soluble
TCRs lacking transmembrane and cytoplasmic regions, single-chain
TCRs containing variable regions of TCRs attached by a flexible
linker, TCR chains linked by an engineered disulfide bond,
monospecific TCRs, multi-specific TCRs (including bispecific TCRs),
TCR fusions, human TCRs, humanized TCRs, chimeric TCRs,
recombinantly produced TCRs, and synthetic TCRs. The term
encompasses wild-type TCRs and genetically engineered TCRs (e.g., a
chimeric TCR comprising a chimeric TCR chain which includes a first
portion from a TCR of a first species and a second portion from a
TCR of a second species).
[0146] As used herein, the terms "major histocompatibility complex"
and "MHC" are used interchangeably and refer to an MHC class I
molecule and/or an MHC class II molecule.
[0147] As used herein, the term "peptide-MHC complex" refers to an
MHC molecule (MHC class I or MHC class II) with a peptide bound in
the art-recognized peptide binding pocket of the MHC.
[0148] As used herein, the term "treat," "treating," and
"treatment" refer to therapeutic or preventative measures described
herein. The methods of "treatment" employ administration of an
antibody to a subject having a disease or disorder, or predisposed
to having such a disease or disorder, in order to prevent, cure,
delay, reduce the severity of, or ameliorate one or more symptoms
of the disease or disorder or recurring disease or disorder, or in
order to prolong the survival of a subject beyond that expected in
the absence of such treatment.
[0149] As used herein, the term "effective amount" in the context
of the administration of a therapy to a subject refers to the
amount of a therapy that achieves a desired prophylactic or
therapeutic effect.
[0150] As used herein, the term "subject" includes any human or
non-human animal. In one embodiment, the subject is a human or
non-human mammal. In one embodiment, the subject is a human.
[0151] The determination of "percent identity" between two
sequences (e.g., amino acid sequences or nucleic acid sequences)
can be accomplished using a mathematical algorithm. A specific,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin S &
Altschul S F (1990) PNAS 87: 2264-2268, modified as in Karlin S
& Altschul S F (1993) PNAS 90: 5873-5877, each of which is
herein incorporated by reference in its entirety. Such an algorithm
is incorporated into the NBLAST and XBLAST programs of Altschul S F
et al., (1990) J Mol Biol 215: 403, which is herein incorporated by
reference in its entirety. BLAST nucleotide searches can be
performed with the NBLAST nucleotide program parameters set, e.g.,
for score=100, wordlength=12 to obtain nucleotide sequences
homologous to a nucleic acid molecules described herein. BLAST
protein searches can be performed with the XBLAST program
parameters set, e.g., to score 50, wordlength=3 to obtain amino
acid sequences homologous to a protein molecule described herein.
To obtain gapped alignments for comparison purposes, Gapped BLAST
can be utilized as described in Altschul S F et al., (1997) Nuc
Acids Res 25: 3389-3402, which is herein incorporated by reference
in its entirety. Alternatively, PSI BLAST can be used to perform an
iterated search which detects distant relationships between
molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast
programs, the default parameters of the respective programs (e.g.,
of XBLAST and NBLAST) can be used (see, e.g., National Center for
Biotechnology Information (NCBI) on the worldwide web,
ncbi.nlm.nih.gov). Another specific, non-limiting example of a
mathematical algorithm utilized for the comparison of sequences is
the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is
herein incorporated by reference in its entirety. Such an algorithm
is incorporated in the ALIGN program (version 2.0) which is part of
the GCG sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used.
[0152] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, typically only
exact matches are counted.
5.2 Anti-TIGIT Antibodies
[0153] In one aspect, the instant disclosure provides antibodies
that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) and antagonize TIGIT function. The amino acid sequences of
exemplary antibodies are set forth in Table 1, herein.
TABLE-US-00001 TABLE 1 Amino acid sequences of exemplary anti-TIGIT
antibodies. SEQ Amino Acid ID Description Sequence NO: BA002 Kabat
CDRH1 SYGIS 1 BA002 Alternate GYTFASY 2 CDRH1 BA002 Kabat CDRH2
GITPFFNRVDVAERFQG 3 BA002 Alternate TPFFNR 4 CDRH2 BA002 Kabat
CDRH3 DLRRGGVGDAFDI 5 BA002 Kabat CDRL1 TGTSSDVGSHNYVS 6 BA002
Kabat CDRL2 EVSYRPS 7 BA002 Kabat CDRL3 SSYTPSSATV 8 BA002 VH
XVQLVQSGAEVERPGASVRV 9 SCRASGYTFASYGISWVRQA PGQGLEWMGGITPFFNRVDV
AERFQGRVTITADTSTNTVY IELSSLTSEDTAVYYCARDL RRGGVGDAFDIWGRGTLVTV SS,
wherein X is glutamate (E) or pyroglutamate (pE) BA002 VL
XSALTQPRSVSGSPGQSVTI 10 SCTGTSSDVGSHNYVSWYQQ HPGRAPQLMIYEVSYRPSEI
SNRFSGSRSGNTASLTISGL QPEDEADYYCSSYTPSSATV FGAGTRLTVL, wherein X is
glutamine(Q) or pyroglutamate(pE) BA002 full length
XVQLVQSGAEVERPGASVRV 11 heavy chain (IgG1) SCRASGYTFASYGISWVRQA
PGQGLEWMGGITPFFNRVDV AERFQGRVTITADTSTNTVY IELSSLTSEDTAVYYCARDL
RRGGVGDAFDIWGRGTLVTV SSASTRGPSVFPLAPSSRST SGGTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRV
EPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNH YTQKSLSLSPG, wherein X is
glutamate(E) or pyroglutamate (pE) BA003 full length
XVQLVQSGAEVEKPGASVKV 12 heavy chain (N297A SCKASGYTFASYGISWVRQA
variant of BA002, PGQGLEWMGGITPFFNRVDV numbered according
AEKFQGRVTITADTSTNTVY to the EU IELSSLTSEDTAVYYCARDL numbering
RRGGVGDAFDIWGRGTLVTV system) SSASTKGPSVFPLAPSSKST
SGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISR
TPEVTCVWDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQY ASTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSD
IAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHY
TQKSLSLSPG, Wherein X is glutamate(E) or pyroglutamate(pE) BA004
full length XVQLVQSGAEVEKPGASVKV 13 heavy chain
SCKASGYTFASYGISWVRQA (L234F/L235F/N297A PGQGLEWMGGITPFFNRVDV
variant of BA002, AEKFQGRVTITADTSTNTVY numbered
IELSSLTSEDTAVYYCARDL according to RRGGVGDAFDIWGRGTLVTV the EU
numbering SSASTKGPSVFPLAPSSKST system) SGGTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRV
EPKSCDKTHTCPPCPAPEFF GGPSVFLFPPKPKDTLMISR TPEVTCVWDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQY ASTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTI
SKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHY TQKSLSLSPG, wherein X is
glutamate(E) or pyroglutamate (pE) BA005 full length
XVQLVQSGAEVEKPGASVKV 14 heavy chain SCKASGYTFASYGISWVRQA
(S239D/I332E variant PGQGLEWMGGITPFFNRVDV of BA002, numbered
AEKFQGRVTITADTSTNTVY according to the EU IELSSLTSEDTAVYYCARDL
numbering system) RRGGVGDAFDIWGRGTLVTV SSASTKGPSVFPLAPSSKST
SGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELL GGPDVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPEEKT ISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNH
YTQKSLSLSPG, wherein X is glutamate (E)or pyroglutamate (pE) BA006
full length XVQLVQSGAEVEKPGASVKV 15 heavy chain
SCKASGYTFASYGISWVRQA (S239D/A330L/I332E PGQGLEWMGGITPFFNRVDV
variant of BA002, AEKFQGRVTITADTSTNTVY numbered
IELSSLTSEDTAVYYCARDL according to RRGGVGDAFDIWGRGTLVTV the EU
numbering SSASTKGPSVFPLAPSSKST system) SGGTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRV
EPKSCDKTHTCPPCPAPELL GGPDVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPLPEEKT
ISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNH YTQKSLSLSPG, wherein X is
glutamate (E) or pyroglutamate (pE) BA007 full length
XVQLVQSGAEVEKPGASVKV 16 heavy chain SCKASGYTFASYGISWVRQA
(L235V/F243L/ PGQGLEWMGGITPFFNRVDV R292P/Y300L/P396L
AEKFQGRVTITADTSTNTVY variant of BA002, IELSSLTSEDTAVYYCARDL
numbered RRGGVGDAFDIWGRGTLVTV according to SSASTKGPSVFPLAPSSKST the
EU SGGTAALGCLVKDYFPEPVT numbering VSWNSGALTSGVHTFPAVLQ system)
SSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELV
GGPSVFLLPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPPEEQ
YNSTLRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTP LVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNH YTQKSLSLSPG, wherein X is glutamate (E) or
pyroglutamate (pE) BA008 full length XVQLVQSGAEVEKPGASVKV 17 heavy
chain SCKASGYTFASYGISWVRQA (S267E/L328F variant
PGQGLEWMGGITPFFNRVDV of BA002, numbered AEKFQGRVTITADTSTNTVY
according to the EU IELSSLTSEDTAVYYCARDL numbering system)
RRGGVGDAFDIWGRGTLVTV SSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRV
EPKSCDKTHTCPPCPAPELL GGPSVFLFPPKP KDTLMISRTPEVTCVVVDVE
HEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNG
KEYKCKVSNKAFPAPIEKTI SKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSP G, wherein X is glutamate (E) or pyroglutamate
(pE) BA009 full length XVQLVQSGAEVEKPGASVKV 18 heavy chain (IgG4
SCKASGYTFASYGISWVRQA S228P variant of PGQGLEWMGGITPFFNRVDV BA002,
numbered AEKFQGRVTITADTSTNTVY according to the EU
IELSSLTSEDTAVYYCARDL numbering system) RRGGVGDAFDIWGRGTLVTV
SSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGT
KTYTCNVDHKPSNTKVDKRV ESKYGPPCPPCPAPEFLGGP SVFLFPPKPKDTLMISRTPE
VTCVWDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEY
KCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQK
SLSLSLG, Wherein X is glutamate (E) or pyroglutamate(pE) BA002
heavy chain ASTKGPSVFPLAPSSKSTSG 19 constant region
GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQK
SLSLSPG ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYA STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT BA003 heavy chain
QKSLSLSPG 20 constant region BA004 heavy chain ASTKGPSVFPLAPSSKSTSG
21 constant region GTAALGQLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPEFFGG
PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYA
STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE
MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYT QKSLSLSPG BA005 heavy chain
ASTKGPSVFPLAPSSKSTSG 22 constant region GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPELLGG PDVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPEEKTIS
KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG BA006 heavy
chain ASTKGPSVFPLAPSSKSTSG 23 constant region GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPELLGG PDVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPLPEEKTIS
KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG BA007 heavy
chain ASTKGPSVFPLAPSSKSTSG 24 constant region GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPELVGG PSVFLLPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPPEEQYN STLRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPLV
LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG BA008 heavy
chain ASTKGPSVFPLAPSSKSTSG 25 constant region GTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVEHEDPEVKFNW
YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKAFPAPIEKTIS
KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRW QQGNVESCSVMHEALHNHYT QKSLSLSPG BA009 heavy
chain ASTKGPSVFPLAPCSRSTSE 26 constant region STAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVES
KYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVD
GVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK
GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKS LSLSLG BA002 full length
XSALTQPRSVSGSPGQSVTI 27 light chain SCTGTSSDVGSHNYVSWYQQ
HPGKAPQLMIYEVSYRPSEI SNRFSGSKSGNTASLTISGL QPEDEADYYCSSYTPSSATV
FGAGTKLTVLGQPKAAPSVT LFPPSSEELQANKATLVCLI SDFYPGAVTVAWKADSSPVK
AGVETTTPSKQSNNKYAASS YLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECS, wherein
X is glutamine (Q) or pyroglutamate (pE) BA002 light chain
GQPKAAPSVTLFPPSSEELQ 28 constant region ANKATLVCLISDFYPGAVTV
AWKADSSPVKAGVETTTPSK QSNNKYAASSYLSLTPEQWK SHRSYSCQVTHEGSTVEKTV
APTECS
TABLE-US-00002 TABLE 2 Closest germline genes to the exemplary
anti-TIGIT antibodies. Closest SEQ germline ID gene Amino Acid
Sequence NO: IGHV1-69*01 QVQLVQSGAEVKKPGSSVKV 34 heavy chain
SCKASGGTFSSYAISWVRQA variable PGQGLEWMGGIIPIFGTANY region
AQKFQGRVTITADESTSTAY MELSSLRSEDTAVYYCAR IGHV1-69*06
QVQLVQSGAEVKKPGSSVKV 35 heavy chain SCKASGGTFSSYAISWVRQA variable
PGQGLEWMGGIIPIFGTANY region AQKFQGRVTITADKSTSTAY MELSSLRSEDTAVYYCAR
IGLV2-14*01 QSALTQPASVSGSPGQSITI 37 light chain
SCTGTSSDVGGYNYVSWYQQ variable HPGKAPKLMIYEVSNRPSGV region
SNRFSGSKSGNTASLTISGL QAEDEADYYCSSYTSSSTL IGLV2-14*02
QSALTQPASVSGSPGQSITI 60 light chain SCTGTSSDVGSYNLVSWYQQ variable
HPGKAPKLMIYEGSKRPSGV region SNRFSGSKSGNTASLTISGL
QAEDEADYYCSSYTSSSTL IGLV2-23*02 QSALTQPASVSGSPGQSITI 38 light chain
SCTGTSSDVGSYNLVSWYQQ variable HPGKAPKLMIYEVSKRPSGV region
SNRFSGSKSGNTASLTISGL QAEDEADYYCCSYAGSSTF IGLV2-11*01
QSALTQPRSVSGSPGQSVTI 39 light chain SCTGTSSDVGGYNYVSWYQQ variable
HPGKAPKLMIYDVSKRPSGV region PDRFSGSKSGNTASLTISGL
QAEDEADYYCCSYAGSYTF
TABLE-US-00003 TABLE 3 Exemplary sequences of TIGIT. SEQ Amino Acid
ID Description Sequence NO: Exemplary MRWCLLLIWAQGLRQ 29 immature
TIGIT APLASGMMTGTIETT full length GNISAEKGGSIILQC sequence
HLSSTTAQVTQVNWE QQDQLLAICNADLGW HISPSFKDRVAPGPG LGLTLQSLTVNDTGE
YFCIYHTYPDGTYTG RIFLEVLESSVAEHG ARFQIPLLGAMAATL VVICTAVIVVVALTR
KKKALRIHSVEGDLR RKSAGQEEWSPSAPS PPGSCVQAEAAPAGL CGEQRGEDCAELHDY
FNVLSYRSLGNCSFF TETG Exemplary TIGIT MMTGTIETTGNISAE 30
extracellular KGGSIILQCHLSSTT domain sequence AQVTQVNWEQQDQLL
(epitope sequences AICNADLGWHISPSF indicated in bold)
KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH TYPDGTYTGRIFLEV LESSVAEHGARF TIGIT
epitope YHTYPDGTYTGRIFL 31 sequence #1 E (Residues 89-104 of mature
TIGIT sequence) TIGIT epitope VTQV 32 sequence #2 (Residues 33-36
of mature TIGIT sequence) TIGIT epitope ICNADLGWHISPSF 33 sequence
#3 (Residues 47-60 of mature TIGIT sequence) Exemplary mature
MMTGTIETTGNISAE 40 TIGIT full length KGGSIILQCHLSSTT sequence
AQVTQVNWEQQDQLL AICNADLGWHISPSF KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH
TYPDGTYTGRIFLEV LESSVAEHGARFQIP LLGAMAATLVVICTA VIVVVALTRKKKALR
IHSVEGDLRRKSAGQ EEWSPSAPSPPGSCV QAEAAPAGLCGEQRG EDCAELHDYFNVLSY
RSLGNCSFFTETG Exemplary TIGIT MMTGTIETTGNISAE 41 extracellular
KGGSIILQCHLSSTT domain sequence AQVTQVNWEQQDQLL AICNADLGWHISPSF
KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH TYPDGTYTGRIFLEV LESSVAEHGARFQIP
Exemplary TIGIT MMTGTIETTGNISAE 42 extracellular KGGSIILQCHLSSTT
domain sequence AQVTQVNWEQQDQLL AICNADLGWHISPSF KDRVAPGPGLGLTLQ
SLTVNDTGEYFCIYH TYPDGTYTGRIFLEV LESSVAEHGARFQ Exemplary TIGIT
MMTGTIETTGNISAE 43 extracellular KGGSIILQCHLSSTT domain T34A
AQVAQVNWEQQDQLL AICNADLGWHISPSF KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH
TYPDGTYTGRIFLEV LESSVAEHGARFQ Exemplary TIGIT MMTGTIETTGNISAE 44
extracellular KGGSIILQCHLSSTT domain Q35A AQVTAVNWEQODQLL
AICNADLGWHISPSF KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH TYPDGTYTGRIFLEV
LESSVAEHGARFQ Exemplary TIGIT MMTGTIETTGNISAE 45 extracellular
KGGSIILQCHLSSTT domain I47E AQVTQVNWEQQDQLL AECNADLGWHISPSF
KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH TYPDGTYTGRIFLEV LESSVAEHGARFQ
Exemplary TIGIT MMTGTIETTGNISAE 46 extracellular KGGSIILQCHLSSTT
domain N49A AQVTQVNWEQQDQLL AICAADLGWHISPSF KDRVAPGPGLGLTLQ
SLTVNDTGEYFCIYH TYPDGTYTGRIFLEV LESSVAEHGARFQ Exemplary TIGIT
MMTGTIETTGNISAE 47 extracellular KGGSIILQCHLSSTT domain L52A
AQVTQVNWEQQDQLL AICNADAGWHISPSF KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH
TYPDGTYTGRIFLEV LESSVAEHGARFQ Exemplary TIGIT MMTGTIETTGNISAE 48
extracellular KGGSIILQCHLSSTT domain L52E AQVTQVNWEQQDQLL
AICNADEGWHISPSF KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH TYPDGTYTGRIFLEV
LESSVAEHGARFQ Exemplary TIGIT MMTGTIETTGNISAE 49 extracellular
KGGSIILQCHLSSTT domain H55A AQVTQVNWEQQDQLL AICNADLGWAISPSF
KDRVAPGPGLGLTIQ SLTVNDTGEYFCIYH TYPDGTYTGRIFLEV LESSVAEHGARFQ
Exemplary TIGIT MMTGTIETTGNISAE 50 extracellular KGGSIILQCHLSSTT
domain P58A AQVTQVNWEQQDQLL AICNADLGWHISASF KDRVAPGPGLGLTLQ
SLTVNDTGEYFCIYH TYPDGTYTGRIFLEV LESSVAEHGARFQ Exemplary TIGIT
MMTGTIETTGNISAE 51 extracellular KGGSIILQCHLSSTT domain H90A
AQVTQVNWEQQDQLL AICNADLGWHISPSF KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYA
TYPDGTYTGRIFLEV LESSVAEHGARFQ Exemplary TIGIT MMTGTIETTGNISAE 52
extracellular KGGSIILQCHLSSTT domain T96A AQVTQVNWEQQDQLL
AICNADLGWHISPSF KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH TYPDGAYTGRIFLEV
LESSVAEHGARFQ Exemplary TIGIT MMTGTIETTGNISAE 53 extracellular
KGGSIILQCHLSSTT domain T96I AQVTQVNWEQQDQLL AICNADLGWHISPSF
KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH TYPDGIYTGRIFLEV LESSVAEHGARFQ
Exemplary TIGIT MMTGTIETTGNISAE 54 extracellular KGGSIILQCHLSSTT
domain T98A AQVTQVNWEQQDQLL AICNADLGWHISPSF KDRVAPGPGLGLTLQ
SLTVNDTGEYFCIYF ITYPDGTYAGRIFLE VLESSVAEHGARFQ Exemplary TIGIT
MMTGTIETTGNISAE 55 extracellular KGGSIILQCHLSSTT domain R100A
AQVTQVNWEQQDQLL AICNADLGWHISPSF KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH
TYPDGTYTGAIFLEV LESSVAEHGARFQ Exemplary TIGIT MMTGTIETTGNISAE 56
extracellular KGGSIILQCHLSSTT domain FI02A AQVTQVNWEQQDQLL
AICNADLGWHISPSF KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH TYPDGTYTGRIALEV
LESSVAEHGARFQ Exemplary TIGIT MMTGTIETTGNISAE 57 extracellular
KGGSIILQCHLSSTT domain C48Y, AQVTQVNWEQQDQLL N49S, A50V
AIYSVDLGWHISPSF KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH TYPDGTYTGRIFLEV
LESSVAEHGARFQ Exemplary TIGIT MMTGTIETTGNISAE 36 extracellular
KGGSIILQCHLSSTT domain N49S AQVTQVNWEQQDQLL AICSADLGWHISPSF
KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH TYPDGTYTGRIFLEV LESSVAEHGARFQ
Exemplary TIGIT MMTGTIETTGNISAE 58 extracellular KGGSIILQCHLSSTT
domain I56V, AQVTQVNWEQQDQLL S57A, P58S, S59V AICNADLGWHVASVF
KDRVAPGPGLGLTLQ SLTVNDTGEYFCIYH TYPDGTYTGRIFLEV LESSVAEHGARFQ
Exemplary TIGIT MMTGTIETTGNISAE 59 extracellular KGGSIILQCHLSSTT
domain T96I, T98K AQVTQVNWEQQDQLL AICNADLGWHISPSF KDRVAPGPGLGLTLQ
SLTVNDTGEYFCIYH TYPDGIYKGRIFLEV LESSVAEHGARFQ
[0154] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), the antibody comprising a VH domain
comprising one, two, or all three of the CDRs of a VH domain set
forth in Table 1 herein. In certain embodiments, the antibody
comprises the CDRH1 of a VH domain set forth in Table 1. In certain
embodiments, the antibody comprises the CDRH2 of a VH domain set
forth in Table 1. In certain embodiments, the antibody comprises
the CDRH3 of a VH domain set forth in Table 1.
[0155] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), the antibody comprising a VL domain
comprising one, two, or all three of the CDRs of a VL domain
disclosed in Table 1 herein. In certain embodiments, the antibody
comprises the CDRL1 of a VL domain set forth in Table 1. In certain
embodiments, the antibody comprises the CDRL2 of a VL domain set
forth in Table 1. In certain embodiments, the antibody comprises
the CDRL3 of a VL domain set forth in Table 1.
[0156] In certain embodiments, the CDRs of an antibody can be
determined according to Kabat et al., J. Biol. Chem. 252, 6609-6616
(1977) and Kabat et al., Sequences of protein of immunological
interest (1991), each of which is herein incorporated by reference
in its entirety. In certain embodiments, the light chain CDRs of an
antibody are determined according to Kabat and the heavy chain CDRs
of an antibody are determined according to MacCallum (supra). In
certain embodiments, heavy chain CDRs and/or light chain CDRs are
defined by performing structural analysis of an antibody and
identifying residues in the variable region(s) predicted to make
contact with an epitope region of a target molecule (e.g., human
and/or cynomolgus TIGIT).
[0157] In certain embodiments, the CDRs of an antibody can be
determined according to the Chothia numbering scheme, which refers
to the location of immunoglobulin structural loops (see, e.g.,
Chothia C & Lesk A M, (1987), J Mol Biol 196: 901-917;
Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et
al., (1992) J Mol Biol 227: 799-817; Tramontano A et al., (1990) J
Mol Biol 215(1): 175-82; and U.S. Pat. No. 7,709,226, all of which
are herein incorporated by reference in their entireties).
Typically, when using the Kabat numbering convention, the Chothia
CDRH1 loop is present at heavy chain amino acids 26 to 32, 33, or
34, the Chothia CDRH2 loop is present at heavy chain amino acids 52
to 56, and the Chothia CDRH3 loop is present at heavy chain amino
acids 95 to 102, while the Chothia CDRL1 loop is present at light
chain amino acids 24 to 34, the Chothia CDRL2 loop is present at
light chain amino acids 50 to 56, and the Chothia CDRL3 loop is
present at light chain amino acids 89 to 97. The end of the Chothia
CDRH1 loop when numbered using the Kabat numbering convention
varies between H32 and H34 depending on the length of the loop
(this is because the Kabat numbering scheme places the insertions
at H35A and H35B; if neither 35A nor 35B is present, the loop ends
at 32; if only 35A is present, the loop ends at 33; if both 35A and
35B are present, the loop ends at 34).
[0158] In certain embodiments, the CDRs of an antibody can be
determined according to MacCallum R M et al., (1996) J Mol Biol
262: 732-745, herein incorporated by reference in its entirety. See
also, e.g., Martin A. "Protein Sequence and Structure Analysis of
Antibody Variable Domains," in Antibody Engineering, Kontermann and
Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin
(2001), herein incorporated by reference in its entirety.
[0159] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), the antibody comprising the Chothia V H
CDRs of a VH disclosed in Table 1 herein. In certain embodiments,
the instant disclosure provides an isolated antibody that
specifically binds to TIGIT (e.g., human TIGIT or cynomolgus
TIGIT), the antibody comprising the Chothia VL CDRs of a VL
disclosed in Table 1 herein. In certain embodiments, the instant
disclosure provides an isolated antibody that specifically binds to
TIGIT (e.g., human TIGIT or cynomolgus TIGIT), the antibody
comprising the Chothia VH CDRs and Chothia VL CDRs of an antibody
disclosed in Table 1 herein. In certain embodiments, antibodies
that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) comprise one or more CDRs, in which the Chothia and Kabat
CDRs have the same amino acid sequence. In certain embodiments, the
instant disclosure provides an isolated antibody that specifically
binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and
comprises combinations of Kabat CDRs and Chothia CDRs.
[0160] In certain embodiments, the CDRs of an antibody can be
determined according to the IMGT numbering system as described in
Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et
al., (1999) Nucleic Acids Res 27: 209-212, each of which is herein
incorporated by reference in its entirety. According to the IMGT
numbering scheme, CDRH1 is at positions 26 to 35, CDRH2 is at
positions 51 to 57, CDRH3 is at positions 93 to 102, CDRL1 is at
positions 27 to 32, CDRL2 is at positions 50 to 52, and CDRL3 is at
positions 89 to 97.
[0161] In certain embodiments, the instant disclosure provides
antibodies that specifically bind to TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) and comprise CDRs of an antibody disclosed in
Table 1 herein, as determined by the IMGT numbering system, for
example, as described in Lefranc M-P (1999) supra and Lefranc M-P
et al., (1999) supra.
[0162] In certain embodiments, the CDRs of an antibody can be
determined according to the AbM numbering scheme, which refers to
AbM hypervariable regions, which represent a compromise between the
Kabat CDRs and Chothia structural loops, and are used by Oxford
Molecular's AbM antibody modeling software (Oxford Molecular Group,
Inc.), herein incorporated by reference in its entirety. In a
particular embodiment, the instant disclosure provides antibodies
that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) and comprise CDRs of an antibody disclosed in Table 1 herein
as determined by the AbM numbering scheme.
[0163] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), wherein the antibody comprises a heavy
chain variable region comprising the CDRH1, CDRH2, and CDRH3 region
amino acid sequences of a VH domain set forth in SEQ ID NO: 9, and
a light chain variable region comprising the CDRL1, CDRL2, and
CDRL3 region amino acid sequences of a VL domain set forth in SEQ
ID NO: 10, wherein each CDR is defined in accordance with the
MacCallum definition, the Kabat definition, the Chothia definition,
the IMGT numbering system, the AbM definition of CDR, structural
analysis, or a combination thereof, wherein the structural analysis
identifies residues in the variable region(s) predicted to make
contact with an epitope region of TIGIT (e.g., human TIGIT or
cynomolgus TIGIT). In certain embodiments, the instant disclosure
provides an isolated antibody that specifically binds to TIGIT
(e.g., human TIGIT or cynomolgus TIGIT) and comprises a combination
of CDRs defined by the Kabat definition and CDRs defined by
structural analysis of the antibody, wherein the structural
analysis identifies residues in the variable region(s) predicted to
make contact with an epitope region of TIGIT (e.g., human TIGIT or
cynomolgus TIGIT).
[0164] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), the antibody comprising:
(a) a CDRH1 comprises the amino acid sequence of SYGIS (SEQ ID NO:
1) or GYTFASY (SEQ ID NO: 2); (b) a CDRH2 comprises the amino acid
sequence of GITPFFNRVDVAEKFQG (SEQ ID NO: 3) or TPFFNR (SEQ ID NO:
4); (c) a CDRH3 comprises the amino acid sequence of DLRRGGVGDAFDI
(SEQ ID NO: 5); (d) a CDRL1 comprises the amino acid sequence of
TGTSSDVGSHNYVS (SEQ ID NO: 6); (e) a CDRL2 comprises the amino acid
sequence of EVSYRPS (SEQ ID NO: 7); and/or (f) a CDRL3 comprises
the amino acid sequence of SSYTPSSATV (SEQ ID NO: 8).
[0165] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), the antibody comprising:
(a) a CDRH1 comprises the amino acid sequence of SYGIS (SEQ ID NO:
1) or GYTFASY (SEQ ID NO: 2); (b) a CDRH2 comprises the amino acid
sequence of GITPFFNRVDVAEKFQG (SEQ ID NO: 3) or TPFFNR (SEQ ID NO:
4); (c) a CDRH3 comprises the amino acid sequence of DLRRGGVGDAFDI
(SEQ ID NO: 5); (d) a CDRL1 comprises the amino acid sequence of
TGTSSDVGSHNYVS (SEQ ID NO: 6); (e) a CDRL2 comprises the amino acid
sequence of EVSYRPS (SEQ ID NO: 7); and (f) a CDRL3 comprises the
amino acid sequence of SSYTPSSATV (SEQ ID NO: 8).
[0166] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), wherein the antibody comprises a VH
domain comprising the CDRH1, CDRH2 and CDRH3 amino acid sequences
set forth in SEQ ID NOs: 1, 3, and 5, respectively. In certain
embodiments, the instant disclosure provides an isolated antibody
that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus
TIGIT), wherein the antibody comprises a VH domain comprising the
CDRH1, CDRH2 and CDRH3 amino acid sequences set forth in SEQ ID
NOs: 2, 4, and 5, respectively. In certain embodiments, the instant
disclosure provides an isolated antibody that specifically binds to
TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the antibody
comprises a VL domain comprising the CDRL1, CDRL2 and CDRL3 amino
acid sequences set forth in SEQ ID NOs: 6, 7, and 8,
respectively.
[0167] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), wherein the antibody comprises a heavy
chain variable region comprising CDRH1, CDRH2, and CDRH3 regions,
and a light chain variable region comprising CDRL1, CDRL2, and
CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and
CDRL3 regions comprise the amino acid sequences set forth in SEQ ID
NOs: 1, 3, 5, 6, 7, and 8, respectively. In certain embodiments,
the instant disclosure provides an isolated antibody that
specifically binds to TIGIT (e.g., human TIGIT or cynomolgus
TIGIT), wherein the antibody comprises a heavy chain variable
region comprising CDRH1, CDRH2, and CDRH3 regions, and a light
chain variable region comprising CDRL1, CDRL2, and CDRL3 regions,
wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions
comprise the amino acid sequences set forth in SEQ ID NOs: 2, 4, 5,
6, 7, and 8, respectively.
[0168] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), comprising a heavy chain variable
region comprising an amino acid sequence of SEQ ID NO: 9. In
certain embodiments, the instant disclosure provides an isolated
antibody that specifically binds to TIGIT (e.g., human TIGIT or
cynomolgus TIGIT), comprising a heavy chain variable region
comprising an amino acid sequence that is at least 75%, 80%, 85%,
90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence set
forth in SEQ ID NO: 9. In certain embodiments, the instant
disclosure provides an isolated antibody that specifically binds to
TIGIT (e.g., human TIGIT or cynomolgus TIGIT), comprising a light
chain variable region comprising an amino acid sequence of SEQ ID
NO: 10. In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), comprising a light chain variable
region comprising an amino acid sequence that is at least 75%, 80%,
85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence
set forth in SEQ ID NO: 10.
[0169] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), comprising a heavy chain variable
region comprising an amino acid sequence of SEQ ID NO: 9, and a
light chain variable region comprising an amino acid sequence of
SEQ ID NO: 10. In certain embodiments, the instant disclosure
provides an isolated antibody that specifically binds to TIGIT
(e.g., human TIGIT or cynomolgus TIGIT), comprising a heavy chain
variable region comprising an amino acid sequence that is at least
75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino
acid sequence set forth in SEQ ID NO: 9, and a light chain variable
region comprising an amino acid sequence that is at least 75%, 80%,
85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence
set forth in SEQ ID NO: 10.
[0170] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), comprising a heavy chain variable
region having an amino acid sequence derived from a human IGHV1-69
germline sequence. In certain embodiments, the human IGHV1-69
germline sequence is selected from the group consisting of a human
IGHV1-69*01 germline sequence (e.g., having the amino acid sequence
of SEQ ID NO: 34), a human IGHV1-69*06 germline sequence (e.g.,
having the amino acid sequence of SEQ ID NO: 35), and a human
IGHV1-69*12 germline sequence. One or more regions selected from
framework 1, framework 2, framework 3, CDRH1, and CDRH2 (e.g., two,
three, four or five of these regions) can be derived from a human
IGHV1-69 germline sequence. In one embodiment, framework 1,
framework 2, framework 3, CDRH1, and CDRH2 are all derived from a
human IGHV1-69 germline sequence. In certain embodiments, the heavy
chain variable region comprises a CDRH3 comprising the amino acid
sequence set forth in SEQ ID NO: 5.
[0171] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), comprising a light chain variable
region having an amino acid sequence derived from a human germline
sequence selected from the group consisting of IGLV2-14 (e.g.,
IGLV2-14*01, e.g., having the amino acid sequence of SEQ ID NO: 37,
or IGLV2-14*02, e.g., having the amino acid sequence of SEQ ID NO:
60), IGLV2-23 (e.g., IGLV2-23*02, e.g., having the amino acid
sequence of SEQ ID NO: 38), and IGLV2-11 (e.g., IGLV2-11*01, e.g.,
having the amino acid sequence of SEQ ID NO: 39). One or more
regions selected from framework 1, framework 2, framework 3, CDRL1,
and CDRL2 (e.g., two, three, four or five of these regions) can be
derived from a human germline sequence selected from the group
consisting of IGLV2-14 (e.g., IGLV2-14*01, e.g., having the amino
acid sequence of SEQ ID NO: 37, or IGLV2-14*02, e.g., having the
amino acid sequence of SEQ ID NO: 60), IGLV2-23 (e.g., IGLV2-23*02,
e.g., having the amino acid sequence of SEQ ID NO: 38), and
IGLV2-11 (e.g., IGLV2-11*01, e.g., having the amino acid sequence
of SEQ ID NO: 39). In one embodiment, framework 1, framework 2,
framework 3, CDRL1, and CDRL2 are all derived from a human germline
sequence selected from the group consisting of IGLV2-14 (e.g.,
IGLV2-14*01, e.g., having the amino acid sequence of SEQ ID NO: 37,
or IGLV2-14*02, e.g., having the amino acid sequence of SEQ ID NO:
60), IGLV2-23 (e.g., IGLV2-23*02, e.g., having the amino acid
sequence of SEQ ID NO: 38), and IGLV2-11 (e.g., IGLV2-11*01, e.g.,
having the amino acid sequence of SEQ ID NO: 39). In certain
embodiments, the light chain variable region comprises a CDRL3
comprising the amino acid sequence set forth in SEQ ID NO: 8.
[0172] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), comprising a heavy chain variable
region having an amino acid sequence derived from a human IGHV1-69
germline sequence (e.g., a human IGHV1-69*01 germline sequence
(e.g., having the amino acid sequence of SEQ ID NO: 34), a human
IGHV1-69*06 germline sequence (e.g., having the amino acid sequence
of SEQ ID NO: 35), or a human IGHV1-69*12 germline sequence); and a
light chain variable region having an amino acid sequence derived
from a human germline sequence selected from the group consisting
of IGLV2-14 (e.g., IGLV2-14*01, e.g., having the amino acid
sequence of SEQ ID NO: 37, or IGLV2-14*02, e.g., having the amino
acid sequence of SEQ ID NO: 60), IGLV2-23 (e.g., IGLV2-23*02, e.g.,
having the amino acid sequence of SEQ ID NO: 38), and IGLV2-11
(e.g., IGLV2-11*01, e.g., having the amino acid sequence of SEQ ID
NO: 39). In certain embodiments, the heavy chain variable region
comprises a CDRH3 comprising the amino acid sequence set forth in
SEQ ID NO: 5, and the light chain variable region comprises a CDRL3
comprising the amino acid sequence set forth in SEQ ID NO: 8.
[0173] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to human TIGIT, the
antibody comprising a heavy chain variable region comprising an
amino acid region that is at least 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ
ID NO: 34 or 35. In certain embodiments, the instant disclosure
provides an isolated antibody that specifically binds to human
TIGIT, the antibody comprising a light chain variable region
comprising an amino acid region that is at least 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid
sequence of any one of SEQ ID NOs: 37-39 and 60.
[0174] In certain embodiments, the instant disclosure provides an
isolated antibody that cross-competes for binding to TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT) with an antibody comprising the
heavy and light chain variable region amino acid sequences set
forth in SEQ ID NOs: 9 and 10, respectively.
[0175] In another aspect, the instant disclosure provides an
antibody or isolated antibody that binds, e.g., specifically binds,
to the same epitope of human TIGIT as an antibody of the present
invention. In certain embodiments, the epitope is determined by
hydrogen-deuterium exchange (HDX), for example as described in the
examples, or by protein mutagenesis, for example as described in
the examples.
[0176] In certain embodiments, the instant disclosure provides an
isolated antibody that binds to the same or an overlapping epitope
of TIGIT (e.g., an epitope of human TIGIT or an epitope of
cynomolgus TIGIT) as an antibody described herein, e.g., an
antibody comprising the heavy and light chain variable region amino
acid sequences set forth in SEQ ID NOs: 9 and 10, respectively. In
certain embodiments, the epitope of an antibody can be determined
by, e.g., NMR spectroscopy, surface plasmon resonance
(BIAcore.RTM.), X-ray diffraction crystallography studies, ELISA
assays, hydrogen/deuterium exchange coupled with mass spectrometry
(e.g., liquid chromatography electrospray mass spectrometry),
array-based oligo-peptide scanning assays, and/or mutagenesis
mapping (e.g., site-directed mutagenesis mapping). For X-ray
crystallography, crystallization may be accomplished using any of
the known methods in the art (e.g., Giege R et al., (1994) Acta
Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A
(1990) Eur J Biochem 189: 1-23; Chayen N E (1997) Structure 5:
1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, all of
which are herein incorporated by reference in their entireties).
Antibody:antigen crystals may be studied using well known X-ray
diffraction techniques and may be refined using computer software
such as X-PLOR (Yale University, 1992, distributed by Molecular
Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 &
115, eds Wyckoff H W et al.; U.S. Patent Application No.
2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D
Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol
276A: 361-423, ed Carter C W; Roversi P et al., (2000) Acta
Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323, all of which
are herein incorporated by reference in their entireties).
Mutagenesis mapping studies may be accomplished using any method
known to one of skill in the art. See, e.g., Champe M et al.,
(1995) supra and Cunningham B C & Wells J A (1989) supra for a
description of mutagenesis techniques, including alanine scanning
mutagenesis techniques. In a specific embodiment, the epitope of an
antibody is determined using alanine scanning mutagenesis studies.
In addition, antibodies that recognize and bind to the same or
overlapping epitopes of TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) can be identified using routine techniques such as an
immunoassay, for example, by showing the ability of one antibody to
block the binding of another antibody to a target antigen, i.e., a
competitive binding assay. Competition binding assays also can be
used to determine whether two antibodies have similar binding
specificity for an epitope. Competitive binding can be determined
in an assay in which the immunoglobulin under test inhibits
specific binding of a reference antibody to a common antigen, such
as TIGIT (e.g., human TIGIT or cynomolgus TIGIT). Numerous types of
competitive binding assays are known, for example: solid phase
direct or indirect radioimmunoassay (MA), solid phase direct or
indirect enzyme immunoassay (EIA), sandwich competition assay (see
Stahli C et al., (1983) Methods Enzymol 9: 242-253); solid phase
direct biotin-avidin EIA (see Kirkland T N et al., (1986) J Immunol
137: 3614-9); solid phase direct labeled assay, solid phase direct
labeled sandwich assay (see Harlow E & Lane D, (1988)
Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid
phase direct label RIA using 1-125 label (see Morel G A et al.,
(1988) Mol Immunol 25(1): 7-15); solid phase direct biotin-avidin
EIA (see Cheung R C et al., (1990) Virology 176: 546-52); and
direct labeled RIA (see Moldenhauer G et al., (1990) Scand J
Immunol 32: 77-82), all of which are herein incorporated by
reference in their entireties. Typically, such an assay involves
the use of purified antigen (e.g., TIGIT, such as human TIGIT or
cynomolgus TIGIT) bound to a solid surface or cells bearing either
of these, an unlabeled test immunoglobulin and a labeled reference
immunoglobulin. Competitive inhibition can be measured by
determining the amount of label bound to the solid surface or cells
in the presence of the test immunoglobulin. Usually the test
immunoglobulin is present in excess. Usually, when a competing
antibody is present in excess, it will inhibit specific binding of
a reference antibody to a common antigen by at least 50-55%,
55-60%, 60-65%, 65-70%, 70-75% or more. A competition binding assay
can be configured in a large number of different formats using
either labeled antigen or labeled antibody. In a common version of
this assay, the antigen is immobilized on a 96-well plate. The
ability of unlabeled antibodies to block the binding of labeled
antibodies to the antigen is then measured using radioactive or
enzyme labels. For further details see, for example, Wagener C et
al., (1983) J Immunol 130: 2308-2315; Wagener C et al., (1984) J
Immunol Methods 68: 269-274; Kuroki M et al., (1990) Cancer Res 50:
4872-4879; Kuroki M et al., (1992) Immunol Invest 21: 523-538;
Kuroki M et al., (1992) Hybridoma 11: 391-407 and Antibodies: A
Laboratory Manual, Ed Harlow E & Lane D editors supra, pp.
386-389, all of which are herein incorporated by reference in their
entireties.
[0177] In certain embodiments, the instant disclosure provides an
isolated antibody that binds to an epitope located within a region
of human TIGIT comprising the amino acid sequence set forth in SEQ
ID NO: 31, 32, or 33. In certain embodiments, the isolated antibody
binds to an epitope located within a region of human TIGIT
consisting essentially of the amino acid sequence set forth in SEQ
ID NO: 31, 32, or 33. In certain embodiments, the isolated antibody
binds to an epitope located within a region of human TIGIT, the
amino acid sequence of the region consisting of the amino acid
sequence set forth in SEQ ID NO: 31, 32, or 33. In certain
embodiments, the isolated antibody binds to a discontinuous epitope
located within a region of human TIGIT comprising a plurality of
amino acid sequences, each of the plurality of amino acid sequences
consisting of, consisting essentially of, or comprising the amino
acid sequence set forth in SEQ ID NO: 31, 32, or 33 (e.g., SEQ ID
NOs: 31 and 32, SEQ ID NOs: 31 and 33, SEQ ID NOs: 32 and 33, or
SEQ ID NOs: 31, 32, and 33).
[0178] In certain embodiments, the isolated antibody binds to an
epitope located within a region of human TIGIT comprising,
consisting essentially of, or consisting of the amino acid sequence
set forth in SEQ ID NO: 31. In another aspect, the instant
disclosure provides an antibody that, when bound to a human TIGIT
protein or fragment thereof, reduces hydrogen/deuterium exchange in
a region consisting of the amino acid sequence set forth in SEQ ID
NO: 31 relative to hydrogen/deuterium exchange in the region
consisting of the amino acid sequence set forth in SEQ ID NO: 31 in
the absence of the antibody, as determined by a hydrogen/deuterium
exchange assay. In certain embodiments, the reduction in
hydrogen/deuterium exchange is measured using hydrogen-deuterium
exchange (HDX), for example as described herein in the
examples.
[0179] In certain embodiments, the isolated antibody binds to an
epitope located within a region of human TIGIT comprising,
consisting essentially of, or consisting of the amino acid sequence
set forth in SEQ ID NO: 32. In another aspect, the instant
disclosure provides an antibody that, when bound to a human TIGIT
protein or fragment thereof, reduces hydrogen/deuterium exchange in
a region consisting of the amino acid sequence set forth in SEQ ID
NO: 32 relative to hydrogen/deuterium exchange in the region
consisting of the amino acid sequence set forth in SEQ ID NO: 32 in
the absence of the antibody, as determined by a hydrogen/deuterium
exchange assay. In certain embodiments, the reduction in
hydrogen/deuterium exchange is measured using hydrogen-deuterium
exchange (HDX), for example as described herein in the
examples.
[0180] In certain embodiments, the isolated antibody binds to an
epitope located within a region of human TIGIT comprising,
consisting essentially of, or consisting of the amino acid sequence
set forth in SEQ ID NO: 33. In another aspect, the instant
disclosure provides an antibody that, when bound to a human TIGIT
protein or fragment thereof, reduces hydrogen/deuterium exchange in
a region consisting of the amino acid sequence set forth in SEQ ID
NO: 33 relative to hydrogen/deuterium exchange in the region
consisting of the amino acid sequence set forth in SEQ ID NO: 33 in
the absence of the antibody, as determined by a hydrogen/deuterium
exchange assay. In certain embodiments, the reduction in
hydrogen/deuterium exchange is measured using hydrogen-deuterium
exchange (HDX), for example as described herein in the
examples.
[0181] In certain embodiments, the antibody binds to a
conformational epitope located within the amino acid sequences of
SEQ ID NOs: 31 and 32; 31 and 33; or 32 and 33. In certain
embodiments, the antibody binds to a conformational epitope located
within the amino acid sequences of 31, 32, and 33.
[0182] In certain embodiments, the antibody binds to an epitope
(e.g., conformational epitope) comprising one or more amino acid
residues selected from the group consisting of Q35, I47, N49, H90,
and T96, numbered according to the amino acid sequence of SEQ ID
NO: 40. In certain embodiments, the antibody binds to an epitope
(e.g., conformational epitope) comprising the amino acid residue of
Q35, numbered according to the amino acid sequence of SEQ ID NO:
40. In certain embodiments, the antibody binds to an epitope (e.g.,
conformational epitope) comprising the amino acid residue of I47,
numbered according to the amino acid sequence of SEQ ID NO: 40. In
certain embodiments, the antibody binds to an epitope (e.g.,
conformational epitope) comprising the amino acid residue of N49,
numbered according to the amino acid sequence of SEQ ID NO: 40. In
certain embodiments, the antibody binds to an epitope (e.g.,
conformational epitope) comprising the amino acid residue of H90,
numbered according to the amino acid sequence of SEQ ID NO: 40. In
certain embodiments, the antibody binds to an epitope (e.g.,
conformational epitope) comprising the amino acid residue of T96,
numbered according to the amino acid sequence of SEQ ID NO: 40. In
certain embodiments, the antibody binds to an epitope (e.g.,
conformational epitope) comprising two or more, three or more, or
four or more amino acid residues selected from the group consisting
of Q35, I47, N49, H90, and T96, numbered according to the amino
acid sequence of SEQ ID NO: 40. In certain embodiments, the
antibody binds to an epitope (e.g., conformational epitope)
comprising the amino acid residues of Q35, I47, N49, H90, and T96,
numbered according to the amino acid sequence of SEQ ID NO: 40.
[0183] In certain embodiments, the antibody binds to an epitope
(e.g., conformational epitope) comprising one or more amino acid
residues selected from the group consisting of Q35, 147, and T96,
numbered according to the amino acid sequence of SEQ ID NO: 40. In
certain embodiments, the antibody binds to an epitope (e.g.,
conformational epitope) comprising two or more amino acid residues
selected from the group consisting of Q35, I47, and T96, numbered
according to the amino acid sequence of SEQ ID NO: 40. In certain
embodiments, the antibody binds to an epitope (e.g., conformational
epitope) comprising the amino acid residues of Q35, I47, and T96,
numbered according to the amino acid sequence of SEQ ID NO: 40.
[0184] In certain embodiments, the epitope (e.g., conformational
epitope) of the antibody does not comprise at least one of the
amino acid residues selected from the group consisting of T34, L52,
H55, I56, S57, P58, S59, T98, R100, and F102, numbered according to
the amino acid sequence of SEQ ID NO: 40. In certain embodiments,
the epitope (e.g., conformational epitope) of the antibody does not
comprise the amino acid residue of T34, numbered according to the
amino acid sequence of SEQ ID NO: 40. In certain embodiments, the
epitope (e.g., conformational epitope) of the antibody does not
comprise the amino acid residue of L52, numbered according to the
amino acid sequence of SEQ ID NO: 40. In certain embodiments, the
epitope (e.g., conformational epitope) of the antibody does not
comprise the amino acid residue of H55, numbered according to the
amino acid sequence of SEQ ID NO: 40. In certain embodiments, the
epitope (e.g., conformational epitope) of the antibody does not
comprise the amino acid residue of I56, numbered according to the
amino acid sequence of SEQ ID NO: 40. In certain embodiments, the
epitope (e.g., conformational epitope) of the antibody does not
comprise the amino acid residue of S57, numbered according to the
amino acid sequence of SEQ ID NO: 40. In certain embodiments, the
epitope (e.g., conformational epitope) of the antibody does not
comprise the amino acid residue of P58, numbered according to the
amino acid sequence of SEQ ID NO: 40. In certain embodiments, the
epitope (e.g., conformational epitope) of the antibody does not
comprise the amino acid residue of S59, numbered according to the
amino acid sequence of SEQ ID NO: 40. In certain embodiments, the
epitope (e.g., conformational epitope) of the antibody does not
comprise the amino acid residue of T98, numbered according to the
amino acid sequence of SEQ ID NO: 40. In certain embodiments, the
epitope (e.g., conformational epitope) of the antibody does not
comprise the amino acid residue of R100, numbered according to the
amino acid sequence of SEQ ID NO: 40. In certain embodiments, the
epitope (e.g., conformational epitope) of the antibody does not
comprise the amino acid residue of F102, numbered according to the
amino acid sequence of SEQ ID NO: 40. In certain embodiments, the
epitope (e.g., conformational epitope) of the antibody does not
comprise at least two, at least three, at least four, at least
five, at least six, at least seven, at least eight, or at least
nine of the amino acid residues selected from the group consisting
of T34, L52, H55, I56, S57, P58, S59, T98, R100, and F102, numbered
according to the amino acid sequence of SEQ ID NO: 40. In certain
embodiments, the epitope (e.g., conformational epitope) of the
antibody does not comprise any one of the amino acid residues of
T34, L52, H55, I56, S57, P58, S59, T98, R100, and F102, numbered
according to the amino acid sequence of SEQ ID NO: 40.
[0185] In certain embodiments, the epitope (e.g., conformational
epitope) of the antibody does not comprise at least one of the
amino acid residues selected from the group consisting of L52, H55,
I56, S57, P58, S59, and F102, numbered according to the amino acid
sequence of SEQ ID NO: 40. In certain embodiments, the epitope
(e.g., conformational epitope) of the antibody does not comprise at
least two, at least three, at least four, at least five, or at
least six of the amino acid residues selected from the group
consisting of L52, H55, I56, S57, P58, S59, and F102, numbered
according to the amino acid sequence of SEQ ID NO: 40. In certain
embodiments, the epitope of the antibody does not comprise any one
of the amino acid residues of L52, H55, I56, S57, P58, S59, and
F102, numbered according to the amino acid sequence of SEQ ID NO:
40.
[0186] In certain embodiments, the epitope (e.g., conformational
epitope) of the antibody does not comprise at least one of the
amino acid residues selected from the group consisting of L52, H55,
and F102, numbered according to the amino acid sequence of SEQ ID
NO: 40. In certain embodiments, the epitope (e.g., conformational
epitope) of the antibody does not comprise at least two of the
amino acid residues selected from the group consisting of L52, H55,
and F102, numbered according to the amino acid sequence of SEQ ID
NO: 40. In certain embodiments, the epitope of the antibody does
not comprise any one of the amino acid residues of L52, H55, and
F102, numbered according to the amino acid sequence of SEQ ID NO:
40.
[0187] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), wherein the antibody does not
substantially bind to a TIGIT protein or an extracellular domain
thereof comprising an amino acid mutation selected from the group
consisting of Q35A, I47E, N49A, L52E, H90A, T96A, T96I,
C48Y/N49S/A50V, and T96I/T98K. The binding affinity can be assessed
by any method known in the art (e.g., the method disclosed in the
Example 5 herein). In certain embodiments, the antibody does not
substantially bind to a TIGIT protein or an extracellular domain
thereof comprising a Q35A mutation. In certain embodiments, the
binding affinity of the antibody to the TIGIT protein or the
extracellular domain thereof comprising a Q35A mutation is at least
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%
lower than the binding affinity of the antibody to a wild-type
TIGIT protein (e.g., comprising the amino acid sequence of SEQ ID
NO: 40) or a corresponding extracellular domain thereof. In certain
embodiments, the amino acid sequence of the extracellular domain of
the TIGIT protein comprising a Q35A mutation consists of or
consists essentially of the amino acid sequence of SEQ ID NO: 44,
and the amino acid sequence of the corresponding extracellular
domain of the wild-type TIGIT protein consists of or consists
essentially of the amino acid sequence of SEQ ID NO: 42. In certain
embodiments, the antibody does not substantially bind to a TIGIT
protein comprising an I47E mutation. In certain embodiments, the
binding affinity of the antibody to the TIGIT protein or the
extracellular domain thereof comprising a I47E mutation is at least
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%
lower than the binding affinity of the antibody to a wild-type
TIGIT protein (e.g., comprising the amino acid sequence of SEQ ID
NO: 40) or a corresponding extracellular domain thereof. In certain
embodiments, the amino acid sequence of the extracellular domain of
the TIGIT protein comprising an I47E mutation consists of or
consists essentially of the amino acid sequence of SEQ ID NO: 45,
and the amino acid sequence of the corresponding extracellular
domain of the wild-type TIGIT protein consists of or consists
essentially of the amino acid sequence of SEQ ID NO: 42. In certain
embodiments, the antibody does not substantially bind to a TIGIT
protein comprising an N49A mutation. In certain embodiments, the
binding affinity of the antibody to the TIGIT protein or the
extracellular domain thereof comprising an N49A mutation is at
least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
or 90% lower than the binding affinity of the antibody to a
wild-type TIGIT protein (e.g., comprising the amino acid sequence
of SEQ ID NO: 40) or a corresponding extracellular domain thereof.
In certain embodiments, the amino acid sequence of the
extracellular domain of the TIGIT protein comprising an N49A
mutation consists of or consists essentially of the amino acid
sequence of SEQ ID NO: 46, and the amino acid sequence of the
corresponding extracellular domain of the wild-type TIGIT protein
consists of or consists essentially of the amino acid sequence of
SEQ ID NO: 42. In certain embodiments, the antibody does not
substantially bind to a TIGIT protein comprising an L52E mutation.
In certain embodiments, the binding affinity of the antibody to the
TIGIT protein or the extracellular domain thereof comprising an
L52E mutation is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, or 90% lower than the binding affinity of the
antibody to a wild-type TIGIT protein (e.g., comprising the amino
acid sequence of SEQ ID NO: 40) or a corresponding extracellular
domain thereof. In certain embodiments, the amino acid sequence of
the extracellular domain of the TIGIT protein comprising an L52E
mutation consists of or consists essentially of the amino acid
sequence of SEQ ID NO: 48, and the amino acid sequence of the
corresponding extracellular domain of the wild-type TIGIT protein
consists of or consists essentially of the amino acid sequence of
SEQ ID NO: 42. In certain embodiments, the antibody does not
substantially bind to a TIGIT protein comprising an H90A mutation.
In certain embodiments, the binding affinity of the antibody to the
TIGIT protein or the extracellular domain thereof comprising an
H90A mutation is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, or 90% lower than the binding affinity of the
antibody to a wild-type TIGIT protein (e.g., comprising the amino
acid sequence of SEQ ID NO: 40) or a corresponding extracellular
domain thereof. In certain embodiments, the amino acid sequence of
the extracellular domain of the TIGIT protein comprising an H90A
mutation consists of or consists essentially of the amino acid
sequence of SEQ ID NO: 51, and the amino acid sequence of the
corresponding extracellular domain of the wild-type TIGIT protein
consists of or consists essentially of the amino acid sequence of
SEQ ID NO: 42. In certain embodiments, the antibody does not
substantially bind to a TIGIT protein comprising a T96A mutation.
In certain embodiments, the binding affinity of the antibody to the
TIGIT protein or the extracellular domain thereof comprising a T96A
mutation is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90% lower than the binding affinity of the
antibody to a wild-type TIGIT protein (e.g., comprising the amino
acid sequence of SEQ ID NO: 40) or a corresponding extracellular
domain thereof. In certain embodiments, the amino acid sequence of
the extracellular domain of the TIGIT protein comprising a T96A
mutation consists of or consists essentially of the amino acid
sequence of SEQ ID NO: 52, and the amino acid sequence of the
corresponding extracellular domain of the wild-type TIGIT protein
consists of or consists essentially of the amino acid sequence of
SEQ ID NO: 42. In certain embodiments, the antibody does not
substantially bind to a TIGIT protein comprising a T96I mutation.
In certain embodiments, the binding affinity of the antibody to the
TIGIT protein or the extracellular domain thereof comprising a T96I
mutation is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, or 90% lower than the binding affinity of the
antibody to a wild-type TIGIT protein (e.g., comprising the amino
acid sequence of SEQ ID NO: 40) or a corresponding extracellular
domain thereof. In certain embodiments, the amino acid sequence of
the extracellular domain of the TIGIT protein comprising a T96I
mutation consists of or consists essentially of the amino acid
sequence of SEQ ID NO: 53, and the amino acid sequence of the
corresponding extracellular domain of the wild-type TIGIT protein
consists of or consists essentially of the amino acid sequence of
SEQ ID NO: 42. In certain embodiments, the antibody does not
substantially bind to a TIGIT protein comprising a C48Y/N49S/A50V
mutation. In certain embodiments, the binding affinity of the
antibody to the TIGIT protein or the extracellular domain thereof
comprising a C48Y/N49S/A50V mutation is at least 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% lower than the
binding affinity of the antibody to a wild-type TIGIT protein
(e.g., comprising the amino acid sequence of SEQ ID NO: 40) or a
corresponding extracellular domain thereof. In certain embodiments,
the amino acid sequence of the extracellular domain of the TIGIT
protein comprising a C48Y/N49S/A50V mutation consists of or
consists essentially of the amino acid sequence of SEQ ID NO: 57,
and the amino acid sequence of the corresponding extracellular
domain of the wild-type TIGIT protein consists of or consists
essentially of the amino acid sequence of SEQ ID NO: 42. In certain
embodiments, the antibody does not substantially bind to a TIGIT
protein comprising a T96I/T98K mutation. In certain embodiments,
the binding affinity of the antibody to the TIGIT protein or the
extracellular domain thereof comprising a T96I/T98K mutation is at
least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
or 90% lower than the binding affinity of the antibody to a
wild-type TIGIT protein (e.g., comprising the amino acid sequence
of SEQ ID NO: 40) or a corresponding extracellular domain thereof.
In certain embodiments, the amino acid sequence of the
extracellular domain of the TIGIT protein comprising a T96I/T98K
mutation consists of or consists essentially of the amino acid
sequence of SEQ ID NO: 59, and the amino acid sequence of the
corresponding extracellular domain of the wild-type TIGIT protein
consists of or consists essentially of the amino acid sequence of
SEQ ID NO: 42. In certain embodiments, the amino acid sequence of
the TIGIT protein comprising a T96I/T98K mutation consists of or
consists essentially of the amino acid sequence of SEQ ID NO:
59.
[0188] In certain embodiments, the antibody specifically and/or
substantially binds to a TIGIT protein comprising an amino acid
mutation selected from the group consisting of T34A, L52A, H55A,
P58A, T98A, R100A, F102A, and I56V/S57A/P58S/S59V. The binding
affinity can be assessed by any method known in the art (e.g., the
method disclosed in the Example 5 herein). In certain embodiments,
the antibody specifically and/or substantially binds to a TIGIT
protein comprising a T34A mutation. In certain embodiments, the
binding affinity of the antibody to the TIGIT protein or the
extracellular domain thereof comprising a T34A mutation is greater
than or equal to 70%, 75%, 80%, 85%, 90%, or 95% of the binding
affinity of the antibody to a wild-type TIGIT protein (e.g.,
comprising the amino acid sequence of SEQ ID NO: 40) or a
corresponding extracellular domain thereof. In certain embodiments,
the amino acid sequence of the extracellular domain of a TIGIT
protein comprising a T34A mutation consists of or consists
essentially of the amino acid sequence of SEQ ID NO: 43, and the
amino acid sequence of the corresponding extracellular domain of
the wild-type TIGIT protein consists of or consists essentially of
the amino acid sequence of SEQ ID NO: 42. In certain embodiments,
the antibody specifically and/or substantially binds to a TIGIT
protein comprising an L52A mutation. In certain embodiments, the
binding affinity of the antibody to the TIGIT protein or the
extracellular domain thereof comprising an L52A mutation is greater
than or equal to 70%, 75%, 80%, 85%, 90%, or 95% of the binding
affinity of the antibody to a wild-type TIGIT protein (e.g.,
comprising the amino acid sequence of SEQ ID NO: 40) or a
corresponding extracellular domain thereof. In certain embodiments,
the amino acid sequence of the extracellular domain of a TIGIT
protein comprising an L52A mutation consists of or consists
essentially of the amino acid sequence of SEQ ID NO: 47, and the
amino acid sequence of the corresponding extracellular domain of
the wild-type TIGIT protein consists of or consists essentially of
the amino acid sequence of SEQ ID NO: 42. In certain embodiments,
the antibody specifically and/or substantially binds to a TIGIT
protein comprising an H55A mutation. In certain embodiments, the
binding affinity of the antibody to the TIGIT protein or the
extracellular domain thereof comprising an H55A mutation is greater
than or equal to 70%, 75%, 80%, 85%, 90%, or 95% of the binding
affinity of the antibody to a wild-type TIGIT protein (e.g.,
comprising the amino acid sequence of SEQ ID NO: 40) or a
corresponding extracellular domain thereof. In certain embodiments,
the amino acid sequence of the extracellular domain of a TIGIT
protein comprising an H55A mutation consists of or consists
essentially of the amino acid sequence of SEQ ID NO: 49, and the
amino acid sequence of the corresponding extracellular domain of
the wild-type TIGIT protein consists of or consists essentially of
the amino acid sequence of SEQ ID NO: 42. In certain embodiments,
the antibody specifically and/or substantially binds to a TIGIT
protein comprising a P58A mutation. In certain embodiments, the
binding affinity of the antibody to the TIGIT protein or the
extracellular domain thereof comprising a P58A mutation is greater
than or equal to 70%, 75%, 80%, 85%, 90%, or 95% of the binding
affinity of the antibody to a wild-type TIGIT protein (e.g.,
comprising the amino acid sequence of SEQ ID NO: 40) or a
corresponding extracellular domain thereof. In certain embodiments,
the amino acid sequence of the extracellular domain of a TIGIT
protein comprising a P58A mutation consists of or consists
essentially of the amino acid sequence of SEQ ID NO: 50, and the
amino acid sequence of the corresponding extracellular domain of
the wild-type TIGIT protein consists of or consists essentially of
the amino acid sequence of SEQ ID NO: 42. In certain embodiments,
the antibody specifically and/or substantially binds to a TIGIT
protein comprising a T98A mutation. In certain embodiments, the
binding affinity of the antibody to the TIGIT protein or the
extracellular domain thereof comprising a T98A mutation is greater
than or equal to 70%, 75%, 80%, 85%, 90%, or 95% of the binding
affinity of the antibody to a wild-type TIGIT protein (e.g.,
comprising the amino acid sequence of SEQ ID NO: 40) or a
corresponding extracellular domain thereof. In certain embodiments,
the amino acid sequence of the extracellular domain of a TIGIT
protein comprising a T98A mutation consists of or consists
essentially of the amino acid sequence of SEQ ID NO: 54, and the
amino acid sequence of the corresponding extracellular domain of
the wild-type TIGIT protein consists of or consists essentially of
the amino acid sequence of SEQ ID NO: 42. In certain embodiments,
the antibody specifically and/or substantially binds to a TIGIT
protein comprising an R100A mutation. In certain embodiments, the
binding affinity of the antibody to the TIGIT protein or the
extracellular domain thereof comprising an R100A mutation is
greater than or equal to 70%, 75%, 80%, 85%, 90%, or 95% of the
binding affinity of the antibody to a wild-type TIGIT protein
(e.g., comprising the amino acid sequence of SEQ ID NO: 40) or a
corresponding extracellular domain thereof. In certain embodiments,
the amino acid sequence of the extracellular domain of a TIGIT
protein comprising an R100A mutation consists of or consists
essentially of the amino acid sequence of SEQ ID NO: 55, and the
amino acid sequence of the corresponding extracellular domain of
the wild-type TIGIT protein consists of or consists essentially of
the amino acid sequence of SEQ ID NO: 42. In certain embodiments,
the antibody specifically and/or substantially binds to a TIGIT
protein comprising an F102A mutation. In certain embodiments, the
binding affinity of the antibody to the TIGIT protein or the
extracellular domain thereof comprising an F102A mutation is
greater than or equal to 70%, 75%, 80%, 85%, 90%, or 95% of the
binding affinity of the antibody to a wild-type TIGIT protein
(e.g., comprising the amino acid sequence of SEQ ID NO: 40) or a
corresponding extracellular domain thereof. In certain embodiments,
the amino acid sequence of the extracellular domain of a TIGIT
protein comprising an F102A mutation consists of or consists
essentially of the amino acid sequence of SEQ ID NO: 56, and the
amino acid sequence of the corresponding extracellular domain of
the wild-type TIGIT protein consists of or consists essentially of
the amino acid sequence of SEQ ID NO: 42. In certain embodiments,
the antibody specifically and/or substantially binds to a TIGIT
protein comprising an I56V/S57A/P58S/S59V mutation. In certain
embodiments, the binding affinity of the antibody to the TIGIT
protein or the extracellular domain thereof comprising an
I56V/S57A/P58S/S59V mutation is greater than or equal to 70%, 75%,
80%, 85%, 90%, or 95% of the binding affinity of the antibody to a
wild-type TIGIT protein (e.g., comprising the amino acid sequence
of SEQ ID NO: 40) or a corresponding extracellular domain thereof.
In certain embodiments, the amino acid sequence of the
extracellular domain of a TIGIT protein comprising an
I56V/S57A/P58S/S59V mutation consists of or consists essentially of
the amino acid sequence of SEQ ID NO: 58, and the amino acid
sequence of the corresponding extracellular domain of the wild-type
TIGIT protein consists of or consists essentially of the amino acid
sequence of SEQ ID NO: 42.
[0189] In certain embodiments, the antibody inhibits the binding of
human TIGIT to human PVR, PVRL2, and/or PVRL3. In certain
embodiments, the binding of human TIGIT to human PVR is reduced by
more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, or 99% in the presence of the antibody relative to the
binding of human TIGIT to human PVR in the absence of the antibody.
In certain embodiments, the binding of human TIGIT to human PVRL2
is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% in the presence of the antibody
relative to the binding of human TIGIT to human PVRL2 in the
absence of the antibody.
[0190] In certain embodiments, the antibody inhibits a soluble
fragment of human TIGIT from binding to a soluble fragment of human
PVR, PVRL2, and/or PVRL3. In certain embodiments, the binding of a
soluble fragment of human TIGIT to a soluble fragment of human PVR
is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% in the presence of the antibody
relative to the binding of a soluble fragment of human TIGIT to a
soluble fragment of human PVR in the absence of the antibody. In
certain embodiments, the binding of a soluble fragment of human
TIGIT to a soluble fragment of human PVRL2 is reduced by more than
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% in the presence of the antibody relative to the binding of a
soluble fragment of human TIGIT to a soluble fragment of human
PVRL2 in the absence of the antibody.
[0191] In certain embodiments, the antibody inhibits a
TIGIT-expressing cell from binding to a soluble fragment of human
PVR, PVRL2, and/or PVRL3. In certain embodiments, the binding of a
TIGIT-expressing cell to a soluble fragment of human PVR is reduced
by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, or 99% in the presence of the antibody relative to the
binding of a TIGIT-expressing cell to a soluble fragment of human
PVR in the absence of the antibody. In certain embodiments, the
binding of a TIGIT-expressing cell to a soluble fragment of human
PVRL2 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the
antibody relative to the binding of a TIGIT-expressing cell to a
soluble fragment of human PVRL2 in the absence of the antibody.
[0192] In certain embodiments, the antibody inhibits a
TIGIT-expressing cell from binding to a cell expressing human PVR,
PVRL2, and/or PVRL3. In certain embodiments, the binding of a
TIGIT-expressing cell to a PVR-expressing cell is reduced by more
than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% in the presence of the antibody relative to the binding
of a TIGIT-expressing cell to a PVR-expressing cell in the absence
of the antibody. In certain embodiments, the binding of a
TIGIT-expressing cell to a PVRL2-expressing cell is reduced by more
than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% in the presence of the antibody relative to the binding
of a TIGIT-expressing cell to a PVRL2-expressing cell in the
absence of the antibody.
[0193] In certain embodiments, the antibody does not bind
specifically to CD226 (e.g., human CD226). In certain embodiments,
the binding affinity of the antibody to TIGIT is stronger by at
least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% than the binding
affinity of the antibody to CD226, as assessed by methods described
herein and/or known to one of skill in the art. In certain
embodiments, the binding affinity of the antibody to TIGIT is
stronger by at least 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2
fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold,
7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40
fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, or
more, than the binding affinity of the antibody to CD226, as
assessed by methods described herein and/or known to one of skill
in the art. In certain embodiments, the K.sub.D that represents the
affinity of the antibody to CD226 is higher than 1, 2, 5, 10, 20,
50, or 100 .mu.g/ml.
[0194] In certain embodiments, the antibody does not bind
specifically to CD96 (e.g., human CD96). In certain embodiments,
the binding affinity of the antibody to TIGIT is stronger by at
least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% than the binding
affinity of the antibody to CD96, as assessed by methods described
herein and/or known to one of skill in the art. In certain
embodiments, the binding affinity of the antibody to TIGIT is
stronger by at least 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2
fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold,
7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40
fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, or
more, than the binding affinity of the antibody to CD96, as
assessed by methods described herein and/or known to one of skill
in the art. In certain embodiments, the K.sub.D that represents the
affinity of the antibody to CD96 is higher than 1, 2, 5, 10, 20,
50, or 100 .mu.g/mL.
[0195] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), the antibody comprising a heavy chain
comprising the amino acid sequence set forth in SEQ ID NO: 11, 12,
13, 14, 15, 16, 17, or 18. In certain embodiments, the antibody
comprises a heavy chain comprising the amino acid sequence set
forth in SEQ ID NO: 11. In certain embodiments, the antibody
comprises a heavy chain comprising the amino acid sequence set
forth in SEQ ID NO: 12. In certain embodiments, the antibody
comprises a heavy chain comprising the amino acid sequence set
forth in SEQ ID NO: 13. In certain embodiments, the antibody
comprises a heavy chain comprising the amino acid sequence set
forth in SEQ ID NO: 14. In certain embodiments, the antibody
comprises a heavy chain comprising the amino acid sequence set
forth in SEQ ID NO: 15. In certain embodiments, the antibody
comprises a heavy chain comprising the amino acid sequence set
forth in SEQ ID NO: 16. In certain embodiments, the antibody
comprises a heavy chain comprising the amino acid sequence set
forth in SEQ ID NO: 17. In certain embodiments, the antibody
comprises a heavy chain comprising the amino acid sequence set
forth in SEQ ID NO: 18. In certain embodiments, the amino acid
sequence of the heavy chain consists of the amino acid sequence set
forth in SEQ ID NO: 11. In certain embodiments, the amino acid
sequence of the heavy chain consists of the amino acid sequence set
forth in SEQ ID NO: 12. In certain embodiments, the amino acid
sequence of the heavy chain consists of the amino acid sequence set
forth in SEQ ID NO: 13. In certain embodiments, the amino acid
sequence of the heavy chain consists of the amino acid sequence set
forth in SEQ ID NO: 14. In certain embodiments, the amino acid
sequence of the heavy chain consists of the amino acid sequence set
forth in SEQ ID NO: 15. In certain embodiments, the amino acid
sequence of the heavy chain consists of the amino acid sequence set
forth in SEQ ID NO: 16. In certain embodiments, the amino acid
sequence of the heavy chain consists of the amino acid sequence set
forth in SEQ ID NO: 17. In certain embodiments, the amino acid
sequence of the heavy chain consists of the amino acid sequence set
forth in SEQ ID NO: 18.
[0196] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), the antibody comprising a light chain
comprising the amino acid sequence set forth in SEQ ID NO: 27. In
certain embodiments, the amino acid sequence of the light chain
consists of the amino acid sequence set forth in SEQ ID NO: 27.
[0197] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), the antibody comprising a heavy chain
comprising the amino acid sequence of SEQ ID NO: 11; and a light
chain comprising the amino acid sequence of SEQ ID NO: 27. In
certain embodiments, the instant disclosure provides an isolated
antibody that specifically binds to TIGIT (e.g., human TIGIT or
cynomolgus TIGIT), the antibody comprising a heavy chain comprising
the amino acid sequence of SEQ ID NO: 12; and a light chain
comprising the amino acid sequence of SEQ ID NO: 27. In certain
embodiments, the instant disclosure provides an isolated antibody
that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus
TIGIT), the antibody comprising a heavy chain comprising the amino
acid sequence of SEQ ID NO: 13; and a light chain comprising the
amino acid sequence of SEQ ID NO: 27. In certain embodiments, the
instant disclosure provides an isolated antibody that specifically
binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), the
antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO: 14; and a light chain comprising the amino
acid sequence of SEQ ID NO: 27. In certain embodiments, the instant
disclosure provides an isolated antibody that specifically binds to
TIGIT (e.g., human TIGIT or cynomolgus TIGIT), the antibody
comprising a heavy chain comprising the amino acid sequence of SEQ
ID NO: 15; and a light chain comprising the amino acid sequence of
SEQ ID NO: 27. In certain embodiments, the instant disclosure
provides an isolated antibody that specifically binds to TIGIT
(e.g., human TIGIT or cynomolgus TIGIT), the antibody comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO: 16;
and a light chain comprising the amino acid sequence of SEQ ID NO:
27. In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), the antibody comprising a heavy chain
comprising the amino acid sequence of SEQ ID NO: 17; and a light
chain comprising the amino acid sequence of SEQ ID NO: 27. In
certain embodiments, the instant disclosure provides an isolated
antibody that specifically binds to TIGIT (e.g., human TIGIT or
cynomolgus TIGIT), the antibody comprising a heavy chain comprising
the amino acid sequence of SEQ ID NO: 18; and a light chain
comprising the amino acid sequence of SEQ ID NO: 27.
[0198] In certain embodiments, the amino acid sequences of the
heavy chain and light chain consist of the amino acid sequences of
SEQ ID NOs: 11 and 27, respectively. In certain embodiments, the
amino acid sequences of the heavy chain and light chain consist of
the amino acid sequences of SEQ ID NOs: 12 and 27, respectively. In
certain embodiments, the amino acid sequences of the heavy chain
and light chain consist of the amino acid sequences of SEQ ID NOs:
13 and 27, respectively. In certain embodiments, the amino acid
sequences of the heavy chain and light chain consist of the amino
acid sequences of SEQ ID NOs: 14 and 27, respectively. In certain
embodiments, the amino acid sequences of the heavy chain and light
chain consist of the amino acid sequences of SEQ ID NOs: 15 and 27,
respectively. In certain embodiments, the amino acid sequences of
the heavy chain and light chain consist of the amino acid sequences
of SEQ ID NOs: 16 and 27, respectively. In certain embodiments, the
amino acid sequences of the heavy chain and light chain consist of
the amino acid sequences of SEQ ID NOs: 17 and 27, respectively. In
certain embodiments, the amino acid sequences of the heavy chain
and light chain consist of the amino acid sequences of SEQ ID NOs:
18 and 27, respectively.
[0199] Any antibody format can be used in the antibodies disclosed
herein. In certain embodiments, the antibody is a single chain
antibody or single-chain Fv (scFv). In certain embodiments, the
antibody is a scFv fused with an Fc region (scFv-Fc). In certain
embodiments, the antibody is a Fab fragment. In certain
embodiments, the antibody is a F(ab').sub.2 fragment.
[0200] In certain embodiments, the antibody disclosed herein is a
multispecific antibody (e.g., a bispecific antibody) which
specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
and a second antigen.
[0201] In certain embodiments, the antibody disclosed herein is
conjugated to a second antibody that specifically binds to a second
antigen. In certain embodiments, the antibody disclosed herein is
covalently conjugated to a second antibody. In certain embodiments,
the antibody disclosed herein is non-covalently conjugated to a
second antibody. In certain embodiments, the antibody disclosed
herein is cross-linked to a second antibody. In certain
embodiments, the second antigen is a tumor-associated antigen
(e.g., a polypeptide overexpressed in a tumor, a polypeptide
derived from an oncovirus, a polypeptide comprising a
post-translational modification specific to a tumor, a polypeptide
specifically mutated in a tumor). In certain embodiments, the
tumor-associated antigen is EGFR (e.g., human EGFR), optionally
wherein the second antibody is cetuximab. In certain embodiments,
the tumor-associated antigen is Her2 (e.g., human Her2), optionally
wherein the second antibody is trastuzumab. In certain embodiments,
the tumor-associated antigen is CD20 (e.g., human CD20).
[0202] In certain embodiments, the antibody disclosed herein is
conjugated to a cytotoxic agent, cytostatic agent, toxin,
radionuclide, or detectable label. In certain embodiments, the
cytotoxic agent is able to induce death or destruction of a cell in
contact therewith. In certain embodiments, the cytostatic agent is
able to prevent or substantially reduce proliferation and/or
inhibits the activity or function of a cell in contact therewith.
In certain embodiments, the cytotoxic agent or cytostatic agent is
a chemotherapeutic agent. In certain embodiments, the radionuclide
is selected from the group consisting of the isotopes .sup.3H,
.sup.14C, .sup.32P, .sup.35S, .sup.36Cl, .sup.51Cr, .sup.57Co,
.sup.58Co, .sup.59Fe, .sup.67Cu, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.117Lu, .sup.121I, .sup.124I, .sup.125I, .sup.131I, .sup.198Au,
.sup.211At, .sup.213Bi, .sup.225Ac and .sup.186Re. In certain
embodiments, the detectable label comprises a fluorescent moiety or
a click chemistry handle.
[0203] Any immunoglobulin (Ig) constant region can be used in the
antibodies disclosed herein. In certain embodiments, the Ig region
is a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule,
any class (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4,
IgA.sub.1, and IgA.sub.2), or any subclass (e.g., IgG.sub.2a and
IgG.sub.2b) of immunoglobulin molecule.
[0204] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), the antibody comprising a heavy chain
constant region comprising the amino acid sequence of SEQ ID NO:
19, 20, 21, 22, 23, 24, 25, or 26. In certain embodiments, the
instant disclosure provides an isolated antibody that specifically
binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), the
antibody comprising a light chain constant region comprising the
amino acid sequence of SEQ ID NO: 28.
[0205] In certain embodiments, one, two, or more mutations (e.g.,
amino acid substitutions) are introduced into the Fc region of an
antibody described herein (e.g., CH2 domain (residues 231-340 of
human IgG.sub.1) and/or CH3 domain (residues 341-447 of human
IgG.sub.1) and/or the hinge region, numbered according to the EU
numbering system, to alter one or more functional properties of the
antibody, such as serum half-life, complement fixation, Fc receptor
binding, and/or antigen-dependent cellular cytotoxicity.
[0206] In certain embodiments, one, two, or more mutations (e.g.,
amino acid substitutions) are introduced into the hinge region of
the Fc region (CH1 domain) such that the number of cysteine
residues in the hinge region are altered (e.g., increased or
decreased) as described in, e.g., U.S. Pat. No. 5,677,425, herein
incorporated by reference in its entirety. The number of cysteine
residues in the hinge region of the CH1 domain may be altered to,
e.g., facilitate assembly of the light and heavy chains, or to
alter (e.g., increase or decrease) the stability of the
antibody.
[0207] In a specific embodiment, one, two, or more amino acid
mutations (e.g., substitutions, insertions or deletions) are
introduced into an IgG constant domain, or FcRn-binding fragment
thereof (preferably an Fc or hinge-Fc domain fragment) to alter
(e.g., decrease or increase) half-life of the antibody in vivo.
See, e.g., International Publication Nos. WO 02/060919; WO
98/23289; and WO 97/34631; and U.S. Pat. Nos. 5,869,046, 6,121,022,
6,277,375 and 6,165,745, all of which are herein incorporated by
reference in their entireties, for examples of mutations that will
alter (e.g., decrease or increase) the half-life of an antibody in
vivo. In certain embodiments, one, two or more amino acid mutations
(e.g., substitutions, insertions, or deletions) are introduced into
an IgG constant domain, or FcRn-binding fragment thereof
(preferably an Fc or hinge-Fc domain fragment) to decrease the
half-life of the antibody in vivo. In other embodiments, one, two
or more amino acid mutations (e.g., substitutions, insertions or
deletions) are introduced into an IgG constant domain, or
FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain
fragment) to increase the half-life of the antibody in vivo. In a
specific embodiment, the antibodies may have one or more amino acid
mutations (e.g., substitutions) in the second constant (CH2) domain
(residues 231-340 of human IgG.sub.1) and/or the third constant
(CH3) domain (residues 341-447 of human IgG.sub.1), numbered
according to the EU numbering system. In a specific embodiment, the
constant region of the IgG.sub.1 of an antibody described herein
comprises a methionine (M) to tyrosine (Y) substitution in position
252, a serine (S) to threonine (T) substitution in position 254,
and a threonine (T) to glutamic acid (E) substitution in position
256, numbered according to the EU numbering system. See U.S. Pat.
No. 7,658,921, which is herein incorporated by reference in its
entirety. This type of mutant IgG, referred to as "YTE mutant" has
been shown to display fourfold increased half-life as compared to
wild-type versions of the same antibody (see Dall'Acqua W F et al.,
(2006) J Biol Chem 281: 23514-24, which is herein incorporated by
reference in its entirety). In certain embodiments, an antibody
comprises an IgG constant domain comprising one, two, three or more
amino acid substitutions of amino acid residues at positions
251-257, 285-290, 308-314, 385-389, and 428-436, numbered according
to the EU numbering system.
[0208] In certain embodiments, one, two, or more mutations (e.g.,
amino acid substitutions) are introduced into the Fc region of an
antibody described herein (e.g., CH2 domain (residues 231-340 of
human IgG.sub.1) and/or CH3 domain (residues 341-447 of human
IgG.sub.1) and/or the hinge region, numbered according to the EU
numbering system, to increase or decrease the affinity of the
antibody for an Fc receptor (e.g., an activated Fc receptor) on the
surface of an effector cell. Mutations in the Fc region of an
antibody that decrease or increase the affinity of an antibody for
an Fc receptor and techniques for introducing such mutations into
the Fc receptor or fragment thereof are known to one of skill in
the art. Examples of mutations in the Fc receptor of an antibody
that can be made to alter the affinity of the antibody for an Fc
receptor are described in, e.g., Smith P et al., (2012) PNAS 109:
6181-6186, U.S. Pat. No. 6,737,056, and International Publication
Nos. WO 02/060919; WO 98/23289; and WO 97/34631, all of which are
herein incorporated by reference in their entireties.
[0209] In certain embodiments, the antibody comprises a heavy chain
constant region that is a variant of a wild type heavy chain
constant region, wherein the variant heavy chain constant region
binds to Fc.gamma.RIIB with higher affinity than the wild type
heavy chain constant region binds to Fc.gamma.RIIB In certain
embodiments, the variant heavy chain constant region is a variant
human heavy chain constant region, e.g., a variant human IgG.sub.1,
a variant human IgG.sub.2, or a variant human IgG.sub.4 heavy chain
constant region. In certain embodiments, the variant human IgG
heavy chain constant region comprises one or more of the following
amino acid mutations, according to the EU numbering system: G236D,
P238D, S239D, S267E, L328F, and L328E. In certain embodiments, the
variant human IgG heavy chain constant region comprises a set of
amino acid mutations selected from the group consisting of: S267E
and L328F; P238D and L328E; P238D and one or more substitutions
selected from the group consisting of E233D, G237D, H268D, P271G,
and A330R; P238D, E233D, G237D, H268D, P271G, and A330R; G236D and
S267E; S239D and S267E; V262E, S267E, and L328F; and V264E, S267E,
and L328F, according to the EU numbering system. In certain
embodiments, the Fc.gamma.RIIB is expressed on a cell selected from
the group consisting of macrophages, monocytes, B cells, dendritic
cells, endothelial cells, and activated T cells.
[0210] In a further embodiment, one, two, or more amino acid
substitutions are introduced into an IgG constant domain Fc region
to alter the effector function(s) of the antibody. For example, one
or more amino acids selected from amino acid residues 234, 235,
236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330,
332, and 396, numbered according to the EU numbering system, can be
replaced with a different amino acid residue such that the antibody
has an altered affinity for an effector ligand but 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 Cl component of complement. This approach is described in
further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, each of
which is herein incorporated by reference in its entirety. In
certain embodiments, the deletion or inactivation (through point
mutations or other means) of a constant region domain may reduce Fc
receptor binding of the circulating antibody thereby increasing
tumor localization. See, e.g., U.S. Pat. Nos. 5,585,097 and
8,591,886, each of which is herein incorporated by reference in its
entirety, for a description of mutations that delete or inactivate
the constant domain and thereby increase tumor localization. In
certain embodiments, one or more amino acid substitutions may be
introduced into the Fc region of an antibody described herein to
remove potential glycosylation sites on the Fc region, which may
reduce Fc receptor binding (see, e.g., Shields R L et al., (2001) J
Biol Chem 276: 6591-604, which is herein incorporated by reference
in its entirety). In various embodiments, one or more of the
following mutations in the constant region of an antibody described
herein may be made: an N297A substitution; an N297Q substitution;
an L234A substitution; an L234F substitution; an L235A
substitution; an L235F substitution; an L235V substitution; an
L237A substitution; an S239D substitution; an E233P substitution;
an L234V substitution; an L235A substitution; a C236 deletion; a
P238A substitution; an S239D substitution; an F243L substitution; a
D265A substitution; an S267E substitution; an L328F substitution;
an R292P substitution; a Y300L substitution; an A327Q substitution;
a P329A substitution; an A332L substitution; an I332E substitution;
or a P396L substitution, numbered according to the EU numbering
system.
[0211] In certain embodiments, a mutation selected from the group
consisting of D265A, P329A, and a combination thereof, numbered
according to the EU numbering system, may be made in the constant
region of an antibody described herein. In certain embodiments, a
mutation selected from the group consisting of L235A, L237A, and a
combination thereof, numbered according to the EU numbering system,
may be made in the constant region of an antibody described herein.
In certain embodiments, a mutation selected from the group
consisting of S267E, L328F, and a combination thereof, numbered
according to the EU numbering system, may be made in the constant
region of an antibody described herein. In certain embodiments, a
mutation selected from the group consisting of S239D, 1332E,
optionally A330L, and a combination thereof, numbered according to
the EU numbering system, may be made in the constant region of an
antibody described herein. In certain embodiments, a mutation
selected from the group consisting of L235V, F243L, R292P, Y300L,
P396L, and a combination thereof, numbered according to the EU
numbering system, may be made in the constant region of an antibody
described herein. In certain embodiments, a mutation selected from
the group consisting of S267E, L328F, and a combination thereof,
numbered according to the EU numbering system, may be made in the
constant region of an antibody described herein.
[0212] In a specific embodiment, an antibody described herein
comprises the constant domain of an IgG.sub.1 with an N297Q or
N297A amino acid substitution, numbered according to the EU
numbering system. In one embodiment, an antibody described herein
comprises the constant domain of an IgG.sub.1 with a mutation
selected from the group consisting of D265A, P329A, and a
combination thereof, numbered according to the EU numbering system.
In another embodiment, an antibody described herein comprises the
constant domain of an IgG.sub.1 with a mutation selected from the
group consisting of L234A, L235A, and a combination thereof,
numbered according to the EU numbering system. In another
embodiment, an antibody described herein comprises the constant
domain of an IgG.sub.1 with a mutation selected from the group
consisting of L234F, L235F, N297A, and a combination thereof,
numbered according to the EU numbering system. In certain
embodiments, amino acid residues in the constant region of an
antibody described herein in the positions corresponding to
positions L234, L235, and D265 in a human IgG.sub.1 heavy chain,
numbered according to the EU numbering system, are not L, L, and D,
respectively. This approach is described in detail in International
Publication No. WO 14/108483, which is herein incorporated by
reference in its entirety. In a particular embodiment, the amino
acids corresponding to positions L234, L235, and D265 in a human
IgG.sub.1 heavy chain are F, E, and A; or A, A, and A,
respectively, numbered according to the EU numbering system.
[0213] In certain embodiments, one or more amino acids selected
from amino acid residues 329, 331, and 322 in the constant region
of an antibody described herein, numbered according to the EU
numbering system, 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
(Idusogie et al.), which is herein incorporated by reference in its
entirety. In certain embodiments, one or more amino acid residues
within amino acid positions 231 to 238 in the N-terminal region of
the CH2 domain of an antibody described herein are altered to
thereby alter the ability of the antibody to fix complement,
numbered according to the EU numbering system. This approach is
described further in International Publication No. WO 94/29351,
which is herein incorporated by reference in its entirety. In
certain embodiments, the Fc region of an antibody described herein
is modified to increase the ability of the antibody to mediate
antibody dependent cellular cytotoxicity (ADCC) and/or to increase
the affinity of the antibody for an Fc.gamma. receptor by mutating
one or more amino acids (e.g., introducing amino acid
substitutions) at the following positions: 238, 239, 248, 249, 252,
254, 255, 256, 258, 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, 328, 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, numbered
according to the EU numbering system. This approach is described
further in International Publication No. WO 00/42072, which is
herein incorporated by reference in its entirety.
[0214] In certain embodiments, an antibody described herein
comprises a modified constant domain of an IgG.sub.1,wherein the
modification increases the ability of the antibody to mediate
antibody dependent cellular cytotoxicity (ADCC). In certain
embodiments, 0.1, 1, or 10 .mu.g/mL of the antibody is capable of
inducing cell death of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, or 60% of TIGIT-expressing cells within 1, 2, or 3 hours, as
assessed by methods described herein and/or known to a person of
skill in the art. In certain embodiments, the modified constant
domain of an IgG.sub.1 comprises S239D and I332E substitutions,
numbered according to the EU numbering system. In certain
embodiments, the modified constant domain of an IgG.sub.1 comprises
S239D, A330L, and I332E substitutions, numbered according to the EU
numbering system. In certain embodiments, the modified constant
domain of an IgG.sub.1 comprises L235V, F243L, R292P, Y300L, and
P396L substitutions, numbered according to the EU numbering system.
In certain embodiments, the antibody is capable of inducing cell
death in effector T cells and Tregs, wherein the percentage of
Tregs that undergo cell death is higher than the percentage of
effector T cells that undergo cell death by at least 1.2 fold, 1.3
fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2
fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, or 5 fold.
[0215] In certain embodiments, an antibody described herein
comprises the constant region of an IgG.sub.4 antibody and the
serine at amino acid residue 228 of the heavy chain, numbered
according to the EU numbering system, is substituted for proline.
In certain embodiments, the instant disclosure provides an isolated
antibody that specifically binds to TIGIT (e.g., human TIGIT or
cynomolgus TIGIT), the antibody comprising a heavy chain constant
region comprising the amino acid sequence of SEQ ID NO: 26.
[0216] In certain embodiments, any of the constant region mutations
or modifications described herein can be introduced into one or
both heavy chain constant regions of an antibody described herein
having two heavy chain constant regions.
[0217] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) and functions as an antagonist (e.g.,
decreases or inhibits TIGIT activity).
[0218] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) and decreases or inhibits TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT) activity by at least 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 98%, or 99%, as assessed by methods described herein
and/or known to one of skill in the art, relative to TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT) activity without any antibody or
with an unrelated antibody (e.g., an antibody that does not
specifically bind to TIGIT (e.g., human TIGIT or cynomolgus
TIGIT)). In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) and decreases or inhibits TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT) activity by at least about 1.2
fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5
fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10
fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70
fold, 80 fold, 90 fold, 100 fold, or more, as assessed by methods
described herein and/or known to one of skill in the art, relative
to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) activity without
any antibody or with an unrelated antibody (e.g., an antibody that
does not specifically bind to TIGIT (e.g., human TIGIT)).
Non-limiting examples of TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) activity can include TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) signaling; TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
binding to its ligand (e.g., PVR (e.g., human or cynomolgus PVR),
PVRL2 (e.g., human or cynomolgus PVRL2), PVRL3 (e.g., human or
cynomolgus PVRL3), or a fragment and/or fusion protein thereof);
activation of a T cell (e.g., a T cell expressing human TIGIT);
activation of a natural killer (NK) cell; decrease or inhibition of
a Treg; increase of cytokine (e.g., IL-2, IFN-.gamma., and/or
TNF-.alpha.) production; increase of the activity of PVR (e.g.,
human PVR), PVRL2 (e.g., human PVRL2), and/or PVRL3 (e.g., human
PVRL3); and activation of an antigen-presenting cell (APC)
expressing PVR (e.g., human PVR), PVRL2 (e.g., human PVRL2), and/or
PVRL3 (e.g., human PVRL3). In specific embodiments, an increase in
a TIGIT (e.g., human TIGIT or cynomolgus TIGIT) activity is
assessed as described in the Examples, infra.
[0219] In specific embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) and decreases or inhibits TIGIT (e.g.,
human or cynomolgus TIGIT) binding to its ligand (e.g., PVR (e.g.,
human or cynomolgus PVR), PVRL2 (e.g., human or cynomolgus PVRL2),
PVRL3 (e.g., human or cynomolgus PVRL3), or a fragment and/or
fusion protein thereof) by at least about 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 98%, or 99%, as assessed by methods described herein (see the
Examples, infra) or known to one of skill in the art, relative to
TIGIT (e.g., human TIGIT or cynomolgus TIGIT) binding to this
ligand without any antibody or with an unrelated antibody (e.g., an
antibody that does not specifically bind to TIGIT (e.g., human or
cynomolgus TIGIT)). In specific embodiments, the instant disclosure
provides an isolated antibody that specifically binds to TIGIT
(e.g., human or cynomolgus TIGIT) and increases TIGIT (e.g., human
or cynomolgus TIGIT) binding to its ligand (e.g., PVR (e.g., human
or cynomolgus PVR), PVRL2 (e.g., human or cynomolgus PVRL2), PVRL3
(e.g., human or cynomolgus PVRL3), or a fragment and/or fusion
protein thereof) by at least about 1.2 fold, 1.3 fold, 1.4 fold,
1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5
fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30
fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100
fold, as assessed by methods described herein (see the Examples,
infra) or known to one of skill in the art, relative to TIGIT
(e.g., human TIGIT) binding to this ligand without any antibody or
with an unrelated antibody (e.g., an antibody that does not
specifically bind to TIGIT (e.g., human or cynomolgus TIGIT)).
[0220] In specific embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) and activates a T cell (e.g., a T cell
expressing human TIGIT). In certain embodiments, the T cell is a
memory T cell. In certain embodiments, the T cell is a
TIGIT-expressing Jurkat cell. In certain embodiments, the antibody
disclosed herein increases the activity of Nuclear factor of
activated T-cells (NFAT) by at least about 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 98%, or 99%, as assessed by methods described herein (see the
Examples, infra) or known to one of skill in the art, relative to
NFAT activity without any antibody or with an unrelated antibody
(e.g., an antibody that does not specifically bind to TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT)). In certain embodiments, the
antibody disclosed herein increases the activity of NFAT by at
least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5
fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8
fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold,
60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, or more, as
assessed by methods described herein (see the Examples, infra) or
known to one of skill in the art, relative to NFAT activity without
any antibody or with an unrelated antibody (e.g., an antibody that
does not specifically bind to TIGIT (e.g., human TIGIT or
cynomolgus TIGIT)). In certain embodiments, the antibody increases
NFAT activity in the presence of a ligand of TIGIT (e.g., PVR
(e.g., human or cynomolgus PVR), PVRL2 (e.g., human or cynomolgus
PVRL2), PVRL3 (e.g., human or cynomolgus PVRL3), a fragment and/or
fusion protein thereof), and/or a cell expressing a ligand of TIGIT
(e.g., a monocyte, a dendritic cell).
[0221] In specific embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) and increases cytokine production (e.g.,
IL-2, IFN-.gamma. and/or TNF-.alpha.) by at least about 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methods described
herein (see the Examples, infra) or known to one of skill in the
art, relative to cytokine production without any antibody or with
an unrelated antibody (e.g., an antibody that does not specifically
bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)). In specific
embodiments, the instant disclosure provides an isolated antibody
that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) and increases cytokine production (e.g., IL-2, IFN-.gamma.
and/or TNF-.alpha.) by at least about 1.2 fold, 1.3 fold, 1.4 fold,
1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5
fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30
fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100
fold, or more, as assessed by methods described herein (see the
Examples, infra) or known to one of skill in the art, relative to
cytokine production without any antibody or with an unrelated
antibody (e.g., an antibody that does not specifically bind to
TIGIT (e.g., human TIGIT or cynomolgus TIGIT)). In certain
embodiments, the antibody increases cytokine production (e.g.,
IL-2, IFN-.gamma. and/or TNF-.alpha.) in the presence of a ligand
of TIGIT (e.g., PVR (e.g., human or cynomolgus PVR), PVRL2 (e.g.,
human or cynomolgus PVRL2), PVRL3 (e.g., human or cynomolgus
PVRL3), a fragment and/or fusion protein thereof), and/or a cell
expressing a ligand of TIGIT (e.g., a monocyte, a dendritic cell).
In certain embodiments, the antibody increases the production of
IL-2 relative to IL-2 production without any antibody or with an
unrelated antibody (e.g., an antibody that does not specifically
bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)) to a greater
degree than the antibody increases the production of IFN-.gamma.
relative to IFN-.gamma. production without any antibody or with an
unrelated antibody (e.g., an antibody that does not specifically
bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)).
[0222] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) and which either alone or in combination
with an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab),
increases IFN.gamma. and/or IL-2 production in human peripheral
blood mononuclear cells (PBMCs) in response to Staphylococcus
Enterotoxin A (SEA) stimulation by at least about 1.2 fold, 1.3
fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4
fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15
fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80
fold, 90 fold, or 100 fold, as assessed by methods described herein
(see the Examples, infra) or known to one of skill in the art,
relative to IFN.gamma. and/or IL-2 production without any antibody
or with an unrelated antibody (e.g., an antibody that does not
specifically bind to TIGIT (e.g., human TIGIT or cynomolgus
TIGIT)).
[0223] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) and which either alone or in combination
with an anti-CTLA-4 antibody (e.g., ipilimumab), increases
IFN.gamma. and/or IL-2 production in human peripheral blood
mononuclear cells (PBMCs) in response to Staphylococcus Enterotoxin
A (SEA) stimulation by at least about 1.2 fold, 1.3 fold, 1.4 fold,
1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5
fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30
fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100
fold, as assessed by methods described herein (see the Examples,
infra) or known to one of skill in the art, relative to IFN.gamma.
and/or IL-2 production without any antibody or with an unrelated
antibody (e.g., an antibody that does not specifically bind to
TIGIT (e.g., human TIGIT or cynomolgus TIGIT)).
[0224] In certain embodiments, human peripheral blood mononuclear
cells (PBMCs) stimulated with Staphylococcus Enterotoxin A (SEA) in
the presence of an antibody described herein, which specifically
binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), have
increased IFN.gamma. and/or IL-2 production by at least about 1.2
fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5
fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10
fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70
fold, 80 fold, 90 fold, or 100 fold, relative to IFN.gamma. and/or
IL-2 production from PBMCs only stimulated with SEA without any
antibody or with an unrelated antibody (e.g., an antibody that does
not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus
TIGIT)), as assessed by methods described herein (see the Examples,
infra) or known to one of skill in the art.
[0225] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) and increases or promotes memory recall
of a memory T cell. In certain embodiments, the memory T cell is a
CD8 effector memory T cell. In certain embodiments, the memory T
cell is a CD4 effector memory T cell. In certain embodiments, the
antibody increases the number of proliferating memory T cells when
the memory T cells are in contact with their cognate antigen(s) by
at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5
fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8
fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold,
60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, as assessed by
methods described herein (see the Examples, infra) or known to one
of skill in the art, relative to the number of proliferating memory
T cells when the memory T cells are in contact with their cognate
antigen(s) in the absence of any antibody or in the presence of an
unrelated antibody (e.g., an antibody that does not specifically
bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)). In certain
embodiments, the antibody increases the production of a cytokine
(e.g., IFN.gamma., TNF.alpha.) from a memory T cell when the memory
T cell is in contact with its cognate antigen by at least about 1.2
fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5
fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10
fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70
fold, 80 fold, 90 fold, or 100 fold, as assessed by methods
described herein (see the Examples, infra) or known to one of skill
in the art, relative to the production of the cytokine from a
memory T cell when the memory T cell is in contact with its cognate
antigen in the absence of any antibody or in the presence of an
unrelated antibody (e.g., an antibody that does not specifically
bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)).
[0226] In certain embodiments, the instant disclosure provides an
isolated antibody that specifically binds to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) and activates an NK cell. In certain
embodiments, the NK cells are isolated. In certain embodiments, the
NK cells are in a mixed culture of PBMCs. In certain embodiments,
the antibody disclosed herein increases the expression level of
CD107a in NK cells by at least about 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
98%, or 99%, as assessed by methods described herein (see the
Examples, infra) or known to one of skill in the art, relative to
the expression level of CD107a in NK cells without any antibody or
with an unrelated antibody (e.g., an antibody that does not
specifically bind to TIGIT (e.g., human TIGIT or cynomolgus
TIGIT)). In certain embodiments, the antibody disclosed herein
increases the expression level of CD107a in NK cells by at least
about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3
fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9
fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60
fold, 70 fold, 80 fold, 90 fold, or 100 fold, or more, as assessed
by methods described herein (see the Examples, infra) or known to
one of skill in the art, relative to the expression level of CD107a
in NK cells without any antibody or with an unrelated antibody
(e.g., an antibody that does not specifically bind to TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT)). In certain embodiments, the
antibody disclosed herein increases cytokine production (e.g.,
IFN.gamma. and/or TNF.alpha.) from NK cells by at least about 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methods
described herein (see the Examples, infra) or known to one of skill
in the art, relative to cytokine production (e.g., IFN.gamma.
and/or TNF.alpha.) from NK cells without any antibody or with an
unrelated antibody (e.g., an antibody that does not specifically
bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)). In certain
embodiments, the antibody disclosed herein increases cytokine
production (e.g., IFN.gamma. and/or TNF.alpha.) from NK cells by at
least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5
fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8
fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold,
60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, or more, as
assessed by methods described herein (see the Examples, infra) or
known to one of skill in the art, relative to cytokine production
(e.g., IFN.gamma. and/or TNF.alpha.) from NK cells without any
antibody or with an unrelated antibody (e.g., an antibody that does
not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus
TIGIT)).
5.3 Pharmaceutical Compositions
[0227] Provided herein are compositions comprising an anti-TIGIT
(e.g., human TIGIT or cynomolgus TIGIT) antibody disclosed herein
having the desired degree of purity in a physiologically acceptable
carrier, excipient or stabilizer (see, e.g., Remington's
Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.).
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g., Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0228] In a specific embodiment, pharmaceutical compositions
comprise an anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
antibody disclosed herein, and optionally one or more additional
prophylactic or therapeutic agents, in a pharmaceutically
acceptable carrier. In a specific embodiment, pharmaceutical
compositions comprise an effective amount of an antibody described
herein, and optionally one or more additional prophylactic or
therapeutic agents, in a pharmaceutically acceptable carrier. In
certain embodiments, the antibody is the only active ingredient
included in the pharmaceutical composition. Pharmaceutical
compositions described herein can be useful in increasing or
promoting TIGIT (e.g., human TIGIT or cynomolgus TIGIT) activity
and treating a condition, such as cancer or an infectious disease.
In one embodiment, the present invention relates to a
pharmaceutical composition of the present invention comprising an
anti-TIGIT antibody of the present invention for use as a
medicament. In another embodiment, the present invention relates to
a pharmaceutical composition of the present invention for use in a
method for the treatment of cancer or an infectious disease.
[0229] Pharmaceutically acceptable carriers used in parenteral
preparations include aqueous vehicles, nonaqueous vehicles,
antimicrobial agents, isotonic agents, buffers, antioxidants, local
anesthetics, suspending and dispersing agents, emulsifying agents,
sequestering or chelating agents and other pharmaceutically
acceptable substances. Examples of aqueous vehicles include Sodium
Chloride Injection, Ringers Injection, Isotonic Dextrose Injection,
Sterile Water Injection, Dextrose and Lactated Ringers Injection.
Nonaqueous parenteral vehicles include fixed oils of vegetable
origin, cottonseed oil, corn oil, sesame oil and peanut oil.
Antimicrobial agents in bacteriostatic or fungistatic
concentrations can be added to parenteral preparations packaged in
multiple-dose containers which include phenols or cresols,
mercurials, benzyl alcohol, chlorobutanol, methyl and propyl
p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and
benzethonium chloride. Isotonic agents include sodium chloride and
dextrose. Buffers include phosphate and citrate. Antioxidants
include sodium bisulfate. Local anesthetics include procaine
hydrochloride. Suspending and dispersing agents include sodium
carboxymethylcelluose, hydroxypropyl methylcellulose and
polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80
(TWEEN.RTM. 80). A sequestering or chelating agent of metal ions
includes EDTA. Pharmaceutical carriers also include ethyl alcohol,
polyethylene glycol and propylene glycol for water miscible
vehicles; and sodium hydroxide, hydrochloric acid, citric acid or
lactic acid for pH adjustment.
[0230] A pharmaceutical composition may be formulated for any route
of administration to a subject. Specific examples of routes of
administration include intranasal, oral, pulmonary, transdermal,
intradermal, and parenteral. Parenteral administration,
characterized by either subcutaneous, intramuscular or intravenous
injection, is also contemplated herein. Injectables can be prepared
in conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution or suspension in liquid prior to
injection, or as emulsions. The injectables, solutions and
emulsions also contain one or more excipients. Suitable excipients
are, for example, water, saline, dextrose, glycerol or ethanol. In
addition, if desired, the pharmaceutical compositions to be
administered can also contain minor amounts of non-toxic auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents, stabilizers, solubility enhancers, and other such agents,
such as for example, sodium acetate, sorbitan monolaurate,
triethanolamine oleate and cyclodextrins.
[0231] Preparations for parenteral administration of an antibody
include sterile solutions ready for injection, sterile dry soluble
products, such as lyophilized powders, ready to be combined with a
solvent just prior to use, including hypodermic tablets, sterile
suspensions ready for injection, sterile dry insoluble products
ready to be combined with a vehicle just prior to use and sterile
emulsions. The solutions may be either aqueous or nonaqueous.
[0232] If administered intravenously, suitable carriers include
physiological saline or phosphate buffered saline (PBS), and
solutions containing thickening and solubilizing agents, such as
glucose, polyethylene glycol, and polypropylene glycol and mixtures
thereof.
[0233] Topical mixtures comprising an antibody are prepared as
described for the local and systemic administration. The resulting
mixture can be a solution, suspension, emulsions or the like and
can be formulated as creams, gels, ointments, emulsions, solutions,
elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols,
irrigations, sprays, suppositories, bandages, dermal patches or any
other formulations suitable for topical administration.
[0234] An anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
antibody disclosed herein can be formulated as an aerosol for
topical application, such as by inhalation (see, e.g., U.S. Pat.
Nos. 4,044,126, 4,414,209 and 4,364,923, which describe aerosols
for delivery of a steroid useful for treatment of inflammatory
diseases, particularly asthma and are herein incorporated by
reference in their entireties). These formulations for
administration to the respiratory tract can be in the form of an
aerosol or solution for a nebulizer, or as a microtine powder for
insufflations, alone or in combination with an inert carrier such
as lactose. In such a case, the particles of the formulation will,
in one embodiment, have diameters of less than 50 microns, in one
embodiment less than 10 microns.
[0235] An anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
antibody disclosed herein can be formulated for local or topical
application, such as for topical application to the skin and mucous
membranes, such as in the eye, in the form of gels, creams, and
lotions and for application to the eye or for intracisternal or
intraspinal application. Topical administration is contemplated for
transdermal delivery and also for administration to the eyes or
mucosa, or for inhalation therapies. Nasal solutions of the
antibody alone or in combination with other pharmaceutically
acceptable excipients can also be administered.
[0236] Transdermal patches, including iontophoretic and
electrophoretic devices, are well known to those of skill in the
art, and can be used to administer an antibody. For example, such
patches are disclosed in U.S. Pat. Nos. 6,267,983, 6,261,595,
6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134,
5,948,433, and 5,860,957, all of which are herein incorporated by
reference in their entireties.
[0237] In certain embodiments, a pharmaceutical composition
comprising an antibody described herein is a lyophilized powder,
which can be reconstituted for administration as solutions,
emulsions and other mixtures. It may also be reconstituted and
formulated as solids or gels. The lyophilized powder is prepared by
dissolving an antibody described herein, or a pharmaceutically
acceptable derivative thereof, in a suitable solvent. In certain
embodiments, the lyophilized powder is sterile. The solvent may
contain an excipient which improves the stability or other
pharmacological component of the powder or reconstituted solution,
prepared from the powder. Excipients that may be used include, but
are not limited to, dextrose, sorbitol, fructose, corn syrup,
xylitol, glycerin, glucose, sucrose or other suitable agent. The
solvent may also contain a buffer, such as citrate, sodium or
potassium phosphate or other such buffer known to those of skill in
the art at, in one embodiment, about neutral pH. Subsequent sterile
filtration of the solution followed by lyophilization under
standard conditions known to those of skill in the art provides the
desired formulation. In one embodiment, the resulting solution will
be apportioned into vials for lyophilization. Each vial will
contain a single dosage or multiple dosages of the compound. The
lyophilized powder can be stored under appropriate conditions, such
as at about 4.degree. C. to room temperature. Reconstitution of
this lyophilized powder with water for injection provides a
formulation for use in parenteral administration. For
reconstitution, the lyophilized powder is added to sterile water or
other suitable carrier. The precise amount depends upon the
selected compound. Such amount can be empirically determined.
[0238] The anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
antibodies disclosed herein and other compositions provided herein
can also be formulated to be targeted to a particular tissue,
receptor, or other area of the body of the subject to be treated.
Many such targeting methods are well known to those of skill in the
art. All such targeting methods are contemplated herein for use in
the instant compositions. For non-limiting examples of targeting
methods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359,
6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082,
6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252,
5,840,674, 5,759,542 and 5,709,874, all of which are herein
incorporated by reference in their entireties. In a specific
embodiment, an antibody described herein is targeted to a
tumor.
[0239] The compositions to be used for in vivo administration can
be sterile. This is readily accomplished by filtration through,
e.g., sterile filtration membranes.
5.4 Methods of Use and Uses
[0240] In another aspect, the instant disclosure provides a method
of treating a subject using the anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibodies disclosed herein. Any disease or
disorder in a subject that would benefit from decrease of TIGIT
(e.g., human TIGIT or cynomolgus TIGIT) function can be treated
using the anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
antibodies disclosed herein. In certain embodiments, the disease or
disorder is resistant to a checkpoint targeting agent (e.g., an
antagonist anti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody,
an antagonist anti-PD-L2 antibody, or an antagonist anti-PD-1
antibody). In certain embodiments, the disease or disorder is
recurrent after treatment with a checkpoint targeting agent (e.g.,
an antagonist anti-CTLA-4 antibody, an antagonist anti-PD-L1
antibody, an antagonist anti-PD-L2 antibody, or an antagonist
anti-PD-1 antibody).
[0241] The anti-TIGIT (e.g., human TIGIT) antibodies disclosed
herein are particularly useful for inhibiting immune system
tolerance to tumors, and accordingly can be used as an
immunotherapy for subjects with cancer. For example, in certain
embodiments, the instant disclosure provides a method of increasing
T cell (e.g., CD8.sup.+ cytotoxic T cells, CD4.sup.+ helper T
cells, NKT cells, effector T cells, or memory T cells) activation
in response to an antigen in a subject, the method comprising
administering to the subject an effective amount of an anti-TIGIT
(e.g., human TIGIT or cynomolgus TIGIT) antibody or pharmaceutical
composition thereof, as disclosed herein. In certain embodiments,
the instant disclosure provides a method of decreasing or
inhibiting regulatory T cell (Treg) activity in a subject, the
method comprising administering to the subject an effective amount
of an anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT) antibody
or pharmaceutical composition thereof, as disclosed herein. In
certain embodiments, the instant disclosure provides a method of
increasing NK cell activation in response to an antigen in a
subject, the method comprising administering to the subject an
effective amount of an anti-TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) antibody or pharmaceutical composition thereof, as disclosed
herein. In certain embodiments, the instant disclosure provides a
method of treating cancer in a subject, the method comprising
administering to the subject an effective amount of the antibody or
pharmaceutical composition, as disclosed herein.
[0242] Cancers that can be treated with the anti-TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) antibodies or pharmaceutical
compositions disclosed herein include, without limitation, a solid
tumor, a hematological cancer (e.g., leukemia, lymphoma, myeloma,
e.g., multiple myeloma), and a metastatic lesion. In one
embodiment, the cancer is a solid tumor. Examples of solid tumors
include malignancies, e.g., sarcomas and carcinomas, e.g.,
adenocarcinomas of the various organ systems, such as those
affecting the lung, breast, ovarian, lymphoid, gastrointestinal
(e.g., colon), anal, genitals and genitourinary tract (e.g., renal,
urothelial, bladder cells, prostate), pharynx, CNS (e.g., brain,
neural or glial cells), head and neck, skin (e.g., melanoma), and
pancreas, as well as adenocarcinomas which include malignancies
such as colon cancers, rectal cancer, renal-cell carcinoma, liver
cancer, lung cancer (e.g., non-small cell lung cancer or small cell
lung cancer), cancer of the small intestine and cancer of the
esophagus. The cancer may be at an early, intermediate, late stage
or metastatic cancer. In certain embodiments, the cancer is
resistant to a checkpoint targeting agent (e.g., an antagonist
anti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an
antagonist anti-PD-L2 antibody, or an antagonist anti-PD-1
antibody). In certain embodiments, the cancer is recurrent after
treatment with a checkpoint targeting agent (e.g., an antagonist
anti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an
antagonist anti-PD-L2 antibody, or an antagonist anti-PD-1
antibody).
[0243] In one embodiment, the cancer is chosen from lung cancer
(e.g., lung adenocarcinoma or non-small cell lung cancer (NSCLC)
(e.g., NSCLC with squamous and/or non-squamous histology, or NSCLC
adenocarcinoma)), melanoma (e.g., an advanced melanoma), renal
cancer (e.g., a renal cell carcinoma), liver cancer (e.g.,
hepatocellular carcinoma), myeloma (e.g., a multiple myeloma), a
prostate cancer, a breast cancer (e.g., a breast cancer that does
not express one, two or all of estrogen receptor, progesterone
receptor, or Her2/neu, e.g., a triple negative breast cancer), an
ovarian cancer, a colorectal cancer, a pancreatic cancer, a head
and neck cancer (e.g., head and neck squamous cell carcinoma
(HNSCC), anal cancer, gastro-esophageal cancer (e.g., esophageal
squamous cell carcinoma), mesothelioma, nasopharyngeal cancer,
thyroid cancer, cervical cancer, epithelial cancer, peritoneal
cancer, or a lymphoproliferative disease (e.g., a post-transplant
lymphoproliferative disease). In a specific embodiment, the cancer
is a cervical cancer.
[0244] In one embodiment, the cancer is a hematological cancer, for
example, a leukemia, a lymphoma, or a myeloma. In one embodiment,
the cancer is a leukemia, for example, acute lymphoblastic leukemia
(ALL), acute myelogenous leukemia (AML), acute myeloblastic
leukemia (AML), chronic lymphocytic leukemia (CLL), chronic
myelogenous leukemia (CML), chronic myeloid leukemia (CML), chronic
myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL),
or hairy cell leukemia. In one embodiment, the cancer is a
lymphoma, for example, B cell lymphoma, diffuse large B-cell
lymphoma (DLBCL), activated B-cell like (ABC) diffuse large B cell
lymphoma, germinal center B cell (GCB) diffuse large B cell
lymphoma, mantle cell lymphoma, Hodgkin lymphoma, non-Hodgkin
lymphoma, relapsed non-Hodgkin lymphoma, refractory non-Hodgkin
lymphoma, recurrent follicular non-Hodgkin lymphoma, Burkitt
lymphoma, small lymphocytic lymphoma, follicular lymphoma,
lymphoplasmacytic lymphoma, or extranodal marginal zone lymphoma.
In one embodiment the cancer is a myeloma, for example, multiple
myeloma.
[0245] In another embodiment, the cancer is chosen from a carcinoma
(e.g., advanced or metastatic carcinoma), melanoma or a lung
carcinoma, e.g., a non-small cell lung carcinoma.
[0246] In one embodiment, the cancer is a lung cancer, e.g., a lung
adenocarcinoma, non-small cell lung cancer, or small cell lung
cancer.
[0247] In one embodiment, the cancer is a melanoma, e.g., an
advanced melanoma. In one embodiment, the cancer is an advanced or
unresectable melanoma that does not respond to other therapies. In
other embodiments, the cancer is a melanoma with a BRAF mutation
(e.g., a BRAF V600 mutation). In yet other embodiments, the
anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT) antibody or
pharmaceutical composition disclosed herein is administered after
treatment with an anti-CTLA-4 antibody (e.g., ipilimumab) with or
without a BRAF inhibitor (e.g., vemurafenib or dabrafenib).
[0248] In another embodiment, the cancer is a hepatocarcinoma,
e.g., an advanced hepatocarcinoma, with or without a viral
infection, e.g., a chronic viral hepatitis.
[0249] In another embodiment, the cancer is a prostate cancer,
e.g., an advanced prostate cancer.
[0250] In yet another embodiment, the cancer is a myeloma, e.g.,
multiple myeloma.
[0251] In yet another embodiment, the cancer is a renal cancer,
e.g., a renal cell carcinoma (RCC) (e.g., a metastatic RCC, clear
cell renal cell carcinoma (CCRCC) or kidney papillary cell
carcinoma).
[0252] In yet another embodiment, the cancer is chosen from a lung
cancer, a melanoma, a renal cancer, a breast cancer, a colorectal
cancer, a leukemia, or a metastatic lesion of the cancer.
[0253] In certain embodiments, the instant disclosure provides a
method of preventing or treating an infectious disease in a
subject, the method comprising administering to the subject an
effective amount of an anti-TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) antibody or pharmaceutical composition thereof, as disclosed
herein. In one embodiment, provided herein are methods for
preventing and/or treating an infection (e.g., a viral infection, a
bacterial infection, a fungal infection, a protozoal infection, or
a parasitic infection). The infection prevented and/or treated in
accordance with the methods can be caused by an infectious agent
identified herein. In a specific embodiment, an anti-TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT) antibody described herein or a
composition thereof is the only active agent administered to a
subject. In certain embodiments, an anti-TIGIT (e.g., human TIGIT
or cynomolgus TIGIT) antibody described herein or a composition
thereof is used in combination with anti-infective interventions
(e.g., antivirals, antibacterials, antifungals, or
anti-helminthics) for the treatment of infectious diseases.
Therefore, in a one embodiment, the present invention relates to an
antibody and/or pharmaceutical composition of the present invention
for use in a method of preventing and/or treating an infectious
disease, optionally wherein the antibody or pharmaceutical
composition is the only active agent administered to a subject, or
wherein the antibody or pharmaceutical composition is used in
combination with anti-infective interventions.
[0254] Infectious diseases that can be treated and/or prevented by
anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT) antibodies or
pharmaceutical compositions disclosed herein are caused by
infectious agents including but not limited to bacteria, parasites,
fungi, protozae, and viruses. In a specific embodiment, the
infectious disease treated and/or prevented by anti-TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT) antibodies or pharmaceutical
compositions disclosed herein is caused by a virus. Viral diseases
or viral infections that can be prevented and/or treated in
accordance with the methods described herein include, but are not
limited to, those caused by hepatitis type A, hepatitis type B,
hepatitis type C, influenza (e.g., influenza A or influenza B),
varicella, adenovirus, herpes simplex type I (HSV-I), herpes
simplex type II (HSV-II), rinderpest, rhinovirus, echovirus,
rotavirus, respiratory syncytial virus, papilloma virus, papova
virus, cytomegalovirus, echinovirus, arbovirus, huntavirus,
coxsackie virus, mumps virus, measles virus, rubella virus, polio
virus, small pox, Epstein Barr virus, human immunodeficiency virus
type I (HIV-I), human immunodeficiency virus type II (HIV-II), and
agents of viral diseases such as viral meningitis, encephalitis,
dengue or small pox.
[0255] Bacterial infections that can be prevented and/or treated
include infections caused by Escherichia coli, Klebsiella
pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Proteus
vulgaris, Staphylococcus viridans, and Pseudomonas aeruginosa.
Bacterial diseases caused by bacteria (e.g., Escherichia coli,
Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus
faecalis, Proteus vulgaris, Staphylococcus viridans, and
Pseudomonas aeruginosa) that can be prevented and/or treated in
accordance with the methods described herein include, but are not
limited to, Mycobacteria rickettsia, Mycoplasma, Neisseria, S.
pneumonia, Borrelia burgdorferi (Lyme disease), Bacillus antracis
(anthrax), tetanus, Streptococcus, Staphylococcus, mycobacterium,
pertissus, cholera, plague, diptheria, chlamydia, S. aureus and
legionella.
[0256] Protozoal diseases or protozoal infections caused by
protozoa that can be prevented and/or treated in accordance with
the methods described herein include, but are not limited to,
leishmania, coccidiosis, trypanosoma schistosoma or malaria.
Parasitic diseases or parasitic infections caused by parasites that
can be prevented and/or treated in accordance with the methods
described herein include, but are not limited to, chlamydia and
rickettsia.
[0257] Fungal diseases or fungal infections that can be prevented
and/or treated in accordance with the methods described herein
include, but are not limited to, those caused by Candida
infections, zygomycosis, Candida mastitis, progressive disseminated
trichosporonosis with latent trichosporonemia, disseminated
candidiasis, pulmonary paracoccidioidomycosis, pulmonary
aspergillosis, Pneumocystis carinii pneumonia, cryptococcal
meningitis, coccidioidal meningoencephalitis and cerebrospinal
vasculitis, Aspergillus niger infection, Fusarium keratitis,
paranasal sinus mycoses, Aspergillus fumigatus endocarditis, tibial
dyschondroplasia, Candida glabrata vaginitis, oropharyngeal
candidiasis, X-linked chronic granulomatous disease, tinea pedis,
cutaneous candidiasis, mycotic placentitis, disseminated
trichosporonosis, allergic bronchopulmonary aspergillosis, mycotic
keratitis, Cryptococcus neoformans infection, fungal peritonitis,
Curvularia geniculata infection, staphylococcal endophthalmitis,
sporotrichosis, and dermatophytosis.
[0258] In certain embodiments, these methods further comprise
administering an additional therapeutic agent to the subject. In
certain embodiments, the additional therapeutic agent is a
chemotherapeutic, a radiotherapeutic, or a checkpoint targeting
agent. In certain embodiments, the chemotherapeutic agent is a
hypomethylating agent (e.g., azacitidine). In certain embodiments,
the chemotherapeutic agent is a DNA damage-inducing agent (e.g.,
gemcitabine). In certain embodiments, the checkpoint targeting
agent is selected from the group consisting of an antagonist
anti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an
antagonist anti-PD-L2 antibody, an antagonist anti-PD-1 antibody,
an antagonist anti-TIM-3 antibody, an antagonist anti-LAG-3
antibody, an antagonist anti-VISTA antibody, an antagonist
anti-CD96 antibody, an antagonist anti-CEACAM1 antibody, an agonist
anti-CD137 antibody, an agonist anti-GITR antibody, and an agonist
anti-OX40 antibody. In certain embodiments, the checkpoint
targeting agent is selected from the group consisting of an
antagonist anti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody,
an antagonist anti-PD-L2 antibody, and an antagonist anti-PD-1
antibody, wherein the anti-TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) antibodies or pharmaceutical compositions disclosed herein
synergize with the checkpoint targeting agent.
[0259] In one embodiment, the present invention relates to an
antibody and/or pharmaceutical composition of the present invention
for use in a method of the present invention, wherein the method
further comprises administering an additional therapeutic agent to
the subject. In one embodiment, the present invention relates to
(a) an antibody and/or pharmaceutical composition of the present
invention and (b) an additional therapeutic agent for use as a
medicament. In one embodiment, the present invention relates to (a)
an antibody and/or pharmaceutical composition of the present
invention, and (b) an additional therapeutic agent for use in a
method for the treatment of cancer. In a further embodiment, the
present invention relates to a pharmaceutical composition, kit or
kit-of-parts comprising (a) an antibody and/or pharmaceutical
composition of the present invention and (b) an additional
therapeutic agent. In one embodiment, the additional therapeutic
agent is a chemotherapeutic, a radiotherapeutic, or a checkpoint
targeting agent.
[0260] In certain embodiments, an anti-PD-1 antibody is used in
methods disclosed herein. In certain embodiments, the anti-PD-1
antibody is nivolumab, also known as BMS-936558 or MDX1106,
developed by Bristol-Myers Squibb. In certain embodiments, the
anti-PD-1 antibody is pembrolizumab, also known as lambrolizumab or
MK-3475, developed by Merck & Co. In certain embodiments, the
anti-PD-1 antibody is pidilizumab, also known as CT-011, developed
by CureTech. In certain embodiments, the anti-PD-1 antibody is
MEDI0680, also known as AMP-514, developed by Medimmune. In certain
embodiments, the anti-PD-1 antibody is PDR001 developed by Novartis
Pharmaceuticals. In certain embodiments, the anti-PD-1 antibody is
REGN2810 developed by Regeneron Pharmaceuticals. In certain
embodiments, the anti-PD-1 antibody is PF-06801591 developed by
Pfizer. In certain embodiments, the anti-PD-1 antibody is BGB-A317
developed by BeiGene. In certain embodiments, the anti-PD-1
antibody is TSR-042 developed by AnaptysBio and Tesaro. In certain
embodiments, the anti-PD-1 antibody is SHR-1210 developed by
Hengrui.
[0261] Further non-limiting examples of anti-PD-1 antibodies that
may be used in treatment methods disclosed herein are disclosed in
the following patents and patent applications, all of which are
herein incorporated by reference in their entireties for all
purposes: U.S. Pat. Nos. 6,808,710; 7,332,582; 7,488,802;
8,008,449; 8,114,845; 8,168,757; 8,354,509; 8,686,119; 8,735,553;
8,747,847; 8,779,105; 8,927,697; 8,993,731; 9,102,727; 9,205,148;
U.S. Publication No. US 2013/0202623 A1; U.S. Publication No. US
2013/0291136 A1; U.S. Publication No. US 2014/0044738 A1; U.S.
Publication No. US 2014/0356363 A1; U.S. Publication No. US
2016/0075783 A1; and PCT Publication No. WO 2013/033091 A1; PCT
Publication No. WO 2015/036394 A1; PCT Publication No. WO
2014/179664 A2; PCT Publication No. WO 2014/209804 A1; PCT
Publication No. WO 2014/206107 A1; PCT Publication No. WO
2015/058573 A1; PCT Publication No. WO 2015/085847 A1; PCT
Publication No. WO 2015/200119 A1; PCT Publication No. WO
2016/015685 A1; and PCT Publication No. WO 2016/020856 A1.
[0262] In certain embodiments, an anti-PD-L1 antibody is used in
methods disclosed herein. In certain embodiments, the anti-PD-L1
antibody is atezolizumab developed by Genentech. In certain
embodiments, the anti-PD-L1 antibody is durvalumab developed by
AstraZeneca, Celgene and Medimmune. In certain embodiments, the
anti-PD-L1 antibody is avelumab, also known as MSB0010718C,
developed by Merck Serono and Pfizer. In certain embodiments, the
anti-PD-L1 antibody is MDX-1105 developed by Bristol-Myers Squibb.
In certain embodiments, the anti-PD-L1 antibody is AMP-224
developed by Amplimmune and GSK.
[0263] Non-limiting examples of anti-PD-L1 antibodies that may be
used in treatment methods disclosed herein are disclosed in the
following patents and patent applications, all of which are herein
incorporated by reference in their entireties for all purposes:
U.S. Pat. Nos. 7,943,743; 8,168,179; 8,217,149; 8,552,154;
8,779,108; 8,981,063; 9,175,082; U.S. Publication No. US
2010/0203056 A1; U.S. Publication No. US 2003/0232323 A1; U.S.
Publication No. US 2013/0323249 A1; U.S. Publication No. US
2014/0341917 A1; U.S. Publication No. US 2014/0044738 A1; U.S.
Publication No. US 2015/0203580 A1; U.S. Publication No. US
2015/0225483 A1; U.S. Publication No. US 2015/0346208 A1; U.S.
Publication No. US 2015/0355184 A1; and PCT Publication No. WO
2014/100079 A1; PCT Publication No. WO 2014/022758 A1; PCT
Publication No. WO 2014/055897 A2; PCT Publication No. WO
2015/061668 A1; PCT Publication No. WO 2015/109124 A1; PCT
Publication No. WO 2015/195163 A1; PCT Publication No. WO
2016/000619 A1; and PCT Publication No. WO 2016/030350 A1.
[0264] In certain embodiments, an anti-CTLA-4 antibody is used in
methods disclosed herein. In certain embodiments, the anti-CTLA-4
antibody is ipilimumab developed by Bristol-Myers Squibb.
[0265] In certain embodiments, an anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibody disclosed herein is administered to a
subject in combination with a compound that targets an
immunomodulatory enzyme(s) such as IDO
(indoleamine-(2,3)-dioxygenase) and/or TDO (tryptophan
2,3-dioxygenase). Therefore, in one embodiment, the additional
therapeutic agent is a compound that targets an immunomodulatory
enzyme(s), such as an inhibitor of indoleamine-(2,3)-dioxygenase
(IDO). In certain embodiments, such compound is selected from the
group consisting of epacadostat (Incyte Corp; see, e.g., WO
2010/005958 which is herein incorporated by reference in its
entirety), F001287 (Flexus Biosciences/Bristol-Myers Squibb),
indoximod (NewLink Genetics), and NLG919 (NewLink Genetics). In one
embodiment, the compound is epacadostat. In another embodiment, the
compound is F001287. In another embodiment, the compound is
indoximod. In another embodiment, the compound is NLG919. In a
specific embodiment, an anti-TIGIT (e.g., human TIGIT) antibody
disclosed herein is administered to a subject in combination with
an IDO inhibitor for treating cancer. The IDO inhibitor as
described herein for use in treating cancer is present in a solid
dosage form of a pharmaceutical composition such as a tablet, a
pill or a capsule, wherein the pharmaceutical composition includes
an IDO inhibitor and a pharmaceutically acceptable excipient. As
such, the antibody as described herein and the IDO inhibitor as
described herein can be administered separately, sequentially or
concurrently as separate dosage forms. In one embodiment, the
antibody is administered parenterally, and the IDO inhibitor is
administered orally. In particular embodiments, the inhibitor is
selected from the group consisting of epacadostat (Incyte
Corporation), F001287 (Flexus Biosciences/Bristol-Myers Squibb),
indoximod (NewLink Genetics), and NLG919 (NewLink Genetics).
Epacadostat has been described in PCT Publication No. WO
2010/005958, which is herein incorporated by reference in its
entirety for all purposes. In one embodiment, the inhibitor is
epacadostat. In another embodiment, the inhibitor is F001287. In
another embodiment, the inhibitor is indoximod. In another
embodiment, the inhibitor is NLG919.
[0266] In certain embodiments, an anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibody disclosed herein is administered to a
subject in combination with a vaccine. The vaccine can be, e.g., a
peptide vaccine, a DNA vaccine, or an RNA vaccine. In certain
embodiments, the vaccine is a heat shock protein based tumor
vaccine or a heat shock protein based pathogen vaccine. In a
specific embodiment, an anti-TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) antibody disclosed herein is administered to a subject in
combination with a heat shock protein based tumor-vaccine. Heat
shock proteins (HSPs) are a family of highly conserved proteins
found ubiquitously across all species. Their expression can be
powerfully induced to much higher levels as a result of heat shock
or other forms of stress, including exposure to toxins, oxidative
stress or glucose deprivation. Five families have been classified
according to molecular weight: HSP-110, -90, -70, -60 and -28. HSPs
deliver immunogenic peptides through the cross-presentation pathway
in antigen presenting cells (APCs) such as macrophages and
dendritic cells (DCs), leading to T cell activation. HSPs function
as chaperone carriers of tumor-associated antigenic peptides
forming complexes able to induce tumor-specific immunity. Upon
release from dying tumor cells, the HSP-antigen complexes are taken
up by antigen-presenting cells (APCs) wherein the antigens are
processed into peptides that bind MHC class I and class II
molecules leading to the activation of anti-tumor CD8+ and CD4+ T
cells. The immunity elicited by HSP complexes derived from tumor
preparations is specifically directed against the unique antigenic
peptide repertoire expressed by the cancer of each subject.
Therefore, in one embodiment, the present invention relates to (a)
an antibody and/or pharmaceutical composition of the present
invention and (b) a vaccine for use as a medicament, for example
for use in a method for the treatment of cancer. In one embodiment,
the present invention relates to a pharmaceutical composition, kit
or kit-of-parts comprising (a) an antibody and/or pharmaceutical
composition of the present invention and (b) a vaccine. In one
embodiment, the vaccine is a heat shock protein based tumor
vaccine. In one embodiment, the vaccine is a heat shock protein
based pathogen vaccine. In certain embodiments, the vaccine is as
described in WO 2016/183486, incorporated herein by reference in
its entirety.
[0267] A heat shock protein peptide complex (HSPPC) is a protein
peptide complex consisting of a heat shock protein non-covalently
complexed with antigenic peptides. HSPPCs elicit both innate and
adaptive immune responses. In a specific embodiment, the antigenic
peptide(s) displays antigenicity for the cancer being treated.
HSPPCs are efficiently seized by APCs via membrane receptors
(mainly CD91) or by binding to Toll-like receptors. HSPPC
internalization results in functional maturation of the APCs with
chemokine and cytokine production leading to activation of natural
killer cells (NK), monocytes and Th1 and Th-2-mediated immune
responses. In certain embodiments, HSPPCs used in methods disclosed
herein comprise one or more heat shock proteins from the hsp60,
hsp70, or hsp90 family of stress proteins complexed with antigenic
peptides. In certain embodiments, HSPPCs comprise hsc70, hsp70,
hsp90, hsp110, grp170, gp96, calreticulin, or combinations of two
or more thereof.
[0268] In a specific embodiment, the heat shock protein peptide
complex (HSPPC) comprises recombinant heat shock proteins (e.g.,
hsp70 or hsc70) or a peptide-binding domain thereof complexed with
recombinant antigenic peptides. Recombinant heat shock proteins can
be produced by recombinant DNA technology, for example, using human
hsc70 sequence as described in Dworniczak and Mirault, Nucleic
Acids Res. 15:5181-5197 (1987) and GenBank accession no. P11142
and/or Y00371, each of which is incorporated herein by reference in
its entirety. In certain embodiments, Hsp70 sequences are as
described in Hunt and Morimoto Proc. Natl. Acad. Sci. U.S.A. 82
(19), 6455-6459 (1985) and GenBank accession no. P0DMV8 and/or
M11717, each of which is incorporated herein by reference in its
entirety. Antigenic peptides can also be prepared by recombinant
DNA methods known in the art.
[0269] In certain embodiments, the antigenic peptides comprise a
modified amino acid. In certain embodiments, the modified amino
acid comprises a post-translational modification. In certain
embodiments, the modified amino acid comprises a mimetic of a
post-translational modification. In certain embodiments, the
modified amino acid is a Tyr, Ser, Thr, Arg, Lys, or His that has
been phosphorylated on a side chain hydroxyl or amine. In certain
embodiments, the modified amino acid is a mimetic of a Tyr, Ser,
Thr, Arg, Lys, or His amino acid that has been phosphorylated on a
side chain hydroxyl or amine.
[0270] In a specific embodiment, an anti-TIGIT (e.g., human TIGIT
or cynomolgus TIGIT) antibody disclosed herein is administered to a
subject in combination with a heat shock protein peptide complex
(HSPPC), e.g., heat shock protein peptide complex-96 (HSPPC-96), to
treat cancer. HSPPC-96 comprises a 96 kDa heat shock protein (Hsp),
gp96, complexed to antigenic peptides. HSPPC-96 is a cancer
immunotherapy manufactured from a subject's tumor and contains the
cancer's antigenic "fingerprint." In certain embodiments, this
fingerprint contains unique antigens that are present only in that
particular subject's specific cancer cells and injection of the
vaccine is intended to stimulate the subject's immune system to
recognize and attack any cells with the specific cancer
fingerprint. Therefore, in one embodiment, the present invention
relates to an antibody and/or pharmaceutical composition of the
present invention in combination with a heat shock protein peptide
complex (HSPPC) for use as a medicament and/or for use in a method
for the treatment of cancer.
[0271] In certain embodiments, the HSPPC, e.g., HSPPC-96, is
produced from the tumor tissue of a subject. In a specific
embodiment, the HSPPC (e.g., HSPPC-96) is produced from a tumor of
the type of cancer or metastasis thereof being treated. In another
specific embodiment, the HSPPC (e.g., HSPPC-96) is autologous to
the subject being treated. In certain embodiments, the tumor tissue
is non-necrotic tumor tissue. In certain embodiments, at least 1
gram (e.g., at least 1, at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, or at
least 10 grams) of non-necrotic tumor tissue is used to produce a
vaccine regimen. In certain embodiments, after surgical resection,
non-necrotic tumor tissue is frozen prior to use in vaccine
preparation. In certain embodiments, the HSPPC, e.g., HSPPC-96, is
isolated from the tumor tissue by purification techniques, filtered
and prepared for an injectable vaccine. In certain embodiments, a
subject is administered 6-12 doses of the HSPPC, e.g., HSPCC-96. In
such embodiments, the HSPPC, e.g., HSPPC-96, doses may be
administered weekly for the first 4 doses and then biweekly for the
2-8 additional doses.
[0272] Further examples of HSPPCs that may be used in accordance
with the methods described herein are disclosed in the following
patents and patent applications, all of which are herein
incorporated by reference in their entireties: U.S. Pat. Nos.
6,391,306, 6,383,492, 6,403,095, 6,410,026, 6,436,404, 6,447,780,
6,447,781 and 6,610,659.
[0273] In certain embodiments, an anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibody disclosed herein is administered to a
subject in combination with an adjuvant. Various adjuvants can be
used depending on the treatment context. Non-limiting examples of
appropriate adjuvants include, but not limited to, Complete
Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA),
montanide ISA (incomplete Seppic adjuvant), the Ribi adjuvant
system (RAS), Titer Max, muramyl peptides, Syntex Adjuvant
Formulation (SAF), alum (aluminum hydroxide and/or aluminum
phosphate), aluminum salt adjuvants, Gerbu.RTM. adjuvants,
nitrocellulose absorbed antigen, encapsulated or entrapped antigen,
3 De-O-acylated monophosphoryl lipid A (3 D-MPL), immunostimulatory
oligonucleotides, toll-like receptor (TLR) ligands, mannan-binding
lectin (MBL) ligands, STING agonists, immuno-stimulating complexes
such as saponins, Quil A, QS-21, QS-7, ISCOMATRIX, and others.
Other adjuvants include CpG oligonucleotides and double stranded
RNA molecules, such as poly(A) and poly(U). Combinations of the
above adjuvants may also be used. See, e.g., U.S. Pat. Nos.
6,645,495; 7,029,678; and 7,858,589, all of which are incorporated
herein by reference in their entireties. In one embodiment, the
adjuvant used herein is QS-21 STIMULON.
[0274] In certain embodiments, an anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibody disclosed herein is administered to a
subject in combination with an additional therapeutic agent
comprising a TCR. In certain embodiments, the additional
therapeutic agent is a soluble TCR. In certain embodiments, the
additional therapeutic agent is a cell expressing a TCR. Therefore,
in one embodiment, the present invention relates to an antibody
and/or pharmaceutical composition of the present invention in
combination with an additional therapeutic agent comprising a TCR
for use as a medicament and/or for use in a method for the
treatment of cancer.
[0275] In certain embodiments, an anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibody disclosed herein is administered to a
subject in combination with a cell expressing a chimeric antigen
receptor (CAR). In certain embodiments, the cell is a T cell.
[0276] In certain embodiments, an anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibody disclosed herein is administered to a
subject in combination with a TCR mimic antibody. In certain
embodiments, the TCR mimic antibody is an antibody that
specifically binds to a peptide-MHC complex. For non-limiting
examples of TCR mimic antibodies, see, e.g., U.S. Pat. No.
9,074,000 and U.S. Publication Nos. US 2009/0304679 A1 and US
2014/0134191 A1, all of which are incorporated herein by reference
in their entireties.
[0277] In certain embodiments, an anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibody disclosed herein is administered to a
subject in combination with a bispecific T-cell engager (BiTE)
(e.g., as described in WO2005061547A2, which is incorporated by
reference herein in its entirety) and/or a dual-affinity
re-targeting antibody (DART) (e.g., as described in WO2012162067A2,
which is incorporated by reference herein in its entirety). In
certain embodiments, the BiTE and/or DART specifically binds to a
tumor-associated antigen (e.g., a polypeptide overexpressed in a
tumor, a polypeptide derived from an oncovirus, a polypeptide
comprising a post-translational modification specific to a tumor, a
polypeptide specifically mutated in a tumor) and a molecule on an
effector cell (e.g., CD3 or CD16). In certain embodiments, the
tumor-associated antigen is EGFR (e.g., human EGFR), optionally
wherein the BiTE and/or DART comprises the VH and VL sequences of
cetuximab. In certain embodiments, the tumor-associated antigen is
Her2 (e.g., human Her2), optionally wherein the BiTE and/or DART
comprises the VH and VL sequences of trastuzumab. In certain
embodiments, the tumor-associated antigen is CD20 (e.g., human
CD20).
[0278] The anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
antibody and the additional therapeutic agent (e.g.,
chemotherapeutic, radiotherapeutic, checkpoint targeting agent, IDO
inhibitor, vaccine, adjuvant, a soluble TCR, a cell expressing a
TCR, a cell expressing a chimeric antigen receptor, and/or a TCR
mimic antibody) can be administered separately, sequentially or
concurrently as separate dosage forms. In one embodiment, an
anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT) antibody is
administered parenterally, and an IDO inhibitor is administered
orally.
[0279] An antibody or pharmaceutical composition described herein
may be delivered to a subject by a variety of routes. These
include, but are not limited to, parenteral, intranasal,
intratracheal, oral, intradermal, topical, intramuscular,
intraperitoneal, transdermal, intravenous, intratumoral,
conjunctival, intra-arterial, and subcutaneous routes. Pulmonary
administration can also be employed, e.g., by use of an inhaler or
nebulizer, and formulation with an aerosolizing agent for use as a
spray. In certain embodiments, the antibody or pharmaceutical
composition described herein is delivered subcutaneously or
intravenously. In certain embodiments, the antibody or
pharmaceutical composition described herein is delivered
intra-arterially. In certain embodiments, the antibody or
pharmaceutical composition described herein is delivered
intratumorally. In certain embodiments, the antibody or
pharmaceutical composition described herein is delivered into a
tumor draining lymph node.
[0280] The amount of an antibody or composition which will be
effective in the treatment and/or prevention of a condition will
depend on the nature of the disease, and can be determined by
standard clinical techniques.
[0281] The precise dose to be employed in a composition will also
depend on the route of administration, and the seriousness of the
infection or disease caused by it, and should be decided according
to the judgment of the practitioner and each subject's
circumstances. For example, effective doses may also vary depending
upon means of administration, target site, physiological state of
the patient (including age, body weight and health), whether the
patient is human or an animal, other medications administered, or
whether treatment is prophylactic or therapeutic. Usually, the
patient is a human, but non-human mammals, including transgenic
mammals, can also be treated. Treatment dosages are optimally
titrated to optimize safety and efficacy.
[0282] An anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
antibody described herein can also be used to assay TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT) protein levels in a biological
sample using classical immunohistological methods known to those of
skill in the art, including immunoassays, such as the enzyme linked
immunosorbent assay (ELISA), immunoprecipitation, or Western
blotting. Suitable antibody assay labels are known in the art and
include enzyme labels, such as, glucose oxidase; radioisotopes,
such as iodine (.sup.125I, .sup.121I), carbon (.sup.14C), sulfur
(.sup.35S), tritium (.sup.3H), indium (.sup.121In), and technetium
(.sup.99Tc); luminescent labels, such as luminol; and fluorescent
labels, such as fluorescein and rhodamine, and biotin. Such labels
can be used to label an antibody described herein. Alternatively, a
second antibody that recognizes an anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibody described herein can be labeled and used
in combination with an anti-TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) antibody to detect TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) protein levels. Therefore, in one embodiment, the present
invention relates to the use of an antibody of the present
invention for in vitro detection of TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) protein in a biological sample. In a further
embodiment, the present invention relates to the use of an
anti-TIGIT antibody of the invention, for assaying and/or detecting
TIGIT (e.g., human TIGIT or cynomolgus TIGIT) protein levels in a
biological sample in vitro, optionally wherein the anti-TIGIT
antibody is conjugated to a radionuclide or detectable label,
and/or carries a label described herein, and/or wherein an
immunohistological method is used.
[0283] Assaying for the expression level of TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) protein is intended to include
qualitatively or quantitatively measuring or estimating the level
of TIGIT (e.g., human TIGIT or cynomolgus TIGIT) protein in a first
biological sample either directly (e.g., by determining or
estimating absolute protein level) or relatively (e.g., by
comparing to the disease associated protein level in a second
biological sample). TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
polypeptide expression level in the first biological sample can be
measured or estimated and compared to a standard TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) protein level, the standard being taken,
for example, from a second biological sample obtained from an
individual not having the disorder or being determined by averaging
levels from a population of individuals not having the disorder. As
will be appreciated in the art, once the "standard" TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT) polypeptide level is known, it can
be used repeatedly as a standard for comparison. Therefore, in a
further embodiment, the present invention relates to an in vitro
method for assaying and/or detecting TIGIT protein levels, for
example human TIGIT protein levels, in a biological sample,
comprising qualitatively or quantitatively measuring or estimating
the level of TIGIT protein, for example of human TIGIT protein, in
a biological sample, by an immunohistological method.
[0284] As used herein, the term "biological sample" refers to any
biological sample obtained from a subject, cell line, tissue, or
other source of cells potentially expressing TIGIT (e.g., human
TIGIT or cynomolgus TIGIT). Methods for obtaining tissue biopsies
and body fluids from animals (e.g., humans or cynomolgus monkeys)
are well known in the art. Biological samples include peripheral
blood mononuclear cells (PBMCs).
[0285] An anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
antibody described herein can be used for prognostic, diagnostic,
monitoring and screening applications, including in vitro and in
vivo applications well known and standard to the skilled artisan
and based on the present description. Prognostic, diagnostic,
monitoring and screening assays and kits for in vitro assessment
and evaluation of immune system status and/or immune response may
be utilized to predict, diagnose and monitor to evaluate patient
samples including those known to have or suspected of having an
immune system-dysfunction or with regard to an anticipated or
desired immune system response, antigen response or vaccine
response. The assessment and evaluation of immune system status
and/or immune response is also useful in determining the
suitability of a patient for a clinical trial of a drug or for the
administration of a particular chemotherapeutic agent, a
radiotherapeutic agent, or an antibody, including combinations
thereof, versus a different agent or antibody. This type of
prognostic and diagnostic monitoring and assessment is already in
practice utilizing antibodies against the HER2 protein in breast
cancer (HercepTest.TM., Dako) where the assay is also used to
evaluate patients for antibody therapy using Herceptin.RTM.. In
vivo applications include directed cell therapy and immune system
modulation and radio imaging of immune responses. Therefore, in one
embodiment, the present invention relates to an anti-TIGIT antibody
and/or pharmaceutical composition of the present invention for use
as a diagnostic. In one embodiment, the present invention relates
to an anti-TIGIT antibody and/or pharmaceutical composition of the
present invention for use in a method for the prediction, diagnosis
and/or monitoring of a subject having or suspected to have an
immune system-dysfunction and/or with regard to an anticipated or
desired immune system response, antigen response or vaccine
response. In another embodiment, the present invention relates to
the use of anti-TIGIT antibody of the invention, for predicting,
diagnosing and/or monitoring of a subject having or suspected to
have an immune system-dysfunction and/or with regard to an
anticipated or desired immune system response, antigen response or
vaccine response by assaying and/or detecting human TIGIT protein
levels in a biological sample of the subject in vitro.
[0286] In one embodiment, an anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibody can be used in immunohistochemistry of
biopsy samples. In one embodiment, the method is an in vitro
method. In another embodiment, an anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibody can be used to detect levels of TIGIT
(e.g., human TIGIT or cynomolgus TIGIT), or levels of cells which
contain TIGIT (e.g., human TIGIT or cynomolgus TIGIT) on their
membrane surface, the levels of which can then be linked to certain
disease symptoms. Anti-TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) antibodies described herein may carry a detectable or
functional label and/or may be conjugated to a radionuclide or
detectable label. When fluorescence labels are used, currently
available microscopy and fluorescence-activated cell sorter
analysis (FACS) or combination of both methods procedures known in
the art may be utilized to identify and to quantitate the specific
binding members. Anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
antibodies described herein may carry or may be conjugated to a
fluorescence label. Exemplary fluorescence labels include, for
example, reactive and conjugated probes, e.g., Aminocoumarin,
Fluorescein and Texas red, Alexa Fluor dyes, Cy dyes and DyLight
dyes. An anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
antibody may carry or may be conjugated to a radioactive label or
radionuclide, such as the isotopes .sup.3H, .sup.14C, .sup.32P,
.sup.35S, .sup.36Cl, .sup.51Cr, .sup.57Co, .sup.58Co, .sup.59Fe,
.sup.67Cu, .sup.90Y, .sup.99Tc, .sup.111In, .sup.117Lu, .sup.121I
.sup.124I, .sup.125I, .sup.131I, .sup.198Au, .sup.211At .sup.213Bi,
.sup.225Ac and .sup.186Re. When radioactive labels are used,
currently available counting procedures known in the art may be
utilized to identify and quantitate the specific binding of
anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT) antibody to
TIGIT (e.g., human TIGIT or cynomolgus TIGIT). In the instance
where the label is an enzyme, detection may be accomplished by any
of the presently utilized colorimetric, spectrophotometric,
fluorospectrophotometric, amperometric or gasometric techniques as
known in the art. This can be achieved by contacting a sample or a
control sample with an anti-TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) antibody under conditions that allow for the formation of a
complex between the antibody and TIGIT (e.g., human TIGIT or
cynomolgus TIGIT). Any complexes formed between the antibody and
TIGIT (e.g., human TIGIT or cynomolgus TIGIT) are detected and
compared in the sample and the control. In light of the specific
binding of the antibodies described herein for TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), the antibodies can be used to
specifically detect TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
expression on the surface of cells. The antibodies described herein
can also be used to purify TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) via immunoaffinity purification. Also included herein is an
assay system which may be prepared in the form of a test kit, kit,
or kit-of-parts for the quantitative analysis of the extent of the
presence of, for instance, TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) or TIGIT (e.g., human TIGIT or cynomolgus TIGIT)/TIGIT
(e.g., human TIGIT or cynomolgus TIGIT) ligand complexes. The
system, test kit, kit or kit-of-parts may comprise a labeled
component, e.g., a labeled antibody, and one or more additional
immunochemical reagents.
5.5 Polynucleotides, Vectors and Methods of Producing Anti-TIGIT
Antibodies
[0287] In another aspect, provided herein are polynucleotides
comprising a nucleotide sequence encoding an antibody described
herein or a fragment thereof (e.g., a light chain variable region
and/or heavy chain variable region) that specifically binds to a
TIGIT (e.g., human TIGIT or cynomolgus TIGIT) antigen, and vectors,
e.g., vectors comprising such polynucleotides for recombinant
expression in host cells (e.g., E. coli and mammalian cells).
Provided herein are polynucleotides comprising nucleotide sequences
encoding a heavy and/or light chain of any of the antibodies
provided herein, as well as vectors comprising such polynucleotide
sequences, e.g., expression vectors for their efficient expression
in host cells, e.g., mammalian cells.
[0288] As used herein, an "isolated" polynucleotide or nucleic acid
molecule is one which is separated from other nucleic acid
molecules which are present in the natural source (e.g., in a mouse
or a human) of the nucleic acid molecule. Moreover, an "isolated"
nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically synthesized.
For example, the language "substantially free" includes
preparations of polynucleotide or nucleic acid molecule having less
than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular less
than about 10%) of other material, e.g., cellular material, culture
medium, other nucleic acid molecules, chemical precursors and/or
other chemicals. In a specific embodiment, a nucleic acid
molecule(s) encoding an antibody described herein is isolated or
purified.
[0289] In particular aspects, provided herein are polynucleotides
comprising nucleotide sequences encoding antibodies, which
specifically bind to a TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) polypeptide and comprises an amino acid sequence as
described herein, as well as antibodies which compete with such
antibodies for binding to a TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) polypeptide (e.g., in a dose-dependent manner), or which
binds to the same epitope as that of such antibodies.
[0290] In certain aspects, provided herein are polynucleotides
comprising a nucleotide sequence encoding the light chain or heavy
chain of an antibody described herein. The polynucleotides can
comprise nucleotide sequences encoding a light chain comprising the
VL FRs and CDRs of antibodies described herein (see, e.g., Table 1)
or nucleotide sequences encoding a heavy chain comprising the VH
FRs and CDRs of antibodies described herein (see, e.g., Table
1).
[0291] Also provided herein are polynucleotides encoding an
anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT) antibody that
are optimized, e.g., by codon/RNA optimization, replacement with
heterologous signal sequences, and elimination of mRNA instability
elements. Methods to generate optimized nucleic acids encoding an
anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT) antibody or a
fragment thereof (e.g., light chain, heavy chain, VH domain, or VL
domain) for recombinant expression by introducing codon changes
and/or eliminating inhibitory regions in the mRNA can be carried
out by adapting the optimization methods described in, e.g., U.S.
Pat. Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and
6,794,498, accordingly, all of which are herein incorporated by
reference in their entireties. For example, potential splice sites
and instability elements (e.g., A/T or A/U rich elements) within
the RNA can be mutated without altering the amino acids encoded by
the nucleic acid sequences to increase stability of the RNA for
recombinant expression. The alterations utilize the degeneracy of
the genetic code, e.g., using an alternative codon for an identical
amino acid. In certain embodiments, it can be desirable to alter
one or more codons to encode a conservative mutation, e.g., a
similar amino acid with similar chemical structure and properties
and/or function as the original amino acid. Such methods can
increase expression of an anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibody or fragment thereof by at least 1 fold,
2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold,
50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold or more
relative to the expression of an anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibody encoded by polynucleotides that have not
been optimized.
[0292] In certain embodiments, an optimized polynucleotide sequence
encoding an anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
antibody described herein or a fragment thereof (e.g., VL domain
and/or VH domain) can hybridize to an antisense (e.g.,
complementary) polynucleotide of an unoptimized polynucleotide
sequence encoding an anti-TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) antibody described herein or a fragment thereof (e.g., VL
domain and/or VH domain). In specific embodiments, an optimized
nucleotide sequence encoding an anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibody described herein or a fragment
hybridizes under high stringency conditions to antisense
polynucleotide of an unoptimized polynucleotide sequence encoding
an anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT) antibody
described herein or a fragment thereof. In a specific embodiment,
an optimized nucleotide sequence encoding an anti-TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT) antibody described herein or a
fragment thereof hybridizes under high stringency, intermediate or
lower stringency hybridization conditions to an antisense
polynucleotide of an unoptimized nucleotide sequence encoding an
anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT) antibody
described herein or a fragment thereof. Information regarding
hybridization conditions has been described, see, e.g., U.S. Patent
Application Publication No. US 2005/0048549 (e.g., paragraphs
72-73), which is herein incorporated by reference in its
entirety.
[0293] The polynucleotides can be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. Nucleotide sequences encoding antibodies described herein,
e.g., antibodies described in Table 1, and modified versions of
these antibodies can be determined using methods well known in the
art, i.e., nucleotide codons known to encode particular amino acids
are assembled in such a way to generate a nucleic acid that encodes
the antibody. Such a polynucleotide encoding the antibody can be
assembled from chemically synthesized oligonucleotides (e.g., as
described in Kutmeier G et al., (1994), BioTechniques 17: 242-6,
herein incorporated by reference in its entirety), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligating of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
[0294] Alternatively, a polynucleotide encoding an antibody
described herein can be generated from nucleic acid from a suitable
source (e.g., a hybridoma) using methods well known in the art
(e.g., PCR and other molecular cloning methods). For example, PCR
amplification using synthetic primers hybridizable to the 3' and 5'
ends of a known sequence can be performed using genomic DNA
obtained from hybridoma cells producing the antibody of interest.
Such PCR amplification methods can be used to obtain nucleic acids
comprising the sequence encoding the light chain and/or heavy chain
of an antibody. Such PCR amplification methods can be used to
obtain nucleic acids comprising the sequence encoding the variable
light chain region and/or the variable heavy chain region of an
antibody. The amplified nucleic acids can be cloned into vectors
for expression in host cells and for further cloning, for example,
to generate chimeric and humanized antibodies.
[0295] If a clone containing a nucleic acid encoding a particular
antibody is not available, but the sequence of the antibody
molecule is known, a nucleic acid encoding the immunoglobulin can
be chemically synthesized or obtained from a suitable source (e.g.,
an antibody cDNA library or a cDNA library generated from, or
nucleic acid, preferably poly A+ RNA, isolated from, any tissue or
cells expressing the antibody, such as hybridoma cells selected to
express an antibody described herein) by PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, e.g., a cDNA clone from a
cDNA library that encodes the antibody. Amplified nucleic acids
generated by PCR can then be cloned into replicable cloning vectors
using any method well known in the art.
[0296] DNA encoding anti-TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) antibodies described herein can be readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the anti-TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT) antibodies). Hybridoma cells can
serve as a source of such DNA. Once isolated, the DNA can be placed
into expression vectors, which are then transfected into host cells
such as E. coli cells, simian COS cells, Chinese hamster ovary
(CHO) cells (e.g., CHO cells from the CHO GS System.TM. (Lonza)),
or myeloma cells that do not otherwise produce immunoglobulin
protein, to obtain the synthesis of anti-TIGIT (e.g., human TIGIT
or cynomolgus TIGIT) antibodies in the recombinant host cells.
[0297] To generate whole antibodies, PCR primers including VH or VL
nucleotide sequences, a restriction site, and a flanking sequence
to protect the restriction site can be used to amplify the VH or VL
sequences in scFv clones. Utilizing cloning techniques known to
those of skill in the art, the PCR amplified VH domains can be
cloned into vectors expressing a heavy chain constant region, e.g.,
the human gamma 1 or human gamma 4 constant region, and the PCR
amplified VL domains can be cloned into vectors expressing a light
chain constant region, e.g., human kappa or lambda constant
regions. In certain embodiments, the vectors for expressing the VH
or VL domains comprise an EF-1.alpha. promoter, a secretion signal,
a cloning site for the variable region, constant domains, and a
selection marker such as neomycin. The VH and VL domains can also
be cloned into one vector expressing the necessary constant
regions. The heavy chain conversion vectors and light chain
conversion vectors are then co-transfected into cell lines to
generate stable or transient cell lines that express full-length
antibodies, e.g., IgG, using techniques known to those of skill in
the art.
[0298] The DNA also can be modified, for example, by substituting
the coding sequence for human heavy and light chain constant
domains in place of the murine sequences, or by covalently joining
to the immunoglobulin coding sequence all or part of the coding
sequence for a non-immunoglobulin polypeptide.
[0299] Also provided are polynucleotides that hybridize under high
stringency, intermediate or lower stringency hybridization
conditions to polynucleotides that encode an antibody described
herein. In specific embodiments, polynucleotides described herein
hybridize under high stringency, intermediate or lower stringency
hybridization conditions to polynucleotides encoding a VH domain
and/or VL domain provided herein.
[0300] Hybridization conditions have been described in the art and
are known to one of skill in the art. For example, hybridization
under stringent conditions can involve hybridization to
filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at
about 45.degree. C. followed by one or more washes in
0.2.times.SSC/0.1% SDS at about 50-65.degree. C.; hybridization
under highly stringent conditions can involve hybridization to
filter-bound nucleic acid in 6.times.SSC at about 45.degree. C.
followed by one or more washes in 0.1.times.SSC/0.2% SDS at about
68.degree. C. Hybridization under other stringent hybridization
conditions are known to those of skill in the art and have been
described, see, for example, Ausubel F M et al., eds., (1989)
Current Protocols in Molecular Biology, Vol. I, Green Publishing
Associates, Inc. and John Wiley & Sons, Inc., New York at pages
6.3.1-6.3.6 and 2.10.3, which is herein incorporated by reference
in its entirety.
[0301] In certain aspects, provided herein are cells (e.g., host
cells) expressing (e.g., recombinantly) antibodies described herein
which specifically bind to TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) and related polynucleotides and expression vectors. Provided
herein are vectors (e.g., expression vectors) comprising
polynucleotides comprising nucleotide sequences encoding anti-TIGIT
(e.g., human TIGIT or cynomolgus TIGIT) antibodies or a fragment
for recombinant expression in host cells, preferably in mammalian
cells (e.g., CHO cells). Also provided herein are host cells
comprising such vectors for recombinantly expressing anti-TIGIT
(e.g., human TIGIT or cynomolgus TIGIT) antibodies described herein
(e.g., human or humanized antibody). In a particular aspect,
provided herein are methods for producing an antibody described
herein, comprising expressing such antibody from a host cell.
[0302] Recombinant expression of an antibody described herein
(e.g., a full-length antibody, heavy and/or light chain of an
antibody, or a single chain antibody described herein) that
specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
generally involves construction of an expression vector containing
a polynucleotide that encodes the antibody. Once a polynucleotide
encoding an antibody molecule, heavy and/or light chain of an
antibody, or a fragment thereof (e.g., heavy and/or light chain
variable regions) described herein has been obtained, the vector
for the production of the antibody molecule can be produced by
recombinant DNA technology using techniques well known in the art.
Thus, methods for preparing a protein by expressing a
polynucleotide containing an antibody or antibody fragment (e.g.,
light chain or heavy chain) encoding nucleotide sequence are
described herein. Methods which are well known to those skilled in
the art can be used to construct expression vectors containing
antibody or antibody fragment (e.g., light chain or heavy chain)
coding sequences and appropriate transcriptional and translational
control signals. These methods include, for example, in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. Also provided are replicable vectors
comprising a nucleotide sequence encoding an antibody molecule
described herein, a heavy or light chain of an antibody, a heavy or
light chain variable region of an antibody or a fragment thereof,
or a heavy or light chain CDR, operably linked to a promoter. Such
vectors can, for example, include the nucleotide sequence encoding
the constant region of the antibody molecule (see, e.g.,
International Publication Nos. WO 86/05807 and WO 89/01036; and
U.S. Pat. No. 5,122,464, which are herein incorporated by reference
in their entireties) and variable regions of the antibody can be
cloned into such a vector for expression of the entire heavy, the
entire light chain, or both the entire heavy and light chains.
[0303] An expression vector can be transferred to a cell (e.g.,
host cell) by conventional techniques and the resulting cells can
then be cultured by conventional techniques to produce an antibody
described herein or a fragment thereof. Thus, provided herein are
host cells containing a polynucleotide encoding an antibody
described herein or fragments thereof, or a heavy or light chain
thereof, or fragment thereof, or a single chain antibody described
herein, operably linked to a promoter for expression of such
sequences in the host cell. In certain embodiments, for the
expression of double-chained antibodies, vectors encoding both the
heavy and light chains, individually, can be co-expressed in the
host cell for expression of the entire immunoglobulin molecule, as
detailed below. In certain embodiments, a host cell contains a
vector comprising a polynucleotide encoding both the heavy chain
and light chain of an antibody described herein, or a fragment
thereof. In specific embodiments, a host cell contains two
different vectors, a first vector comprising a polynucleotide
encoding a heavy chain or a heavy chain variable region of an
antibody described herein, or a fragment thereof, and a second
vector comprising a polynucleotide encoding a light chain or a
light chain variable region of an antibody described herein, or a
fragment thereof. In other embodiments, a first host cell comprises
a first vector comprising a polynucleotide encoding a heavy chain
or a heavy chain variable region of an antibody described herein,
or a fragment thereof, and a second host cell comprises a second
vector comprising a polynucleotide encoding a light chain or a
light chain variable region of an antibody described herein. In
specific embodiments, a heavy chain/heavy chain variable region
expressed by a first cell associated with a light chain/light chain
variable region of a second cell to form an anti-TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) antibody described herein. In certain
embodiments, provided herein is a population of host cells
comprising such first host cell and such second host cell.
[0304] In a particular embodiment, provided herein is a population
of vectors comprising a first vector comprising a polynucleotide
encoding a light chain/light chain variable region of an anti-TIGIT
(e.g., human TIGIT or cynomolgus TIGIT) antibody described herein,
and a second vector comprising a polynucleotide encoding a heavy
chain/heavy chain variable region of an anti-TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) antibody described herein.
[0305] A variety of host-expression vector systems can be utilized
to express antibody molecules described herein (see, e.g., U.S.
Pat. No. 5,807,715, which is herein incorporated by reference in
its entirety). Such host-expression systems represent vehicles by
which the coding sequences of interest can be produced and
subsequently purified, but also represent cells which can, when
transformed or transfected with the appropriate nucleotide coding
sequences, express an antibody molecule described herein in situ.
These include but are not limited to microorganisms such as
bacteria (e.g., E. coli and B. subtilis) transformed with, e.g.,
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing antibody coding sequences; yeast (e.g.,
Saccharomyces Pichia) transformed with, e.g., recombinant yeast
expression vectors containing antibody coding sequences; insect
cell systems infected with, e.g., recombinant virus expression
vectors (e.g., baculovirus) containing antibody coding sequences;
plant cell systems (e.g., green algae such as Chlamydomonas
reinhardtii) infected with, e.g., recombinant virus expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or transformed with, e.g., recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or
COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O,
HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, B-W,
L-M, BSC1, BSC40, YB/20 and BMT10 cells) harboring, e.g.,
recombinant expression constructs containing promoters derived from
the genome of mammalian cells (e.g., metallothionein promoter) or
from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5K promoter). In a specific embodiment, cells for
expressing antibodies described herein are Chinese hamster ovary
(CHO) cells, for example CHO cells from the CHO GS System.TM.
(Lonza). In certain embodiments, the heavy chain and/or light chain
of an antibody produced by a CHO cell may have an N-terminal
glutamine or glutamate residue replaced by pyroglutamate. In a
particular embodiment, cells for expressing antibodies described
herein are human cells, e.g., human cell lines. In a specific
embodiment, a mammalian expression vector is pOptiVEC.TM. or
pcDNA3.3. In a particular embodiment, bacterial cells such as
Escherichia coli, or eukaryotic cells (e.g., mammalian cells),
especially for the expression of whole recombinant antibody
molecule, are used for the expression of a recombinant antibody
molecule. For example, mammalian cells such as CHO cells, in
conjunction with a vector such as the major intermediate early gene
promoter element from human cytomegalovirus is an effective
expression system for antibodies (Foecking M K & Hofstetter H
(1986) Gene 45: 101-5; and Cockett M I et al., (1990) Biotechnology
8(7): 662-7, each of which is herein incorporated by reference in
its entirety). In certain embodiments, antibodies described herein
are produced by CHO cells or NSO cells. In a specific embodiment,
the expression of nucleotide sequences encoding antibodies
described herein which specifically bind to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT) is regulated by a constitutive promoter,
inducible promoter or tissue specific promoter.
[0306] In bacterial systems, a number of expression vectors can be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such an antibody is to be produced, for the generation
of pharmaceutical compositions of an antibody molecule, vectors
which direct the expression of high levels of fusion protein
products that are readily purified can be desirable. Such vectors
include, but are not limited to, the E. coli expression vector
pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794),
in which the antibody coding sequence can be ligated individually
into the vector in frame with the lac Z coding region so that a
fusion protein is produced; pIN vectors (Inouye S & Inouye M
(1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster S M
(1989) J Biol Chem 24: 5503-5509); and the like, all of which are
herein incorporated by reference in their entireties. For example,
pGEX vectors can also be used to express foreign polypeptides as
fusion proteins with glutathione 5-transferase (GST). In general,
such fusion proteins are soluble and can easily be purified from
lysed cells by adsorption and binding to matrix glutathione agarose
beads followed by elution in the presence of free glutathione. The
pGEX vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned target gene product can be
released from the GST moiety.
[0307] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV), for example, can be used as a vector to
express foreign genes. The virus grows in Spodoptera frugiperda
cells. The antibody coding sequence can be cloned individually into
non-essential regions (for example the polyhedrin gene) of the
virus and placed under control of an AcNPV promoter (for example
the polyhedrin promoter).
[0308] In mammalian host cells, a number of viral-based expression
systems can be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest can be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene can then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
antibody molecule in infected hosts (e.g., see Logan J & Shenk
T (1984) PNAS 81(12): 3655-9, which is herein incorporated by
reference in its entirety). Specific initiation signals can also be
required for efficient translation of inserted antibody coding
sequences. These signals include the ATG initiation codon and
adjacent sequences. Furthermore, the initiation codon must be in
phase with the reading frame of the desired coding sequence to
ensure translation of the entire insert. These exogenous
translational control signals and initiation codons can be of a
variety of origins, both natural and synthetic. The efficiency of
expression can be enhanced by the inclusion of appropriate
transcription enhancer elements, transcription terminators, etc.
(see, e.g., Bitter G et al., (1987) Methods Enzymol. 153: 516-544,
which is herein incorporated by reference in its entirety).
[0309] In addition, a host cell strain can be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products can be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product can be used. Such mammalian
host cells include but are not limited to CHO, VERO, BHK, Hela,
MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D,
NSO (a murine myeloma cell line that does not endogenously produce
any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS),
PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M,
BSC1, BSC40, YB/20, BMT10 and HsS78Bst cells. In certain
embodiments, anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
antibodies described herein are produced in mammalian cells, such
as CHO cells.
[0310] In a specific embodiment, the antibodies described herein
have reduced fucose content or no fucose content. Such antibodies
can be produced using techniques known one skilled in the art. For
example, the antibodies can be expressed in cells deficient or
lacking the ability of to fucosylate. In a specific example, cell
lines with a knockout of both alleles of a1,6-fucosyltransferase
can be used to produce antibodies with reduced fucose content. The
Potelligent.RTM. system (Lonza) is an example of such a system that
can be used to produce antibodies with reduced fucose content.
[0311] For long-term, high-yield production of recombinant
proteins, stable expression cells can be generated. For example,
cell lines which stably express an anti-TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antibody described herein can be engineered. In
specific embodiments, a cell provided herein stably expresses a
light chain/light chain variable region and a heavy chain/heavy
chain variable region which associate to form an antibody described
herein.
[0312] In certain aspects, rather than using expression vectors
which contain viral origins of replication, host cells can be
transformed with DNA controlled by appropriate expression control
elements (e.g., promoter, enhancer, sequences, transcription
terminators, polyadenylation sites, etc.), and a selectable marker.
Following the introduction of the foreign DNA/polynucleotide,
engineered cells can be allowed to grow for 1-2 days in an enriched
media, and then are switched to a selective media. The selectable
marker in the recombinant plasmid confers resistance to the
selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method can advantageously be
used to engineer cell lines which express an anti-TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT) antibody described herein or a
fragment thereof. Such engineered cell lines can be particularly
useful in screening and evaluation of compositions that interact
directly or indirectly with the antibody molecule.
[0313] A number of selection systems can be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler M et
al., (1977) Cell 11(1): 223-32), hypoxanthineguanine
phosphoribosyltransferase (Szybalska E H & Szybalski W (1962)
PNAS 48(12): 2026-2034) and adenine phosphoribosyltransferase (Lowy
I et al., (1980) Cell 22(3): 817-23) genes in tk-, hgprt- or
aprt-cells, respectively, all of which are herein incorporated by
reference in their entireties. Also, antimetabolite resistance can
be used as the basis of selection for the following genes: dhfr,
which confers resistance to methotrexate (Wigler M et al., (1980)
PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78: 1527-31);
gpt, which confers resistance to mycophenolic acid (Mulligan R C
& Berg P (1981) PNAS 78(4): 2072-6); neo, which confers
resistance to the aminoglycoside G-418 (Wu G Y & Wu C H (1991)
Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol
32: 573-596; Mulligan R C (1993) Science 260: 926-932; and Morgan R
A & Anderson W F (1993) Ann Rev Biochem 62: 191-217; Nabel G J
& Felgner P L (1993) Trends Biotechnol 11(5): 211-5); and
hygro, which confers resistance to hygromycin (Santerre R F et al.,
(1984) Gene 30(1-3): 147-56), all of which are herein incorporated
by reference in their entireties. Methods commonly known in the art
of recombinant DNA technology can be routinely applied to select
the desired recombinant clone and such methods are described, for
example, in Ausubel F M et al., (eds.), Current Protocols in
Molecular Biology, John Wiley & Sons, N Y (1993); Kriegler M,
Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
N Y (1990); and in Chapters 12 and 13, Dracopoli N C et al.,
(eds.), Current Protocols in Human Genetics, John Wiley & Sons,
N Y (1994); Colbere-Garapin F et al., (1981) J Mol Biol 150: 1-14,
all of which are herein incorporated by reference in their
entireties.
[0314] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington C R
& Hentschel C C G, The use of vectors based on gene
amplification for the expression of cloned genes in mammalian cells
in DNA cloning, Vol. 3 (Academic Press, New York, 1987), which is
herein incorporated by reference in its entirety). When a marker in
the vector system expressing antibody is amplifiable, increase in
the level of inhibitor present in culture of host cell will
increase the number of copies of the marker gene. Since the
amplified region is associated with the antibody gene, production
of the antibody will also increase (Crouse G F et al., (1983) Mol
Cell Biol 3: 257-66, which is herein incorporated by reference in
its entirety).
[0315] The host cell can be co-transfected with two or more
expression vectors described herein, the first vector encoding a
heavy chain derived polypeptide and the second vector encoding a
light chain derived polypeptide. The two vectors can contain
identical selectable markers which enable equal expression of heavy
and light chain polypeptides. The host cells can be co-transfected
with different amounts of the two or more expression vectors. For
example, host cells can be transfected with any one of the
following ratios of a first expression vector and a second
expression vector: about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,
1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or
1:50.
[0316] Alternatively, a single vector can be used which encodes,
and is capable of expressing, both heavy and light chain
polypeptides. In such situations, the light chain should be placed
before the heavy chain to avoid an excess of toxic free heavy chain
(Proudfoot N J (1986) Nature 322: 562-565; and Kohler G (1980) PNAS
77: 2197-2199, each of which is herein incorporated by reference in
its entirety). The coding sequences for the heavy and light chains
can comprise cDNA or genomic DNA. The expression vector can be
monocistronic or multicistronic. A multicistronic nucleic acid
construct can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
genes/nucleotide sequences, or in the range of 2-5, 5-10, or 10-20
genes/nucleotide sequences. For example, a bicistronic nucleic acid
construct can comprise, in the following order, a promoter, a first
gene (e.g., heavy chain of an antibody described herein), and a
second gene and (e.g., light chain of an antibody described
herein). In such an expression vector, the transcription of both
genes can be driven by the promoter, whereas the translation of the
mRNA from the first gene can be by a cap-dependent scanning
mechanism and the translation of the mRNA from the second gene can
be by a cap-independent mechanism, e.g., by an IRES.
[0317] Once an antibody molecule described herein has been produced
by recombinant expression, it can be purified by any method known
in the art for purification of an immunoglobulin molecule, for
example, by chromatography (e.g., ion exchange, affinity,
particularly by affinity for the specific antigen after Protein A,
and sizing column chromatography), centrifugation, differential
solubility, or by any other standard technique for the purification
of proteins. Further, the antibodies described herein can be fused
to heterologous polypeptide sequences described herein or otherwise
known in the art to facilitate purification.
[0318] In specific embodiments, an antibody described herein is
isolated or purified. Generally, an isolated antibody is one that
is substantially free of other antibodies with different antigenic
specificities than the isolated antibody. For example, in a
particular embodiment, a preparation of an antibody described
herein is substantially free of cellular material and/or chemical
precursors. The language "substantially free of cellular material"
includes preparations of an antibody in which the antibody is
separated from cellular components of the cells from which it is
isolated or recombinantly produced. Thus, an antibody that is
substantially free of cellular material includes preparations of
antibody having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or
0.1% (by dry weight) of heterologous protein (also referred to
herein as a "contaminating protein") and/or variants of an
antibody, for example, different post-translational modified forms
of an antibody or other different versions of an antibody (e.g.,
antibody fragments). When the antibody is recombinantly produced,
it is also generally substantially free of culture medium, i.e.,
culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%,
or 0.1% of the volume of the protein preparation. When the antibody
is produced by chemical synthesis, it is generally substantially
free of chemical precursors or other chemicals, i.e., it is
separated from chemical precursors or other chemicals which are
involved in the synthesis of the protein. Accordingly, such
preparations of the antibody have less than about 30%, 20%, 10%, or
5% (by dry weight) of chemical precursors or compounds other than
the antibody of interest. In a specific embodiment, antibodies
described herein are isolated or purified.
[0319] Antibodies or fragments thereof that specifically bind to
TIGIT (e.g., human TIGIT or cynomolgus TIGIT) can be produced by
any method known in the art for the synthesis of antibodies, for
example, by chemical synthesis or by recombinant expression
techniques. The methods described herein employ, unless otherwise
indicated, conventional techniques in molecular biology,
microbiology, genetic analysis, recombinant DNA, organic chemistry,
biochemistry, PCR, oligonucleotide synthesis and modification,
nucleic acid hybridization, and related fields within the skill of
the art. These techniques are described, for example, in the
references cited herein and are fully explained in the literature.
See, e.g., Maniatis T et al., (1982) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J
et al., (1989), Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al.,
(2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel F M et al.,
Current Protocols in Molecular Biology, John Wiley & Sons (1987
and annual updates); Current Protocols in Immunology, John Wiley
& Sons (1987 and annual updates) Gait (ed.) (1984)
Oligonucleotide Synthesis: A Practical Approach, IRL Press;
Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical
Approach, IRL Press; Birren B et al., (eds.) (1999) Genome
Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
all of which are herein incorporated by reference in their
entireties.
[0320] In a specific embodiment, an antibody described herein is an
antibody (e.g., recombinant antibody) prepared, expressed, created
or isolated by any means that involves creation, e.g., via
synthesis, genetic engineering of DNA sequences. In certain
embodiments, such an antibody comprises sequences (e.g., DNA
sequences or amino acid sequences) that do not naturally exist
within the antibody germline repertoire of an animal or mammal
(e.g., human) in vivo.
[0321] In one aspect, provided herein is a method of making an
antibody which specifically binds to TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) comprising culturing a cell or host cell
described herein. In one embodiment, the method is performed in
vitro. In a certain aspect, provided herein is a method of making
an antibody which specifically binds to TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) comprising expressing (e.g., recombinantly
expressing) the antibody using a cell or host cell described herein
(e.g., a cell or a host cell comprising polynucleotides encoding an
antibody described herein). In a particular embodiment, the cell is
an isolated cell. In a particular embodiment, the exogenous
polynucleotides have been introduced into the cell. In a particular
embodiment, the method further comprises the step of purifying the
antibody obtained from the cell or host cell.
[0322] Methods for producing polyclonal antibodies are known in the
art (see, for example, Chapter 11 in: Short Protocols in Molecular
Biology, (2002) 5th Ed., Ausubel F M et al., eds., John Wiley and
Sons, New York, which is herein incorporated by reference in its
entirety).
[0323] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow E & Lane D, Antibodies: A Laboratory
Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
Hammerling G J et al., in: Monoclonal Antibodies and T-Cell
Hybridomas 563 681 (Elsevier, N.Y., 1981), each of which is herein
incorporated by reference in its entirety. The term "monoclonal
antibody" as used herein is not limited to antibodies produced
through hybridoma technology. For example, monoclonal antibodies
can be produced recombinantly from host cells exogenously
expressing an antibody described herein or a fragment thereof, for
example, light chain and/or heavy chain of such antibody.
[0324] In specific embodiments, a "monoclonal antibody," as used
herein, is an antibody produced by a single cell (e.g., hybridoma
or host cell producing a recombinant antibody), wherein the
antibody specifically binds to TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) as determined, e.g., by ELISA or other
antigen-binding or competitive binding assay known in the art or in
the examples provided herein. In particular embodiments, a
monoclonal antibody can be a chimeric antibody or a humanized
antibody. In certain embodiments, a monoclonal antibody is a
monovalent antibody or multivalent (e.g., bivalent) antibody. In
particular embodiments, a monoclonal antibody is a monospecific or
multispecific antibody (e.g., bispecific antibody). Monoclonal
antibodies described herein can, for example, be made by the
hybridoma method as described in Kohler G & Milstein C (1975)
Nature 256: 495, which is herein incorporated by reference in its
entirety, or can, e.g., be isolated from phage libraries using the
techniques as described herein, for example. Other methods for the
preparation of clonal cell lines and of monoclonal antibodies
expressed thereby are well known in the art (see, for example,
Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th
Ed., Ausubel F M et al., supra).
[0325] As used herein, an antibody binds to an antigen
multivalently (e.g., bivalently) when the antibody comprises at
least two (e.g., two or more) monovalent binding domains, each
monovalent binding domain capable of binding to an epitope on the
antigen. Each monovalent binding domain can bind to the same or
different epitopes on the antigen.
[0326] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
For example, in the hybridoma method, a mouse or other appropriate
host animal, such as a sheep, goat, rabbit, rat, hamster or macaque
monkey, is immunized to elicit lymphocytes that produce or are
capable of producing antibodies that will specifically bind to the
protein (e.g., TIGIT (e.g., human TIGIT or cynomolgus TIGIT)) used
for immunization. Alternatively, lymphocytes may be immunized in
vitro. Lymphocytes then are fused with myeloma cells using a
suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell (Goding J W (Ed), Monoclonal Antibodies: Principles
and Practice, pp. 59-103 (Academic Press, 1986), herein
incorporated by reference in its entirety). Additionally, a RIMMS
(repetitive immunization multiple sites) technique can be used to
immunize an animal (Kilpatrick K E et al., (1997) Hybridoma
16:381-9, herein incorporated by reference in its entirety).
[0327] In certain embodiments, mice (or other animals, such as
rats, monkeys, donkeys, pigs, sheep, hamster, or dogs) can be
immunized with an antigen (e.g., TIGIT (e.g., human TIGIT or
cynomolgus TIGIT)) and once an immune response is detected, e.g.,
antibodies specific for the antigen are detected in the mouse
serum, the mouse spleen is harvested and splenocytes isolated. The
splenocytes are then fused by well-known techniques to any suitable
myeloma cells, for example, cells from cell line SP20 available
from the American Type Culture Collection (ATCC.RTM.) (Manassas,
Va.), to form hybridomas. Hybridomas are selected and cloned by
limited dilution. In certain embodiments, lymph nodes of the
immunized mice are harvested and fused with NSO myeloma cells.
[0328] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0329] Specific embodiments employ myeloma cells that fuse
efficiently, support stable high-level production of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. Among these myeloma cell lines are
murine myeloma lines, such as the NSO cell line or those derived
from MOPC-21 and MPC-11 mouse tumors available from the Salk
Institute Cell Distribution Center, San Diego, Calif., USA, and
SP-2 or X63-Ag8.653 cells available from the American Type Culture
Collection, Rockville, Md., USA. Human myeloma and mouse-human
heteromyeloma cell lines also have been described for the
production of human monoclonal antibodies (Kozbor D (1984) J
Immunol 133: 3001-5; Brodeur et al., Monoclonal Antibody Production
Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New
York, 1987), each of which is herein incorporated by reference in
its entirety).
[0330] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
TIGIT (e.g., human TIGIT or cynomolgus TIGIT). The binding
specificity of monoclonal antibodies produced by hybridoma cells is
determined by methods known in the art, for example,
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA).
[0331] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding J W (Ed), Monoclonal Antibodies:
Principles and Practice, supra). Suitable culture media for this
purpose include, for example, D-MEM or RPMI 1640 medium. In
addition, the hybridoma cells may be grown in vivo as ascites
tumors in an animal.
[0332] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0333] Antibodies described herein include, e.g., antibody
fragments which recognize a specific TIGIT (e.g., human TIGIT or
cynomolgus TIGIT), and which can be generated by any technique
known to those of skill in the art. For example, Fab and
F(ab').sub.2 fragments described herein can be produced by
proteolytic cleavage of immunoglobulin molecules, using enzymes
such as papain (to produce Fab fragments) or pepsin (to produce
F(ab').sub.2 fragments). A Fab fragment corresponds to one of the
two identical arms of an antibody molecule and contains the
complete light chain paired with the VH and CH1 domains of the
heavy chain. A F(ab').sub.2 fragment contains the two
antigen-binding arms of an antibody molecule linked by disulfide
bonds in the hinge region.
[0334] Further, the antibodies described herein can also be
generated using various phage display methods known in the art. In
phage display methods, functional antibody domains are displayed on
the surface of phage particles which carry the polynucleotide
sequences encoding them. In particular, DNA sequences encoding VH
and VL domains are amplified from animal cDNA libraries (e.g.,
human or murine cDNA libraries of affected tissues). The DNA
encoding the VH and VL domains are recombined together with a scFv
linker by PCR and cloned into a phagemid vector. The vector is
electroporated in E. coli and the E. coli is infected with helper
phage. Phage used in these methods are typically filamentous phage
including fd and M13, and the VH and VL domains are usually
recombinantly fused to either the phage gene III or gene VIII.
Phage expressing an antigen binding domain that binds to a
particular antigen can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Examples of phage display methods that can be used
to make the antibodies described herein include those disclosed in
Brinkman U et al., (1995) J Immunol Methods 182: 41-50; Ames R S et
al., (1995) J Immunol Methods 184: 177-186; Kettleborough C A et
al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene
187: 9-18; Burton D R & Barbas C F (1994) Advan Immunol 57:
191-280; PCT Application No. PCT/GB91/001134; International
Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO
92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and WO 97/13844;
and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717,
5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637,
5,780,225, 5,658,727, 5,733,743 and 5,969,108, all of which are
herein incorporated by reference in their entireties.
[0335] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described below. Techniques to
recombinantly produce antibody fragments such as Fab, Fab' and
F(ab').sub.2 fragments can also be employed using methods known in
the art such as those disclosed in PCT publication No. WO 92/22324;
Mullinax R L et al., (1992) BioTechniques 12(6): 864-9; Sawai H et
al., (1995) Am J Reprod Immunol 34: 26-34; and Better M et al.,
(1988) Science 240: 1041-1043, all of which are herein incorporated
by reference in their entireties.
[0336] In certain embodiments, to generate whole antibodies, PCR
primers including VH or VL nucleotide sequences, a restriction
site, and a flanking sequence to protect the restriction site can
be used to amplify the VH or VL sequences from a template, e.g.,
scFv clones. Utilizing cloning techniques known to those of skill
in the art, the PCR amplified VH domains can be cloned into vectors
expressing a VH constant region, and the PCR amplified VL domains
can be cloned into vectors expressing a VL constant region, e.g.,
human kappa or lambda constant regions. The VH and VL domains can
also be cloned into one vector expressing the necessary constant
regions. The heavy chain conversion vectors and light chain
conversion vectors are then co-transfected into cell lines to
generate stable or transient cell lines that express full-length
antibodies, e.g., IgG, using techniques known to those of skill in
the art.
[0337] A chimeric antibody is a molecule in which different
portions of the antibody are derived from different immunoglobulin
molecules. For example, a chimeric antibody can contain a variable
region of a mouse or rat monoclonal antibody fused to a constant
region of a human antibody. Methods for producing chimeric
antibodies are known in the art. See, e.g., Morrison S L (1985)
Science 229: 1202-7; Oi V T & Morrison S L (1986) BioTechniques
4: 214-221; Gillies S D et al., (1989) J Immunol Methods 125:
191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, 4,816,397, and
6,331,415, all of which are herein incorporated by reference in
their entireties.
[0338] A humanized antibody is capable of binding to a
predetermined antigen and which comprises a framework region having
substantially the amino acid sequence of a human immunoglobulin and
CDRs having substantially the amino acid sequence of a non-human
immunoglobulin (e.g., a murine immunoglobulin). In particular
embodiments, a humanized antibody also comprises at least a portion
of an immunoglobulin constant region (Fc), typically that of a
human immunoglobulin. The antibody also can include the CH1, hinge,
CH2, CH3, and CH4 regions of the heavy chain. A humanized antibody
can be selected from any class of immunoglobulins, including IgM,
IgG, IgD, IgA and IgE, and any isotype, including IgG.sub.1,
IgG.sub.2, IgG.sub.3 and IgG.sub.4. Humanized antibodies can be
produced using a variety of techniques known in the art, including
but not limited to, CDR-grafting (European Patent No. EP 239400;
International Publication No. WO 91/09967; and U.S. Pat. Nos.
5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing
(European Patent Nos. EP 592106 and EP 519596; Padlan E A (1991)
Mol Immunol 28(4/5): 489-498; Studnicka G M et al., (1994) Prot
Engineering 7(6): 805-814; and Roguska M A et al., (1994) PNAS 91:
969-973), chain shuffling (U.S. Pat. No. 5,565,332), and techniques
disclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886,
International Publication No. WO 93/17105; Tan P et al., (2002) J
Immunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5):
353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et
al., (1997) J Biol Chem 272(16): 10678-84; Roguska M A et al.,
(1996) Protein Eng 9(10): 895 904; Couto J R et al., (1995) Cancer
Res. 55 (23 Supp): 5973s-5977s; Couto J R et al., (1995) Cancer Res
55(8): 1717-22; Sandhu J S (1994) Gene 150(2): 409-10 and Pedersen
J T et al., (1994) J Mol Biol 235(3): 959-73, all of which are
herein incorporated by reference in their entireties. See also U.S.
Application Publication No. US 2005/0042664 A1 (Feb. 24, 2005),
which is herein incorporated by reference in its entirety.
[0339] Methods for making multispecific (e.g., bispecific
antibodies) have been described, see, for example, U.S. Pat. Nos.
7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830; 5,869,620;
6,132,992 and 8,586,713, all of which are herein incorporated by
reference in their entireties.
[0340] Single domain antibodies, for example, antibodies lacking
the light chains, can be produced by methods well known in the art.
See Riechmann L & Muyldermans S (1999) J Immunol 231: 25-38;
Nuttall S D et al., (2000) Curr Pharm Biotechnol 1(3): 253-263;
Muyldermans S, (2001) J Biotechnol 74(4): 277-302; U.S. Pat. No.
6,005,079; and International Publication Nos. WO 94/04678, WO
94/25591 and WO 01/44301, all of which are herein incorporated by
reference in their entireties.
[0341] Further, antibodies that specifically bind to a TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT) antigen can, in turn, be utilized
to generate anti-idiotype antibodies that "mimic" an antigen using
techniques well known to those skilled in the art. See, e.g.,
Greenspan N S & Bona C A (1989) FASEB J 7(5): 437-444; and
Nissinoff A (1991) J Immunol 147(8): 2429-2438, each of which is
herein incorporated by reference in its entirety.
[0342] In particular embodiments, an antibody described herein,
which binds to the same epitope of TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) as an anti-TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) antibody described herein, is a human antibody. In
particular embodiments, an antibody described herein, which
competitively blocks (e.g., in a dose-dependent manner) any one of
the antibodies described herein, from binding to TIGIT (e.g., human
TIGIT or cynomolgus TIGIT), is a human antibody. Human antibodies
can be produced using any method known in the art. For example,
transgenic mice which are incapable of expressing functional
endogenous immunoglobulins, but which can express human
immunoglobulin genes, can be used. In particular, the human heavy
and light chain immunoglobulin gene complexes can be introduced
randomly or by homologous recombination into mouse embryonic stem
cells. Alternatively, the human variable region, constant region,
and diversity region can be introduced into mouse embryonic stem
cells in addition to the human heavy and light chain genes. The
mouse heavy and light chain immunoglobulin genes can be rendered
non-functional separately or simultaneously with the introduction
of human immunoglobulin loci by homologous recombination. In
particular, homozygous deletion of the J.sub.H region prevents
endogenous antibody production. The modified embryonic stem cells
are expanded and microinjected into blastocysts to produce chimeric
mice. The chimeric mice are then bred to produce homozygous
offspring which express human antibodies. The transgenic mice are
immunized in the normal fashion with a selected antigen, e.g., all
or a portion of an antigen (e.g., TIGIT (e.g., human TIGIT or
cynomolgus TIGIT)). Monoclonal antibodies directed against the
antigen can be obtained from the immunized, transgenic mice using
conventional hybridoma technology. The human immunoglobulin
transgenes harbored by the transgenic mice rearrange during B cell
differentiation, and subsequently undergo class switching and
somatic mutation. Thus, using such a technique, it is possible to
produce therapeutically useful IgG, IgA, IgM and IgE antibodies.
For an overview of this technology for producing human antibodies,
see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93,
herein incorporated by reference in its entirety. For a detailed
discussion of this technology for producing human antibodies and
human monoclonal antibodies and protocols for producing such
antibodies, see, e.g., International Publication Nos. WO 98/24893,
WO 96/34096 and WO 96/33735; and U.S. Pat. Nos. 5,413,923,
5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318
and 5,939,598, all of which are herein incorporated by reference in
their entireties. Examples of mice capable of producing human
antibodies include the Xenomouse.TM. (Abgenix, Inc.; U.S. Pat. Nos.
6,075,181 and 6,150,184), the HuAb-Mouse.TM. (Mederex, Inc./Gen
Pharm; U.S. Pat. Nos. 5,545,806 and 5,569,825), the Trans Chromo
Mouse.TM.(Kirin) and the KM Mouse.TM. (Medarex/Kirin), all of which
are herein incorporated by reference in their entireties.
[0343] Human antibodies that specifically bind to TIGIT (e.g.,
human TIGIT or cynomolgus TIGIT) can be made by a variety of
methods known in the art including the phage display methods
described above using antibody libraries derived from human
immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887,
4,716,111, and 5,885,793; and International Publication Nos. WO
98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO
96/33735, and WO 91/10741, all of which are herein incorporated by
reference in their entireties.
[0344] In certain embodiments, human antibodies can be produced
using mouse-human hybridomas. For example, human peripheral blood
lymphocytes transformed with Epstein-Barr virus (EBV) can be fused
with mouse myeloma cells to produce mouse-human hybridomas
secreting human monoclonal antibodies, and these mouse-human
hybridomas can be screened to determine ones which secrete human
monoclonal antibodies that specifically bind to a target antigen
(e.g., TIGIT (e.g., human TIGIT or cynomolgus TIGIT)). Such methods
are known and are described in the art, see, e.g., Shinmoto H et
al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005)
Human Antibodies 14: 27-31, each of which is herein incorporated by
reference in its entirety.
5.6 Kits
[0345] Also provided are kits comprising one or more antibodies
described herein, or pharmaceutical compositions or conjugates
thereof. In a specific embodiment, provided herein is a
pharmaceutical pack or kit comprising one or more containers filled
with one or more of the ingredients of the pharmaceutical
compositions described herein, such as one or more antibodies
provided herein. In certain embodiments, the kits contain a
pharmaceutical composition described herein and any prophylactic or
therapeutic agent, such as those described herein. In certain
embodiments, the kits may contain a T cell mitogen, such as, e.g.,
phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA),
or a TCR complex stimulating antibody, such as an anti-CD3 antibody
and anti-CD28 antibody. Optionally associated with such
container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration.
[0346] Also provided, are kits that can be used in the above
methods. In one embodiment, a kit comprises an antibody described
herein, preferably a purified antibody, in one or more containers.
In a specific embodiment, kits described herein contain a
substantially isolated TIGIT (e.g., human TIGIT or cynomolgus
TIGIT) antigen as a control. In another specific embodiment, the
kits described herein further comprise a control antibody which
does not react with a TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
antigen. In another specific embodiment, kits described herein
contain one or more elements for detecting the binding of an
antibody to a TIGIT (e.g., human TIGIT or cynomolgus TIGIT) antigen
(e.g., the antibody can be conjugated to a detectable substrate
such as a fluorescent compound, an enzymatic substrate, a
radioactive compound or a luminescent compound, or a second
antibody which recognizes the first antibody can be conjugated to a
detectable substrate). In specific embodiments, a kit provided
herein can include a recombinantly produced or chemically
synthesized TIGIT (e.g., human TIGIT or cynomolgus TIGIT) antigen.
The TIGIT (e.g., human TIGIT or cynomolgus TIGIT) antigen provided
in the kit can also be attached to a solid support. In a more
specific embodiment, the detecting means of the above described kit
includes a solid support to which a TIGIT (e.g., human TIGIT or
cynomolgus TIGIT) antigen is attached. Such a kit can also include
a non-attached reporter-labeled anti-human antibody or
anti-mouse/rat antibody. In this embodiment, binding of the
antibody to the TIGIT (e.g., human TIGIT or cynomolgus TIGIT)
antigen can be detected by binding of the said reporter-labeled
antibody. In one embodiment, the present invention relates to the
use of a kit of the present invention for in vitro assaying and/or
detecting TIGIT antigen (e.g., human TIGIT or cynomolgus TIGIT) in
a biological sample.
6. EXAMPLES
[0347] The examples in this Section (i.e., Section 6) are offered
by way of illustration and not by way of limitation.
6.1 Example 1: Characterization of Anti-TIGIT Antibody BA002
[0348] This example describes the characterization of BA002, an
antibody that specifically binds to human TIGIT. The amino acid
sequences of the heavy and light chains of BA002 are provided in
Table 1.
6.1.1 Anti-Human TIGIT Antibody BA002 Binds to Purified Human and
Cynomolgus TIGIT Proteins
[0349] The ability of the BA002 antibody to bind to purified TIGIT
protein was assessed by surface plasmon resonance (SPR).
[0350] Briefly, surface plasmon resonance experiments were
performed using a Biacore T200 instrument, and the association rate
(K.sub.a), dissociation rate (K.sub.d), and dissociation constant
(K.sub.D) were calculated from each experiment using a 1:1 binding
model with Biacore T200 Evaluation Software.
[0351] To measure the binding affinity of human and cynomolgus
TIGIT to captured BA002, BA002 was captured at a flow rate of 10
.mu.L/min on flow-cell 2, keeping flow-cell 1 as reference, on a
CM5 chip on which an anti-human Fab antibody had been immobilized
by amine coupling. Human and cynomolgus TIGIT fused to Fc
("TIGIT-Fc"), a dimeric form of TIGIT, were independently run over
all the flow-cells at the concentrations of 20, 6.66, 2.22 and 0.74
nM at 50 .mu.l/min for 90 seconds, followed by a dissociation phase
of 400 seconds. Traces of response units vs. time after protein
injection for each concentration tested are shown in FIGS. 1A
(human TIGIT-Fc) and 1B (cynomolgus TIGIT-Fc), respectively. Based
on these data, captured BA002 bound to human TIGIT-Fc with a
calculated K.sub.a of 1.29.times.10.sup.6 M.sup.-1s.sup.-1, a
calculated K.sub.d of 1.60.times.10.sup.-4 s.sup.-1, and a
calculated K.sub.D of 0.12 nM. Captured BA002 bound to cynomolgus
TIGIT-Fc with a calculated K.sub.a of 4.28.times.10.sup.6
M.sup.-1s.sup.-1, a calculated K.sub.d of 3.02.times.10.sup.-3
s.sup.-1, and a calculated K.sub.D of 0.70 nM.
[0352] In a similar experiment assessing binding of a monomeric
form of human TIGIT fused to a polyhistidine tag ("TIGIT-His") to
BA002, BA002 was captured on flow-cell 2 of a Protein A chip,
keeping flow-cell 1 as reference, at a flow rate of 10 .mu.L/min.
TIGIT-His was run over both flow-cells at the concentrations of
125, 25, 5, and 1 nM at 30 .mu.L/min for 240 seconds, followed by a
dissociation phase of 900 seconds. Traces of response units vs.
time for each concentration tested are shown in FIG. 1C. Based on
these data, captured BA002 bound to human TIGIT-His with a
calculated K.sub.a of 4.1.times.10.sup.6 M.sup.-1s.sup.-1, a
calculated K.sub.d of 3.6.times.10.sup.-2 s.sup.-1, and a
calculated K.sub.D of 8.6 nM. The higher calculated dissociation
rate between monomeric human TIGIT (TIGIT-His) and BA002, relative
to the calculated dissociation rate between dimeric human TIGIT
(TIGIT-Fc) and BA002 described above, is consistent with the
presence of an avidity effect with the dimeric form of TIGIT.
[0353] In a similar experiment measuring binding of a monovalent
form of BA002 to human TIGIT-Fc, human TIGIT-Fc was captured on
flow-cell 2 of a CM5 chip, keeping flow-cell 1 as reference, at a
flow rate of 5 .mu.L/min. BA002 in Fab format was run over both
flow cells at the concentrations of 200, 50, 12.5, 3.125, 0.78 and
0.195 nM at 20 .mu.L/min for 120 seconds, followed by a
dissociation phase of 600 seconds. Traces of response units vs.
time for each concentration tested are shown in FIG. 1D. Based on
these data, immobilized human TIGIT-Fc bound to BA002 Fab with a
calculated K.sub.a of 2.8.times.10.sup.6 M.sup.-1s.sup.-1, a
calculated K.sub.d of 1.9.times.10.sup.-2 s.sup.-1, and a
calculated K.sub.D of 6.8 nM. The higher calculated dissociation
rate between human TIGIT-Fc and BA002 Fab, relative to the
calculated dissociation rate between human TIGIT-Fc and full-length
BA002 described above, is consistent with the presence of an
avidity effect with the bivalent form of BA002.
6.1.2 Anti-Human TIGIT Antibody BA002 Binds to Cells Expressing
Human and Cynomolgus Monkey TIGIT
[0354] The capacity of the human anti-TIGIT IgG1 antibody BA002 to
bind to cells expressing human TIGIT or cynomolgus monkey TIGIT was
tested in a variety of cell types.
Human TIGIT-Expressing Jurkat Cells
[0355] The ability of BA002 to bind to human TIGIT expressed on the
surface of Jurkat cells was assessed. Briefly, Jurkat cells were
transfected with a vector encoding human TIGIT, and a clone stably
expressing a high level of TIGIT was selected. This stable cell
line was cultured in RPMI 1640 medium supplemented with 10%
heat-inactivated FBS and 2% Normocin (Invivogen, Cat #ANT-NR-1).
For the antibody binding assay, the cells were seeded in a 96-well
U-bottom tissue culture plate at a density of 1.times.10.sup.5
cells per well and were incubated with Human TruStain FcX.TM. (Fc
Receptor Blocking Solution, Biolegend, Cat #422302) diluted 1:50 in
PBS supplemented with 2% heat-inactivated FBS (FACS Buffer) for 10
minutes at 4.degree. C. The cells were then incubated for 30
minutes at 4.degree. C. with a series dilution of BA002 or isotype
control antibody at concentrations from 50 .mu.g/mL to 0.64 ng/mL
diluted in FACS Buffer. For antibody staining, the cells were
washed twice with cold FACS Buffer and re-suspended in FACS Buffer
containing R-Phycoerythrin AffiniPure F(ab').sub.2 Fragment Donkey
Anti-Human IgG (H+L) (Jackson, Cat #09-116-149) at 1:200 dilution
and LIVE/DEAD.RTM. Fixable Near-IR Dead Cell Stain (Life
Technologies, Cat #L10119). After a 10-minute incubation on ice,
the cells were washed twice with cold FACS Buffer, and the cells
were analyzed by flow cytometry (BD LSR Fortessa Flow Cytometer).
The data were analyzed by the FlowJo software by sequentially
gating on the FSC-A vs. SSC-A, FSC-H vs FSC-A, SSC-A vs. Dead Cell
Stain, and SSC-A vs PE. Mean fluorescence intensity (MFI) values
were calculated, and the data were plotted by GraphPad Prism
software.
[0356] As shown in FIG. 2A, BA002 bound to human TIGIT-expressing
Jurkat cells in a dose-dependent manner.
Activated Primary Human T Cells
[0357] In similar experiments, the capacity of BA002 to bind to
activated human CD4+ or CD8+ T cells was tested. Briefly, a frozen
aliquot of human peripheral blood mononuclear cells (PBMCs) was
retrieved from liquid nitrogen and immediately thawed in 37.degree.
C. water until floating ice was observed. Cells were then
transferred to 9 mL of pre-warmed R10 media. 10 .mu.L was removed
and added to 390 .mu.L viability dye to count cells and check
viability using a Muse apparatus. Samples were centrifuged at 2000
rpm for two minutes and then suspended to a final concentration of
0.1.times.10.sup.6 cells/mL with R10 media. A 1 .mu.g/mL stock
solution SEA was added to the PBMC cells prepared as described
above to a final concentration of 100 ng/mL. 100 of stimulated
cells were pipetted to each well of a 96 well U-bottom tissue
culture plate and incubated in a tissue culture incubator at
37.degree. C. in 5% CO2 for five days.
[0358] A dose range of antibody was prepared in a 96 well round
bottom plate. First, 600 .mu.L of 50 .mu.g/mL of each antibody was
prepared in buffer. Antibodies were then serially diluted 1-to-3 by
pipetting 200 .mu.L of the previous dilution into 400 .mu.L of
sample buffer. A total of 12 dilutions ranging from 50 .mu.g/mL to
0.0002 .mu.g/mL were prepared. After 5 days, the sample plate was
centrifuged for two minutes at 2000 rpm, and supernatants were
discarded. Samples were blocked with Fc.gamma.R Block prepared in
FACs buffer at 5 .mu.L per 100 .mu.L test (550 .mu.L of Fc Receptor
Blocking reagent diluted in 10.45 mL of FACs buffer) for 10
minutes. Sample plates were then centrifuged for two minutes at
2000 rpm, and the supernatant was discarded. The cells were then
re-suspended in 100 .mu.L of anti-TIGIT antibody or a relevant
isotype control at the concentrations shown in FIGS. 2B-2C. Sample
plates were incubated for 20 minutes at 4.degree. C. Cells were
washed by addition of cold sample buffer and centrifuged for two
minutes at 2000 rpm, and the supernatant was discarded. This wash
was repeated once.
[0359] Cells were then resuspended in a cocktail of fluorescently
labeled antibodies. A cocktail of fluorescently labeled antibodies
sufficient for all samples was prepared in FACs buffer. 100 .mu.L
of antibody per well was then added to a round-bottom 96-well
plate. The sample plate was incubated for 20 minutes on ice. Cells
were washed by addition of cold sample buffer, centrifuged for two
minutes at 2000 rpm, and supernatants discarded. This wash was
repeated once. A final cocktail of PE-labeled secondary anti-human
IgG antibody was prepared in 11 mL of FACs buffer. 100 .mu.L of
secondary antibody was added per well to a round-bottom 96-well
plate. The sample plate was incubated for 5 minutes on ice. Cells
were washed by addition of cold sample buffer, centrifuged for two
minutes at 2000 rpm, and the supernatants were discarded. This wash
was repeated once.
[0360] Antibody binding was measured by flow cytometry using a BD
LSR Fortessa Flow Cytometer. Unstained control cells were used to
gate on the lymphocyte population using a plot of forward
scatter-area (FSC-A) versus side scatter area (SSC-A) and another
plot of FSC-A versus FSC-Height (FSC-H) for selection of single
cells. Tubes of cells stained with each individual antibody were
used to calculate compensation of the various colors used in the
experiment. 100,000 events were recorded for each sample. Samples
were analyzed by sequentially gating on the following populations:
FSC-A vs SSC-A, FSC-H vs FSC-A, SSC-A vs LIVE/DEAD, CD4 vs CD8, and
SSC vs CD25. Mean fluorescence intensity (MFI) was calculated.
[0361] As shown in FIGS. 2B and 2C, BA002 bound to activated
primary human CD4.sup.+ T cells (FIG. 2B) and activated primary
human CD8+ T cells (FIG. 2C) in a dose-dependent manner.
CHO Cells Expressing Cynomolgus Monkey TIGIT
[0362] In similar experiments, the capacity of BA002 to bind to
Chinese hamster ovary (CHO) cells engineered to express cynomolgus
monkey TIGIT on their cell surfaces (cynomolgus TIGIT-CHO cells)
was tested. Briefly, a frozen aliquot of cynomolgus TIGIT-CHO cells
was thawed in 37.degree. C. water and then transferred to a tube
containing 9 mL of pre-warmed R10 media. Cells were centrifuged at
2000 rpm for two minutes. The supernatant was discarded and the
cells were resuspended in 20 mL of R10 media. Cells were then
transferred to a T75 flask and incubated in a tissue culture
incubator at 37.degree. C. and 8% CO.sub.2 for 1 day. Cells were
then removed from the incubator and treated with 5 mL of TrypLE
express. Liberated cells were then diluted with 10 mL of R10 media
and centrifuged for two minutes at 2000 RPM. The supernatant was
discarded and the cells were resuspended in 10 mL of R10 media and
assessed for count and viability. Cell samples were then
centrifuged at 2000 rpm for two minutes and re-suspended to a final
concentration of 1.times.10.sup.6 cells/mL with R10 media. 100
.mu.L of cells were then pipetted to each well of a 96 well
U-bottom tissue culture plate for a final concentration of 100,000
cells per well.
[0363] A dose-range of antibody was prepared in 1.2 mL bullet
tubes. First, 600 .mu.L of 50 .mu.g/mL of each antibody was
prepared in FACs buffer. Antibodies were then serially diluted
1-to-5 by pipetting 120 .mu.L of the previous dilution into 600
.mu.L of sample buffer. A total of 12 dilutions ranging from 50
.mu.g/mL to 0.000001024 .mu.g/mL were prepared. Sample plates were
centrifuged for two minutes at 2000 rpm, and the supernatants were
discarded. Samples were washed with twice with FACs buffer. The
cells were then re-suspended in 100 .mu.L of anti-TIGIT antibody
BA002 or an isotype control at the concentrations shown in FIG. 2D.
Sample plates were then incubated for 30 minutes at 4.degree. C.
Cells were washed by addition of cold sample buffer and centrifuged
for two minutes at 2000 rpm, and the supernatant was discarded.
This wash was repeated once. Cells were then resuspended in a
cocktail of live/dead stain and PE-labeled secondary anti-human IgG
antibody. Sample plates were incubated for 10 minutes on ice. Cells
were washed and centrifuged for two minutes at 2000 rpm, and the
supernatants were discarded. This wash was repeated once.
[0364] Antibody binding was measured by flow cytometry using a BD
LSR Fortessa Flow Cytometer. Unstained control cells were used to
gate on the lymphocyte population using a plot of forward
scatter-area (FSC-A) versus side scatter area (SSC-A) and another
plot of FSC-A versus FSC-Height (FSC-H) for selection of single
cells. Tubes of cells stained with each individual antibody were
used to calculate compensation of the various colors used in the
experiment. 100,000 events were recorded for each sample. Samples
were analyzed by sequentially gating on the following populations:
FSC-A vs SSC-A, FSC-H vs FSC-A, SSC-A vs LIVE/DEAD, and SSC-A vs
PE. Mean fluorescence intensity (MFI) was calculated.
[0365] As shown in FIG. 2D, BA002 bound to CHO cells expressing
cynomolgus monkey TIGIT in a dose-dependent manner.
6.1.3 Anti-TIGIT Antibody Selectively Binds to TIGIT
[0366] In this example, the selectivity of BA002 for TIGIT compared
to its related family members CD96 and CD226 was tested.
Specifically, BA002 was tested for binding to two engineered Jurkat
cell lines, one that expressed human TIGIT, CD96, and CD226 on its
cell surface (TIGIT.sup.+ CD96.sup.+ CD226.sup.+), and one that
expressed CD96 and CD226 but not TIGIT (TIGIT.sup.- CD96.sup.+
CD226.sup.+).
[0367] Briefly, frozen aliquots of TIGIT-Jurkat Clone D3 cells and
wild type Jurkat cells were retrieved from liquid nitrogen and
thawed in 37.degree. C. water. Each clone was transferred to a
separate tube containing 9 mL of pre-warmed R10 media. Cells were
centrifuged at 2000 rpm for two minutes. The supernatant was
discarded and the cells were resuspended in 20 mL of R10 media.
Cells were then transferred to a T75 flask and incubated in a
tissue culture incubator at 37.degree. C. and 5% CO2 for 1 day.
After incubation, cells were assessed for count and viability. Cell
samples were then centrifuged at 2000 rpm for two minutes and
re-suspended to a final concentration of 1.times.10.sup.6 cells/mL
with R10 media. Next, 100 .mu.L of cells were pipetted to each well
of a 96 well U-bottom tissue culture plate for a final
concentration of 100,000 cells per well.
[0368] A dose-range of each antibody (i.e., BA002 or isotype
control) was prepared in 1.2 mL bullet tubes. First, 400 .mu.L of
50 .mu.g/mL of each antibody was prepared in FACs buffer.
Antibodies were then serially diluted 1-to-5 by pipetting 80 .mu.L
of the previous dilution into 320 of sample buffer. A total of 8
dilutions ranging from 50 .mu.g/mL to 0.00064 .mu.g/mL were
prepared.
[0369] Sample plates were centrifuged for two minutes at 2000 rpm,
and supernatants were discarded. Samples were blocked with
Fc.gamma.R Block prepared in FACs buffer (550 .mu.L of Fc Receptor
Blocking reagent diluted in 10.45 mL of FACs buffer) for ten
minutes. Sample plates were then centrifuged for two minutes at
2000 rpm, and the supernatant was discarded. The cells were then
resuspended in 100 .mu.L of BA002 or isotype control at the
concentrations shown in FIGS. 3A-3B. Sample plates were incubated
with antibody for 30 minutes at 4.degree. C. Cells were then washed
by addition of cold sample buffer and centrifuged for two minutes
at 2000 rpm and the supernatant discarded. This wash was repeated
once. Cells were then resuspended in a cocktail of live/dead stain
and PE-labeled secondary anti-human IgG antibody was prepared in 20
mL of FACs buffer. Sample plates were incubated for 10 minutes on
ice. Cells were washed by addition of cold FACs buffer, centrifuged
for two minutes at 2000 rpm, and the supernatants were discarded.
This wash was repeated once.
[0370] Antibody binding was measured by flow cytometry using a BD
LSR Fortessa Flow Cytometer. Unstained control cells were used to
gate on the lymphocyte population using a plot of forward scatter
area (FSC-A) versus side scatter area (SSC-A) and another plot of
FSC-A versus FSC-Height (FSC-H) for selection of single cells.
Tubes of cells stained with each individual antibody were used to
calculate compensation of the various colors used in the
experiment. 100,000 events for each sample were recorded. Samples
were analyzed by sequentially gating on the following populations:
FSC-A vs SSC-A, FSC-H vs FSC-A, SSC-A vs LIVE/DEAD, and SSC-A vs
PE. Mean fluorescence intensity (MFI) was calculated.
[0371] As shown in FIGS. 3A and 3B, BA002 strongly bound to Jurkat
cells expressing human TIGIT but showed no cross-reactivity with
the related family members CD96 and CD226.
6.1.4 Anti-TIGIT Antibody Blocks Ligand Binding to TIGIT
TIGIT Binding to CD155/PVR
[0372] In this example, the capacity of BA002 to block binding
between TIGIT and its ligand CD155 (also referred to as PVR) was
tested. Specifically, BA002, a series of reference anti-TIGIT
antibodies, and isotype controls were tested for their ability to
block binding between soluble TIGIT and CD155 in vitro.
[0373] Briefly, a 5.times. concentrated intermediate stock of each
antibody (i.e., BA002, reference anti-TIGIT antibodies #1, 2, 3, 4,
5, and 6, and corresponding isotype controls) sufficient for two
replicates was prepared in 1.2 mL bullet tubes. First, 60 .mu.L of
250 .mu.g/mL of each antibody was prepared in PBS. Antibodies were
then serially diluted 1-to-3 by pipetting 20 .mu.L of the previous
dilution into 40 .mu.L of sample buffer. A total of 12 working
dilutions ranging from 50 .mu.g/mL to 0.00028 .mu.g/mL was
prepared. A solution comprising 4 ng/.mu.L of CD155-His in assay
buffer was prepared. 2 .mu.L 3.times.TIGIT assay buffer, 2 .mu.L
CD155-His solution, and 2 .mu.L distilled water were combined to
produce a master mixture, and then 6 .mu.L of master mixture was
added to each well of an assay plate. A solution comprising 2
ng/.mu.L of biotinylated TIGIT (TIGIT-biotin) in assay buffer was
also prepared, of which 2 .mu.L was added per well and incubated
for 60 minutes. Additionally, Ni Chelate Acceptor beads
(PerkinElmer #AL108C) were diluted 250-fold with 1.times. assay
buffer, and 10 .mu.L of the diluted acceptor bead solution was
added per well. After shaking briefly, the mixture was incubated at
room temperature for 30 minutes. Streptavidin-conjugated donor
beads (PE #6760002S) were then diluted 125-fold with 1.times. assay
buffer, and 10 .mu.L of the diluted donor bead solution was added
per well. The mixture was incubated at room temperature for 30
minutes. Alpha counts were obtained and relative light unit values
were calculated and normalized according to standard methods to
determine percent binding between TIGIT and CD155 in the presence
of each antibody tested.
[0374] As shown in FIGS. 4A-4F, BA002 showed substantial blocking
of TIGIT binding to CD155. The ligand blocking activity of BA002
for CD155 was comparable to or greater than that observed for the
series of reference anti-TIGIT antibodies. For ease of
visualization, the same data for BA002 and isotype control are
shown in each of FIGS. 4A-4F, while data for a different reference
antibody is shown in each Figure.
TIGIT Binding to CD112/PVRL2
[0375] In this example, the capacity of BA002 to block binding
between TIGIT and its ligand CD112 (also referred to as PVRL2) was
tested. Specifically, BA002, a series of reference anti-TIGIT
antibodies, and isotype controls were tested for their ability to
block binding between soluble TIGIT and CD112 in vitro.
[0376] Briefly, a 5.times. concentrated intermediate stock of each
antibody (i.e., BA002, reference anti-TIGIT antibodies #1, 2, 3, 4,
5, and 6, and corresponding isotype controls) sufficient for two
replicates was prepared in 1.2 mL bullet tubes. First, 60 .mu.L of
250 .mu.g/mL of each antibody was prepared in PBS. Antibodies were
then serially diluted 1-to-3 by pipetting 20 .mu.L of the previous
dilution into 40 .mu.L of sample buffer. A total of 12 working
dilutions ranging from 50 .mu.g/mL to 0.00028 .mu.g/mL was
prepared. A solution comprising 4 ng/.mu.L of CD112-Histidine in
assay buffer was prepared. 2 .mu.l 3.times. TIGIT assay buffer, 2
.mu.l CD112-His solution, and 2 .mu.l distilled water were combined
to produce a master mixture, and then 6 .mu.L of master mixture was
added to each well of an assay plate. A solution comprising 2
ng/.mu.L of biotinylated TIGIT (TIGIT-biotin) in assay buffer was
also prepared, of which 2 .mu.L was added per well and incubated
for 60 minutes. Additionally, Ni Chelate Acceptor beads
(PerkinElmer #AL108C) were diluted 250-fold with 1.times. assay
buffer, and 10 .mu.L of the diluted acceptor bead solution was
added per well. After shaking briefly, the mixture was incubated at
room temperature for 30 minutes. Streptavidin-conjugated donor
beads (PE #6760002S) were then diluted 125-fold with 1.times. assay
buffer, and 10 .mu.L of the diluted donor bead solution was added
per well. The mixture was incubated at room temperature for 30
minutes. Alpha counts were obtained and relative light unit values
were calculated and normalized according to standard methods to
determine percent binding between TIGIT and CD112 in the presence
of each antibody tested.
[0377] As shown in FIGS. 5A-5F, BA002 showed substantial blocking
of TIGIT binding to CD112. The ligand blocking activity of BA002
for CD112 was comparable to or greater than that observed for the
series of reference anti-TIGIT antibodies. For ease of
visualization, the same data for BA002 and isotype control are
shown in each of FIGS. 5A-5F, while data for a different reference
antibody is shown in each Figure.
6.2 Example 2: Functionality of Anti-TIGIT Antibody and Combination
Therapies
6.2.1 Anti-TIGIT Antibody Enhances T.sub.H1 Cytokine Secretion by
Primary Cells
Anti-TIGIT Antibody Enhances IFN.gamma. Secretion by Stimulated
PBMCs
[0378] In this example, the capacity of BA002 and a series of
reference anti-TIGIT antibodies to promote secretion of IFN.gamma.
by PBMCs stimulated with Staphylococcal Enterotoxin A (SEA) was
tested. The anti-TIGIT antibodies were also tested for
cooperativity with an anti-PD-1 antibody in this assay.
[0379] A 5.times. concentrated intermediate stock of each antibody
(i.e., BA002, reference anti-TIGIT antibodies #1, 3, 5, or 6, or an
isotype control) was prepared in 1.2 mL bullet tubes. One set of
tubes also received 25 .mu.g/mL of anti-PD-1 antibody, while
another set of tubes also received 25 .mu.g/mL of IgG4 isotype
antibody, each representing a 5.times. concentrated intermediate
stock of anti-PD-1 or IgG4 isotype antibody. First, 400 .mu.L of 50
.mu.g/mL of each anti-TIGIT antibody supplemented with 25 .mu.g/mL
of anti-PD-1 or IgG4 isotype antibody was prepared in R10 media. 20
.mu.L of antibody was then added per well to a round-bottom 96-well
plate. Frozen aliquots of human PBMCs were retrieved from liquid
nitrogen and immediately thawed in 37.degree. C. water until
floating ice was observed. Cells were transferred to 9 mL of
pre-warmed R10 media and immediately centrifuged at 2000 rpm for
two minutes. To count cells and check viability, 10 of sample was
removed and added to 390 .mu.L of viability dye, mixed, and read
using a Muse apparatus.
[0380] Samples were centrifuged at 2000 rpm for two minutes and
resuspended to an intermediate concentration. An intermediate stock
concentration of SEA was made by adding 10 of 1000 .mu.g/mL SEA to
90 .mu.L R10 to make an intermediate concentration of 100 .mu.g/mL.
To stimulate the cells, 12 .mu.L of a 100 .mu.g/mL intermediate
stock of SEA was added to the 9.60 mL of cells prepared above. 80
.mu.L of cells and SEA mixture was added into corresponding wells
and incubated in tissue culture incubator at 37.degree. C. and 5%
CO2 within a humidified chamber for four days. A total of
0.1.times.10.sup.6 cells/well and final concentration of 100 ng/mL
of SEA was used.
[0381] After four days of incubation, plates were removed from the
incubator and gently agitated by hand. The plates were then
centrifuged for two minutes at 2000 rpm. 5 .mu.L of supernatant was
transferred to a 384-well AlphaLISA plate for cytokine analysis.
AlphaLISA kits (Perkin Elmer) were used for measurement of
IFN.gamma. secretion. Briefly, assay buffer was prepared by
pipetting 2.5 mL of 10.times. AlphaLISA HiBlock Buffer to 22.5 mL
water. Human IFN.gamma. analyte was used to prepare a standard
dilution according to manufacturer instructions. A mixture of
1.6.times. AlphaLISA anti-IFN.gamma. acceptor beads and
biotinylated anti-IFN.gamma. antibody was prepared in assay buffer.
8 .mu.L was added to each well and incubated in darkness at room
temperature, rotating at 500 rpm for 90 minutes. A 2.3.times.
Streptavidin Donor Bead intermediate stock was prepared in assay
buffer. 10 .mu.L were added to each well and incubated in darkness
at room temperature, rotating at 500 rpm for 20 minutes. AlphaLISA
plates were briefly centrifuged at 2000 rpm. Relative light units
(RLU) were measured using the AlphaScreen protocol on an EnVision
Plate Reader.
[0382] As shown in FIG. 6, BA002 enhanced IFN.gamma. secretion by
SEA-stimulated PBMCs to a greater degree than reference antibodies
or isotype control. In addition, the combination of BA002 and the
anti-PD-1 antibody resulted in a substantial increase in IFN.gamma.
secretion compared to treatment with BA002 alone. This increase was
greater than that seen for the reference anti-TIGIT antibodies.
Anti-TIGIT Antibody Enhances IL-2 Secretion by Stimulated PBMCs
[0383] In this example, the capacity of BA002 and a reference
anti-TIGIT antibody to promote secretion of the cytokine
interleukin-2 (IL-2) by PBMCs stimulated with SEA was tested. The
anti-TIGIT antibodies were also tested for cooperativity with an
anti-CTLA-4 antibody in this assay.
[0384] A 5.times. concentrated intermediate stock of each antibody
(i.e., BA002, reference anti-TIGIT antibody #4, or an isotype
control) was prepared in 1.2 mL bullet tubes. One set of tubes also
received 25 .mu.g/mL of anti-CTLA-4 antibody, while another set of
tubes also received 25 .mu.g/mL of IgG1 isotype antibody, each
representing a 5.times. concentrated intermediate stock of
anti-CTLA-4 or anti-IgG1 isotype antibody. First, 400 .mu.L of 50
.mu.g/mL of each anti-TIGIT antibody supplemented with 25 .mu.g/mL
of anti-CTLA-4 or IgG1 isotype antibody was prepared in R10 media.
20 .mu.L of antibody was then added per well to a round-bottom
96-well plate. Frozen aliquots of human PBMCs were retrieved from
liquid nitrogen and immediately thawed in 37.degree. C. water until
floating ice was observed. Cells were transferred to 9 mL of
pre-warmed R10 media and immediately centrifuged at 2000 rpm for
two minutes. To count cells and check viability, 10 of sample was
removed and added to 390 .mu.L of viability dye, mixed, and read
using a Muse apparatus.
[0385] Samples were centrifuged at 2000 rpm for two minutes and
resuspended to an intermediate concentration. An intermediate stock
concentration of SEA was made by adding 10 .mu.L of 1000 .mu.g/mL
SEA to 90 .mu.L R10 to make an intermediate concentration of 100
.mu.g/mL. To stimulate the cells, 12 .mu.L of a 100 .mu.g/mL
intermediate stock of SEA was added to the 9.60 mL of cells
prepared above. 80 .mu.L of cells and SEA mixture was added into
corresponding wells and incubated in tissue culture incubator at
37.degree. C. and 5% CO2 within a humidified chamber for four days.
A total of 0.1.times.10.sup.6 cells/well and final concentration of
100 ng/mL of SEA was used.
[0386] After four days of incubation, plates were removed from the
incubator and gently agitated by hand. The plates were then
centrifuged for two minutes at 2000 rpm. 5 .mu.L of supernatant was
transferred to a 384-well AlphaLISA plate for cytokine analysis
AlphaLISA kits (Perkin Elmer) were used for measurement of IL-2
secretion. Briefly, assay buffer was prepared by pipetting 2.5 mL
of 10.times. AlphaLISA Immunoassay Buffer to 22.5 mL water. Human
IL-2 analyte was used to prepare a standard dilution. A mixture of
1.6.times. AlphaLISA anti-IL-2 acceptor beads and biotinylated
anti-IL-2 antibody was prepared in assay buffer. 8 .mu.L was added
to each well and incubated in darkness at room temperature,
rotating at 500 rpm for 90 minutes. A 2.3.times. Streptavidin Donor
Bead intermediate stock was prepared in assay buffer. 10 .mu.L was
added to each well and incubated in darkness at room temperature,
rotating at 500 rpm for 20 minutes. AlphaLISA plates were briefly
centrifuged at 2000 rpm. Relative light units (RLU) were measured
using the AlphaScreen protocol on an EnVision Plate Reader.
[0387] As shown in FIGS. 7A-7B, the combination of BA002 and the
anti-CTLA-4 antibody resulted in a substantial increase in IL-2
secretion compared to treatment with BA002 or the anti-CTLA-4
antibody alone. This increase was greater than that seen for the
reference anti-TIGIT antibody tested in this experiment, reference
antibody #4.
6.3 Example 3: Fc Variants of Anti-TIGIT Antibody
[0388] 6.3.1 Characterization of Anti-TIGIT Antibody Variants with
Different Fc Regions
[0389] In this example, the impact of Fc region/Fc.gamma.R
interaction on the binding and functional activity of BA002 was
analyzed. In particular, the VH region of BA002 was expressed with
various Fc backbones, as summarized in Table 4.
TABLE-US-00004 TABLE 4 Fc variants of BA002. Heavy Light Chain
Chain Antibody Antibody Description (numbered SEQ ID SEQ ID Name
according to the EU numbering system) NO: NO: BA002 IgG1 11 27
BA003 N297A variant of BA002 12 27 BA004 L234F/L235F/N297A variant
of BA002 13 27 BA005 S239D/I332E variant of BA002 14 27 BA006
S239D/A330L/I332E variant of BA002 15 27 BA007
L235V/F243L/R292P/Y300L/P396L 16 27 variant of BA002 BA008
S267E/L328F variant of BA002 17 27 BA009 IgG4 S228P variant of
BA002 18 27
[0390] In addition, BA002_AF, an afucosylated version of BA002 with
identical heavy and light chain sequences, was expressed.
[0391] These variants of BA002 were then tested in binding and
functional assays, as described below.
Binding to Activated Primary Human T Cells
[0392] BA006 was tested for its ability to bind to activated
primary CD4.sup.+ T cells, using the same experimental design and
conditions as described for BA002 in Section 6.1.2. As shown in
FIG. 8A, BA006 bound to activated primary CD4.sup.+ T cells in a
dose-dependent manner. Binding to CHO cells expressing cynomolgus
monkey TIGIT
[0393] BA006 was tested for its ability to bind to cynomolgus
monkey TIGIT expressed on the surface of engineered CHO cells,
using the same experimental design and conditions as described for
BA002 in Section 6.1.2. As shown in FIG. 8B, BA006 bound to CHO
cells expressing cynomolgus monkey TIGIT in a dose-dependent
manner.
Cell Binding and Selectivity for Human TIGIT
[0394] The Fc variant anti-TIGIT antibody BA006 was tested for its
ability to bind to human TIGIT, as well as its selectivity for
human TIGIT over its related family members CD96 and CD226.
Specifically, BA006 or isotype control were tested for binding to
(i) TIGIT.sup.+ CD96.sup.+ CD226.sup.+ Jurkat cells, or (ii)
TIGIT.sup.- CD96.sup.+ CD226.sup.+ Jurkat cells, using the same
experimental design and conditions as described for BA002 in
Section 6.1.3. In these experiments, BA006 strongly bound to Jurkat
cells expressing human TIGIT (FIG. 8C), but showed no
cross-reactivity with the related family members CD96 and CD226
(FIG. 8D).
Fc Variants of BA002 Further Enhance IL-2 Secretion by Stimulated
PBMCs Alone and in Combination with Anti-PD-1 Antibody
[0395] In this example, the capability of Fc variants of BA002 to
promote secretion of IL-2 by PBMCs stimulated with SEA was tested.
The anti-TIGIT antibodies were also tested for cooperativity with
an anti-PD-1 antibody in this assay.
[0396] A 5.times. concentrated intermediate stock of each antibody
(i.e., BA002, BA002_AF, BA003, BA005, BA006, BA007, BA008, BA009,
or isotype controls for IgG1 and IgG4) was prepared in 1.2 mL
bullet tubes. One set of tubes also received 25 .mu.g/mL of
anti-PD-1 antibody, while another set of tubes also received 25
.mu.g/mL of IgG4 isotype control antibody, each representing a
5.times. concentrated intermediate stock of anti-PD-1 or IgG4
isotype control antibody. First, 400 .mu.L of 50 .mu.g/mL of each
anti-TIGIT antibody supplemented with 25 .mu.g/mL of anti-PD-1 or
IgG4 isotype antibody was prepared in R10 media. 20 .mu.L of
antibody was then added per well to a round-bottom 96-well plate.
Frozen aliquots of human PBMCs were retrieved from liquid nitrogen
and immediately thawed in 37.degree. C. water until floating ice
was observed. Cells were transferred to 9 mL of pre-warmed R10
media and immediately centrifuged at 2000 rpm for two minutes.
Cells were counted and checked for viability.
[0397] Samples were centrifuged at 2000 rpm for two minutes and
resuspended to an intermediate concentration. An intermediate stock
concentration of SEA was made by adding 10 of 1000 .mu.g/mL SEA to
90 .mu.L R10 to make an intermediate concentration of 100 .mu.g/mL.
To stimulate the cells, 12 .mu.L of a 100 .mu.g/mL intermediate
stock of SEA was added to the 9.60 mL of cells prepared above. 80
.mu.L of cells and SEA mixture was added into corresponding wells
and incubated in tissue culture incubator at 37.degree. C. and 5%
CO2 within a humidified chamber for four days. A total of
0.1.times.10.sup.6 cells/well and final concentration of 100 ng/mL
of SEA was used.
[0398] After four days of incubation, plates were removed from the
incubator and gently agitated by hand. The plates were then
centrifuged for two minutes at 2000 rpm. 5 .mu.L of supernatant was
transferred to a 384-well AlphaLISA plate for cytokine analysis.
AlphaLISA kits (Perkin Elmer) were used for measurement of IL-2
secretion. Briefly, assay buffer was prepared by pipetting 2.5 mL
of 10.times. AlphaLISA Immunoassay Buffer to 22.5 mL water. Human
IL-2 analyte was used to prepare a standard dilution. A mixture of
1.6.times. AlphaLISA anti-IL-2 acceptor beads and biotinylated
anti-IL-2 antibody was prepared in assay buffer. 8 .mu.L was added
to each well and incubated in darkness at room temperature,
rotating at 500 rpm for 90 minutes. A 2.3.times. Streptavidin Donor
Bead intermediate stock was prepared in assay buffer. 10 .mu.L was
added to each well and incubated in darkness at room temperature,
rotating at 500 rpm for 20 minutes. AlphaLISA plates were briefly
centrifuged at 2000 rpm. Relative light units (RLU) were measured
using the AlphaScreen protocol on an EnVision Plate Reader. This
experiment was run for four replicates using PBMCs obtained from
two different donors.
[0399] As shown in FIGS. 9A and 9B, Fc variants of BA002 further
enhanced IL-2 secretion by SEA-stimulated PBMCs beyond the effect
observed for BA002. In particular, BA002_AF, BA005, BA006, and
BA007 each induced greater IL-2 secretion than BA002, which in turn
induced greater IL-2 secretion than BA003, BA008, BA009, or isotype
control. Combining Fc variants of BA002 with an anti-PD-1 antibody
also produced a further improvement in IL-2 secretion by
SEA-stimulated PBMCs.
Fc Variants of BA002 Further Enhanced Activation of CD4.sup.+ and
CD8.sup.+ T Cells Alone and in Combination with Anti-PD-1
Antibody
[0400] In this example, the capability of Fc variants of BA002 to
promote T cell activation was tested. The anti-TIGIT antibodies
were also tested for cooperativity with an anti-PD-1 antibody in
this assay.
[0401] A 5.times. concentrated intermediate stock of antibody
(i.e., BA002, BA002_AF, BA003, BA005, BA006, BA007, BA008, BA009,
isotype IgG1 control, or isotype IgG4 control, each with a matching
quantity of either an anti-PD-1 antibody or an isotype IgG4
control) sufficient for four replicates for two donors was prepared
in 1.2 mL bullet tubes. First, 400 .mu.L of 50 .mu.g/mL of each
antibody was prepared in R10 media. 20 .mu.L of antibody solution
per well was then added to a round-bottom 96-well plate. Frozen
aliquots of indicated human PBMC donors were retrieved from liquid
nitrogen and thawed in 37.degree. C. water. Cells were transferred
to 9 mL of pre-warmed R10 media and immediately centrifuged at 2000
rpm for two minutes. Cell were then counted and viability was
assessed. Cell samples were then centrifuged at 2000 rpm for two
minutes and resuspended to an intermediate concentration. An
intermediate stock concentration of SEA was made by diluting 10
.mu.L of 1000 .mu.g/mL of SEA in 90 .mu.L of R10 medium to make an
intermediate concentration of 100 .mu.g/mL. To stimulate the cells,
12 .mu.L of a 100 .mu.g/mL intermediate stock of SEA was added to
7.20 mL of the cells prepared above. 60 .mu.L of cells and SEA
mixture was added into corresponding wells and incubated in a
humidified chamber at 37.degree. C. and 5% CO2 for five days. A
total of 0.1.times.10.sup.6 cells/well and final concentration of
100 ng/mL of SEA was used.
[0402] After 5 days, the sample plate was centrifuged for two
minutes at 2000 rpm, and supernatants were discarded. Samples were
washed twice and blocked with Fc.gamma.R block at 5 per 100 .mu.L
test (i.e., 550 .mu.L of Fc Receptor Blocking reagent diluted in
10.45 mL of FACs buffer) for 10 minutes. Sample plates were then
centrifuged for two minutes at 2000 rpm and the supernatant was
discarded. A cocktail of fluorescent-labeled antibodies sufficient
for all samples was prepared in 11 mL of FACs buffer. 100 .mu.L of
fluorescent antibody cocktail was then added per well to a
round-bottom 96-well plate using a multi-channel a pipette. The
sample plate was incubated for 20 minutes on ice. Cells were washed
by addition of cold sample buffer, centrifuged for two minutes at
2000 rpm, and the supernatants were discarded. This wash was
repeated once before proceeding to flow cytometry analysis. Samples
were analyzed by sequentially gating on the following populations:
FSC-A vs SSC-A, FSC-H vs FSC-A, SSC-A vs LIVE/DEAD, CD4 vs CD8, and
SSC vs CD25. Mean fluorescence intensity (MFI) of CD4+CD25+ T cells
or CD8+CD25+ T cells were calculated and exported to Excel for
analysis. GraphPad Prism was used to plot the data.
[0403] As shown in FIGS. 9C and 9D, the Fc variants BA005, BA006,
and BA007, and an afucosylated form of BA002 (BA002_AF), enhanced
CD4+ and CD8+ T cell activation to a substantially greater degree
than isotype controls. This enhancement was further increased when
these antibodies were combined with an anti-PD-1 antibody.
Anti-TIGIT Antibodies Show Dose-Dependent Enhancement of IL-2
Secretion by SEA-Stimulated PBMCs Alone and in Combination with an
Anti-PD-1 Antibody
[0404] In a further example, BA002 and several Fc variants thereof
(i.e., BA005, BA006, and BA002_AF) were each tested for their
ability to promote IL-2 secretion by SEA-stimulated PBMCs from
different donors at various antibody concentrations. In one
experiment, a dose titration was performed for antibodies BA002,
BA002_AF, BA005, BA006, and isotype control, each alone (FIG. 10A).
In a second experiment, a dose titration was performed for
antibodies BA002, BA002_AF, BA006, and isotype control, each in
combination with an anti-PD-1 antibody (FIG. 10B). In a third
experiment, a dose titration was performed for antibodies BA002,
BA006, and isotype control in PBMCs obtained from a third donor in
the presence of CD155-Fc, each antibody alone (FIG. 10C).
[0405] For each of the first two experiments described above in
this section, a 5.times. concentrated intermediate stock of
antibody sufficient for three replicates per donor was prepared in
1.2 mL bullet tubes. First, 400 .mu.L of 250 .mu.g/mL of each
antibody was prepared in R10 media. Antibodies were then serially
diluted 1-to-4 by pipetting 100 .mu.L of the previous dilution into
300 .mu.L of sample buffer. A total of 8 dilutions ranging from
50-0.003052 .mu.g/mL were prepared (see concentrations shown in
FIGS. 10A-10C). 20 .mu.l of each antibody mixture was then added
per well of a round-bottom 96-well plate. For the second experiment
(i.e., the combination of anti-TIGIT antibody and anti-PD-1
antibody), either anti-PD-1 antibody or an isotype IgG4 control
antibody were prepared as a 5.times. concentrated intermediate
stock. 20 .mu.l of anti-PD-1 antibody or isotype IgG4 control
mixture was then added per well to a round-bottom 96-well
plate.
[0406] Frozen aliquots of indicated human PBMCs were retrieved from
liquid nitrogen and immediately thawed in 37.degree. C. water.
Cells were transferred to 9 mL of pre-warmed R10 media and
centrifuged at 2000 rpm for two minutes. Cells were counted and
assessed for viability. Samples were centrifuged at 2000 rpm for
two minutes and resuspended. An intermediate stock concentration of
SEA was made by diluting 10 .mu.L of 1000 .mu.g/mL of SEA to 90
.mu.L of R10 to make an intermediate concentration of 100 .mu.g/mL.
To stimulate the cells, 50 .mu.L of a 100 .mu.g/mL intermediate
stock of SEA was added to the 30 mL of cells prepared above. 60
.mu.L of the cell and SEA mixture was added into corresponding
wells and incubated in a humidified chamber at 37.degree. C. and 5%
CO2 for four days. A total of 0.1.times.10.sup.6 cells/well and a
final concentration of 100 ng/mL of SEA was used.
[0407] After four days of incubation, the plates were removed from
incubator, gently agitated by hand, and centrifuged for two minutes
at 2000 rpm. 5 .mu.L of the supernatant was transferred to a
384-well AlphaLISA plate (Perkin Elmer) for cytokine analysis.
AlphaLISA kits were used for the measurements of IL-2 in accordance
with manufacturer instructions. Briefly, assay buffer was prepared
by pipetting 2.5 mL of 10.times. AlphaLISA Immunoassay Buffer to
22.5 mL water. Human IL-2 analyte was used to prepare a standard
dilution. A mixture of 1.6.times. AlphaLISA anti-IL-2 acceptor
beads and biotinylated antibody anti-IL-2 was prepared in assay
buffer. 8 .mu.L was added to each well and incubated in darkness at
room temperature, rotating at 500 rpm for 90 minutes. A 2.3.times.
Streptavidin donor bead intermediate stock was prepared in assay
buffer. 10 .mu.L were added to each well and incubated in darkness
at room temperature, rotating at 500 rpm for 20 minutes. AlphaLISA
plates were briefly centrifuged at 2000 rpm. Relative light units
(RLU) were measured using the AlphaScreen protocol on an EnVision
Plate Reader.
[0408] As shown in FIG. 10A, BA006 showed the highest enhancement
of IL-2 secretion by SEA-stimulated PBMCs when administered alone.
BA005, BA002_AF, and BA002 also showed enhancement of IL-2
secretion when administered alone. As shown in FIG. 10B, BA006,
BA002, and BA002 AF each also showed enhancement of IL-2 secretion
by SEA-stimulated PBMCs when combined with an anti-PD-1 antibody,
with BA006 inducing the strongest level of IL-2 secretion.
[0409] For the third experiment described above in this section,
the ability of BA006 and BA002 to enhance IL-2 secretion by PBMCs
in the presence of plate-coated CD155-Fc was assessed across a
range of antibody concentrations.
[0410] To prepare plates coated with CD155-Fc, 50 .mu.g of
recombinant human CD155-Fc protein was reconstituted in 100 .mu.L
PBS to make a stock concentration of 500 .mu.g/mL. The
reconstituted protein was then diluted to a working concentration
of 1 .mu.g/mL by adding 24 .mu.L of the 500 .mu.g/mL CD155-Fc stock
solution to 11.976 mL of PBS. A 96-well high-binding plate was then
coated with CD155-Fc protein by adding 100 .mu.L of the working
concentration of CD155-Fc protein solution to each well of the
96-well plate. The plate was then sealed with an adhesive and
incubated overnight at 4.degree. C. The next day, the plate was
centrifuged at 2000 rpm for two minutes. The supernatant was
discarded and antibodies were added as described below.
[0411] A 5.times. concentrated intermediate stock of antibody
sufficient for three replicates per donor was prepared in 1.2 mL
bullet tubes. First, 420 .mu.L of 500 .mu.g/mL of each antibody was
prepared in R10 media. Antibodies were then serially diluted 1-to-3
by transferring 140 .mu.L of the previous dilution into 280 .mu.L
of R10 media. A total of eight dilutions ranging from 100-0.045725
.mu.g/mL were prepared. 20 .mu.l of antibody mixture was then added
to corresponding wells of a round-bottom 96-well plate.
[0412] Frozen aliquots of human PBMCs were retrieved from liquid
nitrogen and immediately thawed in 37.degree. C. water. Cells were
transferred to 9 mL of pre-warmed R10 media and immediately
centrifuged at 2000 rpm for two minutes. Cells were then counted
and viability was assessed. Cells were centrifuged at 2000 rpm for
two minutes and resuspended. An intermediate stock concentration of
SEA was made by diluting 10 .mu.L of 1000 .mu.g/mL of SEA in 90
.mu.L of R10 to make an intermediate concentration of 100 .mu.g/mL.
To stimulate the cells, 12 of the 100 .mu.g/mL intermediate stock
of SEA was added to 9.60 mL of cells. 80 .mu.L of cells and SEA
mixture was added into corresponding wells and incubated in a
humidified chamber at 37.degree. C. and 5% CO2 for four days. A
total of 0.1.times.10.sup.6 cells/well and a final concentration of
100 ng/mL of SEA was used.
[0413] After four days of incubation, plates were removed from the
incubator, gently agitated by hand, and then centrifuged for two
minutes at 2000 rpm. 5 .mu.L of the supernatant was transferred to
a 384-well AlphaLISA plate (Perkin Elmer) for cytokine analysis.
AlphaLISA kits were used for the measurements of IL-2 in accordance
with manufacturer instructions. Briefly, assay buffer was prepared
by adding 2.5 mL of 10.times. AlphaLISA Immunoassay Buffer to 22.5
mL water. Human IL-2 analyte was used to prepare a standard
dilution in accordance with manufacturer instructions. A mixture of
1.6.times. AlphaLISA anti-IL-2 acceptor beads and biotinylated
antibody anti-IL-2 mix was prepared in assay buffer. 8 .mu.L was
added to each well and incubated in darkness at room temperature,
rotating at 500 rpm for 90 minutes. A 2.3.times. streptavidin donor
bead intermediate stock was prepared in assay buffer. 10 .mu.L was
added to each well and incubated in darkness at room temperature,
rotating at 500 rpm for 20 minutes. AlphaLISA plates were briefly
centrifuged at 2000 rpm. Relative light units (RLU) were measured
using the AlphaScreen protocol on an EnVision Plate Reader.
[0414] As shown in FIG. 10C, BA006 and BA002 enhanced IL-2
secretion in a dose-dependent manner from SEA-stimulated PBMCs
co-cultured with plate-bound CD155-Fc. BA006 enhanced IL-2
secretion to a greater degree than BA002, which in turn increased
IL-2 secretion relative to isotype control.
[0415] In further experiments, the activation of PBMCs by BA006 in
the presence of a lower concentration of SEA was tested. The
experiment was set up similarly as provided in section 6.2.1,
except that a 10 .mu.g/mL intermediate stock of SEA was used. The
final cell culture contained a total of 1.2.times.10.sup.5
cells/well and a final concentration of 10 ng/mL of SEA peptide.
After four days of incubation, IL-2 production from the cells was
measured by AlphaLISA kit (Perkin Elmer).
[0416] As shown in FIGS. 10D and 10E, BA006 enhanced IL-2 secretion
in a dose-dependent manner in PBMCs from two different donors in
the presence of 10 ng/mL SEA. The EC.sub.50 values measured from
these two experiments were 68 ng/mL and 56 ng/mL, respectively.
Thus, BA006 effectively increased the sensitivity of PBMCs to the
SEA antigen.
Fc Variants of Anti-TIGIT Antibody Stimulate IFN.gamma. Secretion
by Stimulated PBMCs
[0417] In this example, the capability of Fc variants of BA002 to
promote secretion of IFN.gamma. by PBMCs stimulated with SEA was
tested.
[0418] A 5.times. concentrated intermediate stock of each antibody
(i.e., BA002, BA002_AF, BA003, BA005, BA006, BA007, BA008, BA009,
or isotype controls for IgG1 and IgG4) was prepared in 1.2 mL
bullet tubes. First, 400 .mu.L of 50 .mu.g/mL of each antibody was
prepared in R10 media. 20 .mu.L of antibody was then added per well
to a round-bottom 96-well plate. Frozen aliquots of human PBMCs
were retrieved from liquid nitrogen and immediately thawed in
37.degree. C. water until floating ice was observed. Cells were
transferred to 9 mL of pre-warmed R10 media and immediately
centrifuged at 2000 rpm for two minutes. To count cells and check
viability, 10 of sample was removed and added to 390 .mu.L of
viability dye, mixed, and read using a Muse apparatus.
[0419] Samples were centrifuged at 2000 rpm for two minutes and
resuspended to an intermediate concentration. An intermediate stock
concentration of SEA was made by adding 10 of 1000 .mu.g/mL SEA to
90 .mu.L R10 to make an intermediate concentration of 100 .mu.g/mL.
To stimulate the cells, 12 .mu.L of a 100 .mu.g/mL intermediate
stock of SEA was added to the 7.20 mL of cells prepared above. 60
.mu.L of cells and SEA mixture was added into corresponding wells
and incubated in tissue culture incubator at 37.degree. C. and 5%
CO.sub.2 within a humidified chamber for four days. A total of
0.1.times.10.sup.6 cells/well and final concentration of 100 ng/mL
of SEA was used.
[0420] After four days of incubation, plates were removed from the
incubator and gently agitated by hand. The plates were then
centrifuged for two minutes at 2000 rpm. 5 .mu.L of supernatant was
transferred to a 384-well AlphaLISA plate for cytokine analysis.
AlphaLISA kits (Perkin Elmer) were used for measurement of
IFN.gamma. secretion. Briefly, assay buffer was prepared by
pipetting 2.5 mL of 10.times. AlphaLISA HiBlock Buffer to 22.5 mL
water. Human IFN.gamma. analyte was used to prepare a standard
dilution according to manufacturer instructions. A mixture of
1.6.times. AlphaLISA anti-IFN.gamma. acceptor beads and
biotinylated anti-IFN.gamma. antibody was prepared in assay buffer.
8 .mu.L was added to each well and incubated in darkness at room
temperature, rotating at 500 rpm for 90 minutes. A 2.3.times.
Streptavidin Donor Bead intermediate stock was prepared in assay
buffer. 10 .mu.L were added to each well and incubated in darkness
at room temperature, rotating at 500 rpm for 20 minutes. AlphaLISA
plates were briefly centrifuged at 2000 rpm. Relative light units
(RLU) were measured using the AlphaScreen protocol on an EnVision
Plate Reader.
[0421] As shown in FIGS. 11A-11B, BA002 and its Fc variants
enhanced IFN.gamma. secretion by SEA-stimulated PBMCs from two
different donors.
6.3.2 Anti-TIGIT Antibody Fc Variants Showed Varying Capability to
Signal Through Fc.gamma.RIIA and Fc.gamma.RIIIA
Fc.gamma.RIIA Signaling
[0422] In one example, the capacity of BA002 Fc variants to
activate reporter cells expressing Fc.gamma.RIIA.sup.H131 was
tested. Briefly, target cells (i.e., Jurkat cells engineered to
express human TIGIT) were added to the wells of an ADCP assay plate
(2.4.times.10.sup.6 cells/mL). Serial dilutions of antibody (i.e.,
anti-TIGIT antibody BA002 or Fc variants thereof, or appropriate
isotype controls (Evitria); one antibody per well) were added to
the assay plate wells with ADCP assay buffer. 150,000 effector
cells (i.e., Jurkat NFAT-luciferase reporter cells overexpressing
the Fc.gamma.RIIA CD32A with a high affinity 131 H/H polymorphism,
less than six weeks in culture; Promega) were added to each well,
and the mixtures were then incubated for 20 hours at 37.degree. C.
Binding of antibody/antigen complex on target cell surfaces to
CD32A on effector cell surfaces would result in signaling to the
reporter construct and expression of luciferase.
[0423] The next day, plates were equilibrated to room temperature
for 15 minutes and then 75 .mu.L of Bio-Glo Luciferase Assay
Reagent (Promega Catalog #G7940) was added per well. The mixtures
were then incubated at room temperature for 5-10 minutes, and
luminescence was measured using a plate reader (Envision). Relative
Light Units (RLU) were calculated as the induced RLU-background
RLU.
[0424] As shown in FIG. 12A, for Fc.gamma.RIIA binding and
signaling, BA005 exhibited the highest level of signaling followed
in order by BA008, BA006, BA007, BA002, and BA002_AF. BA003 and
isotype controls showed substantially no signaling.
Fc.gamma.RIIIA Signaling
[0425] In another example, the capacity of BA002 Fc variants to
activate reporter cells expressing Fc.gamma.RIIIA.sup.V158 was
tested. Briefly, target cells (i.e., Jurkat cells engineered to
express human TIGIT) were added to the wells of an ADCC assay plate
(2.4.times.10.sup.6 cells/mL). Serial dilutions of antibody (i.e.,
anti-TIGIT antibody BA002 or Fc variants thereof, or appropriate
isotype controls (Evitria); one antibody per well) were added to
the assay plate wells with ADCC assay buffer. 150,000 effector
cells (i.e., Jurkat NFAT-luciferase reporter cells overexpressing
the Fc.gamma.RIIIA CD16A with a high affinity 158 V/V polymorphism,
less than six weeks in culture; Promega) were added to each well,
and the mixtures were then incubated for 20 hours at 37.degree. C.
Binding of antibody/antigen complex on target cell surfaces to
CD16A on effector cell surfaces would result in signaling to the
reporter construct and expression of luciferase.
[0426] The next day, plates were equilibrated to room temperature
for 15 minutes and then 75 .mu.L of Bio-Glo Luciferase Assay
Reagent (Promega Catalog #G7940) was added per well. The mixtures
were then incubated at room temperature for 5-10 minutes, and
luminescence was measured using a plate reader (Envision). RLU was
calculated as the induced RLU-background RLU.
[0427] As shown in FIG. 12B, for Fc.gamma.RIIIA binding and
signaling, BA006 exhibited the highest level of signaling, followed
in order by BA002_AF, BA005, BA007, and BA002. BA003 and isotype
controls showed substantially no signaling.
6.3.3 Fc Variants of BA002 Enhanced Killing of TIGIT.sup.+ Jurkat
Cells in Co-Culture with CD16+NK Cells
[0428] Fc variants of BA002 were examined for their capacity to
induce antibody-dependent cell-mediated cytotoxicity (ADCC)
activity in a co-culture of TIGIT-expressing Jurkat cells and
CD16-expressing natural killer (NK) cells. Briefly, Jurkat cells
were cultured in RPMI 1640 (Corning Catalog #10-040-CM, Lot
35316005) supplemented with 10% fetal bovine serum (Benchmark
Catalog #100-106, Lot A69E00F) and 1% Pen Strep Glutamine (Gibco
Catalog #10378-016, Lot 1835954). NK cells were cultured in NK MACS
Basal Medium (MACS Catalog #130-107-209) supplemented with 2% NK
MACS Medium Supplement (MACS Catalog #130-107-210, Lot 5160804070),
5% human serum (Sigma Catalog #H4522, Lot SLBQ9160V), 1% Pen Strep
Glutamine (Gibco Catalog #10378-016, Lot 1835954), 100 Units/mL
IL-2 (R&D Systems Catalog #202-16, Lot AE6016102), and 100
Units/mL IL-15 (R&D Systems Catalog #247-ILB, Lot TLM1016102).
Two million Jurkat cells were pelleted by centrifugation for 5
minutes at 1200 rpm. The cells were stained by resuspending the
pellet in 1 mL of 0.5 .mu.M CellTrace Far Red (Invitrogen Catalog
#C34565, Lot 1764050) in PBS (Corning Catalog #21-040-CV, Lot
00217005) and incubating for 30 minutes at 37.degree. C. and 5%
CO2. After incubation, 9 mL of PBS was added and the cells were
pelleted by centrifugation for 5 minutes at 1200 rpm. The cell
pellet was then resuspended in Jurkat culture media. Antibodies
were diluted in Jurkat culture media containing 1 .mu.M CellEvent
Caspase-3/7 Green Detection Reagent (Invitrogen Catalog #C10423,
Lot 1849709) at six times their final concentration. Stained Jurkat
cells were diluted to 0.5 million cells per mL and NK cells to 0.75
million cells per mL. The assay was performed in 384-well
microscopy plates (Greiner, Cat. No. 781936, Lot E161233K) by
pipetting 10 .mu.L of the antibodies (final concentrations: 0.1, 1,
and 10 .mu.g/mL), 30 .mu.L stained Jurkat cells (15000 cells), and
20 .mu.L NK cells (15000 cells) per well.
[0429] Live imaging was performed immediately afterward, using an
ImageXpress Micro Confocal High-Content microscope (Molecular
Devices) under environmental control (37.degree. C., 5% CO.sub.2)
and images were acquired every hour from the Cy5 (CellTrace Far
Red) and FITC (Caspase 3/7) channels for Jurkat cells and Caspase
3/7-positive Jurkat cells, respectively, over the course of three
hours. Image analysis was performed using the MetaXpress analysis
software (Molecular Devices). Jurkat cells were identified from the
Cy5 channel and the amount of Caspase 3/7 signal was quantified per
cell from the FITC channel. Cells with Caspase 3/7 intensity above
the background were designated as apoptotic. The number of
apoptotic cells was normalized against the total cell count per
condition to determine a percent killing measurement.
[0430] As shown in FIG. 13A, Fc variants that exhibited improved
binding to Fc.gamma.RIIIA (BA006, BA007, BA005, and BA002_AF)
promoted killing of TIGIT-expressing Jurkat cells to a greater
degree than BA002, which in turn promoted killing of
TIGIT-expressing Jurkat cells to a greater degree than BA003, which
contains the "Fc-silent" N297A mutation, and isotype control.
6.3.4 BA002 and BA006 Preferentially Kill Regulatory T Cells as
Compared to Effector T Cells
[0431] In one example, BA002 and BA006 were examined for their
capacity to induce ADCC in primary regulatory T cells (Treg) and
effector T cells (Teff). Briefly, antibody BA002, antibody BA006,
and an IgG1 isotype control antibody were examined for ADCC
activity in a co-culture of CD16-expressing NK cells and either (i)
primary effector T cells or (ii) primary regulatory T cells.
Primary T cells were isolated from PBMCs and expanded over 10 days
according to methods known in the art. The identity of the T
effector cells and T regulatory cells was confirmed by flow
cytometric analysis of appropriate markers. Before the ADCC assay,
T effector cells and T regulatory cells were either rested in
X-VIVO 15 media (Lonza Catalog #04-418Q, Lot 0000542070)
supplemented with 50 Units/mL IL-2 (R&D Systems Catalog
#202-16, Lot AE6016102), or stimulated in X-VIVO 15 media
supplemented with 50 Units/mL IL-2 and 25 .mu.L per mL CD3/CD28 T
cell activator (Stemcell Catalog #10971, Lot 16L75402), for 16
hours. NK cells were cultured in NK MACS Basal Medium (MACS Catalog
#130-107-209) supplemented with 2% NK MACS Medium Supplement (MACS
Catalog #130-107-210, Lot 5160804070), 5% human serum (Sigma
Catalog #H4522, Lot SLBQ9160V), 1% Pen Strep Glutamine (Gibco
Catalog #10378-016, Lot 1835954), 100 Units/mL IL-2 (R&D
Systems Catalog #202-16, Lot AE6016102), and 100 Units/mL IL-15
(R&D Systems Catalog #247-ILB, Lot TLM1016102). T cells were
pelleted by centrifugation for 5 minutes at 1200 rpm. The cells
were stained by resuspending the pellet in 1 mL of 0.5 .mu.M
CellTrace Far Red (Invitrogen Catalog #C34565, Lot 1764050) in PBS
(Corning Catalog #21-040-CV, Lot 00217005) and incubated for 30
minutes at 37.degree. C. and 5% CO2. After incubation, 9 mL of PBS
was added and the cells were pelleted by centrifugation for 5
minutes at 1200 rpm. The cell pellet was resuspended in X-VIVO 15
media. Antibodies were diluted in X-VIVO 15 media containing 1
.mu.M CellEvent Caspase-3/7 Green Detection Reagent (Invitrogen
Catalog #C10423, Lot 1849709) at six times their final
concentration. Stained T cells were diluted to 0.5 million cells
per mL, and NK cells to 0.75 million cells per ml. The assay was
performed in 384-well microscopy plates (Greiner Catalog #781936,
Lot E161233K) by pipetting 10 .mu.l of the antibodies (final
concentrations: 1, and 10 .mu.g/mL), 30 .mu.L stained Jurkat cells
(15,000 cells), and 20 .mu.L NK cells (15,000 cells) per well.
[0432] Live imaging was performed immediately afterward using an
ImageXpress Micro Confocal High-Content microscope (Molecular
Devices) under environmental control (37.degree. C., 5% CO2) and
images were acquired every hour from the Cy5 (CellTrace Far Red)
and FITC (Caspase 3/7) channels for T cells and Caspase
3/7-positive T cells, respectively, over the course of three hours.
Image analysis was performed using the MetaXpress analysis software
(Molecular Devices). T cells were identified from the Cy5 channel
and the amount of Caspase 3/7 signal was quantified per cell from
the FITC channel. Cells with Caspase 3/7 intensity above the
background were designated as apoptotic. The number of apoptotic
cells was normalized against the total cell count per condition to
determine a percent killing measurement.
[0433] As shown in FIG. 13B, both the anti-TIGIT antibody BA002 and
its Fc variant, BA006, preferentially killed regulatory T cells as
compared to effector T cells at antibody concentrations of 1
.mu.g/mL and 10 .mu.g/mL. BA006 generally exhibited higher levels
of T cell killing, and preferential regulatory T cell killing, than
did BA002.
[0434] Without wishing to be bound by any particular mechanism or
theory, it is contemplated that BA002 blocks the interaction
between TIGIT and PVR, thereby inhibiting TIGIT-mediated T cell and
NK cell inhibitory mechanisms and promoting CD226-mediated
co-stimulatory signaling. This may result in enhancement of T cell
effector function and TH1 cytokine secretion. It is also
contemplated that BA006 further enhances binding and signaling
through Fc.gamma.RIIIA and thereby promotes stronger interactions
between the T cell and APC. This in turn may enhance T cell
signaling while at the same time maintaining potent antagonism of
TIGIT. Thus, it is contemplated that by strengthening the immune
synapse between the T cell and APC, BA006 may be able to further
enhance T cell effector function and cytokine secretion.
6.4 Example 4: Characterization of an Fc Variant Anti-Human TIGIT
Antibody
[0435] This example describes further characterization of
BA006.
6.4.1 BA006 Promotes Secretion of IL-2 by SEA-Stimulated PBMCs from
a Human Donor
[0436] BA006 was tested for its ability to promote secretion of
IL-2 by SEA-stimulated PBMCs from a human donor.
[0437] A 5.times. concentrated intermediate stock of antibody
BA006, antibody BA002, or isotype control antibodies for BA002 was
prepared in 1.2 mL bullet tubes. Intermediate stocks of a panel of
reference anti-TIGIT antibodies and an isotype control antibody for
BA006 were also prepared. First, 400 .mu.L of 50 .mu.g/mL of each
antibody was prepared in R10 media. 20 .mu.L of antibody was then
added per well to a round-bottom 96-well plate. Frozen aliquots of
human PBMCs were retrieved from liquid nitrogen and immediately
thawed in 37.degree. C. water until floating ice was observed.
Cells were transferred to 9 mL of pre-warmed R10 media and
immediately centrifuged at 2000 rpm for two minutes. Cells were
counted and checked for viability.
[0438] Samples were centrifuged at 2000 rpm for two minutes and
resuspended to an intermediate concentration. An intermediate stock
concentration of SEA was made by adding 10 .mu.L of 1000 .mu.g/mL
SEA to 90 .mu.L R10 to make an intermediate concentration of 100
.mu.g/mL. To stimulate the cells, 12 .mu.L of a 100 .mu.g/mL
intermediate stock of SEA was added to the 7.20 mL of cells
prepared above. 60 .mu.L of cells and SEA mixture was added into
corresponding wells and incubated in tissue culture incubator at
37.degree. C. and 5% CO2 within a humidified chamber for four days.
A total of 0.1.times.10.sup.6 cells/well and final concentration of
100 ng/mL of SEA was used.
[0439] After four days of incubation, plates were removed from the
incubator and gently agitated by hand. The plates were then
centrifuged for two minutes at 2000 rpm. 5 .mu.L of supernatant was
transferred to a 384-well AlphaLISA plate for cytokine analysis.
AlphaLISA kits (Perkin Elmer) were used for measurement of IL-2
secretion. Briefly, assay buffer was prepared by pipetting 2.5 mL
of 10.times. AlphaLISA Immunoassay Buffer to 22.5 mL water. Human
IL-2 analyte was used to prepare a standard dilution. A mixture of
1.6.times. AlphaLISA anti-IL-2 acceptor beads and biotinylated
anti-IL-2 antibody was prepared in assay buffer. 8 .mu.L was added
to each well and incubated in darkness at room temperature,
rotating at 500 rpm for 90 minutes. A 2.3.times. Streptavidin Donor
Bead intermediate stock was prepared in assay buffer. 10 .mu.L was
added to each well and incubated in darkness at room temperature,
rotating at 500 rpm for 20 minutes. AlphaLISA plates were briefly
centrifuged at 2000 rpm. Relative light units (RLU) were measured
using the AlphaScreen protocol on an EnVision Plate Reader. This
experiment was run for four replicates using PBMCs obtained from
two different donors.
[0440] As shown in FIG. 14, the anti-TIGIT antibodies BA002 and
BA006 each enhanced IL-2 secretion by SEA-stimulated PBMCs,
compared to isotype controls and reference antibodies, with BA006
inducing substantially greater IL-2 secretion compared to the other
anti-TIGIT antibodies tested.
6.4.2 Combination of Anti-TIGIT Antibodies with Antibodies that
Modulate Other Immune Checkpoint Molecules
[0441] In this example, BA002 and its Fc variant, BA006, were
tested for their capacity to promote IL-2 secretion by
SEA-stimulated PBMCs when administered alone or in combination with
antibodies targeting various immune checkpoint molecules
(anti-PD-1, anti-PD-L1, anti-CTLA-4, and anti-LAG-3 antagonist
antibodies and anti-CD137 and anti-OX40 agonist antibodies).
[0442] A 5.times. concentrated intermediate stock of each antibody
sufficient for eight replicates for two donors was prepared in 1.2
mL bullet tubes. First, 600 .mu.L of 50 .mu.g/mL of each antibody
was prepared in R10 media. For samples that would receive a
combination of two antibodies (i.e., pairwise combinations between
(i) either BA002 or BA006, and (ii) either an anti-PD-1 antagonist
antibody, anti-PD-L1 antagonist antibody, anti-CD137 agonist
antibody, or anti-OX40 agonist antibody), both antibodies were
prepared in the same 1.2 mL bullet tube. 20 .mu.l of antibody
mixture was then added per well to a round-bottom 96-well plate to
reach final concentrations of 10 .mu.g/mL BA002 or BA006 in
combination with 5 .mu.g/mL anti-PD-1 antibody, anti-PD-L1
antibody, or anti-OX40 antibody, or 5 .mu.g/mL BA002 or BA006 in
combination with 10 .mu.g/mL anti-CTLA-4 antibody, anti-LAG-3
antibody, or anti-CD137 antibody.
[0443] Frozen aliquots of human PBMCs were retrieved from liquid
nitrogen and immediately thawed in 37.degree. C. water. Cells were
transferred to 9 mL of pre-warmed R10 media and immediately
centrifuged at 2000 rpm for two minutes. Cells were counted and
assessed for viability. Samples were centrifuged at 2000 rpm for
two minutes and resuspended to an intermediate concentration. An
intermediate stock concentration of SEA was made by diluting 10
.mu.L of 1000 .mu.g/mL of SEA in 90 .mu.L of R10 to make an
intermediate concentration of 100 .mu.g/mL. To stimulate the cells,
40 .mu.L of the 100 .mu.g/mL intermediate stock of SEA was added to
the 32 mL of cells prepared as described above. 80 .mu.L of cells
and SEA mixture was added into corresponding wells and incubated in
a humidified chamber at 37.degree. C. and 5% CO2 for four days. A
total of 0.1.times.10.sup.6 cells/well and a final concentration of
100 ng/mL of SEA was used.
[0444] After four days of incubation, the plates were removed from
the incubator and gently agitated by hand. The plates were then
centrifuged for two minutes at 2000 rpm. 5 .mu.L of the supernatant
was added to a 384-well AlphaLISA plate (Perkin Elmer) for cytokine
analysis. AlphaLISA kits were used for the measurements of IL-2 in
accordance with manufacturer instructions. Briefly, assay buffer
was prepared by pipetting 2.5 mL of 10.times. AlphaLISA Immunoassay
Buffer to 22.5 mL water. Human IL-2 analyte was used to prepare a
standard dilution. A 1.6.times. AlphaLISA anti-IL-2 Acceptor
beads+biotinylated antibody anti-IL-2 mix was prepared in assay
buffer. 8 .mu.L were added to each well and incubated in darkness
at room temperature, rotating at 500 rpm for 90 minutes. A
2.3.times. Streptavidin Donor Bead intermediate stock was prepared
in assay buffer. 10 .mu.L was added to each well and incubated in
darkness at room temperature, rotating at 500 rpm for 20 minutes.
AlphaLISA plates were briefly centrifuged at 2000 rpm. Relative
light units (RLU) were then measured using the AlphaScreen protocol
on an EnVision Plate Reader.
[0445] As shown in FIGS. 15A-15I, the anti-TIGIT antibodies BA002
and BA006 enhanced IL-2 secretion by SEA-stimulated PBMCs when
provided alone. IL-2 secretion was further enhanced when antibody
BA002 or BA006 was administered in combination with an anti-PD-1
antagonist antibody (FIG. 15A), either one of two anti-PD-L1
antagonist antibodies (FIGS. 15B-15C), an anti-CD137 agonist
antibody (FIG. 15D), an anti-CTLA-4 antagonistic antibody (FIG.
15E), an anti-LAG3 antagonistic antibody tested with cells from two
different donors (FIGS. 15F and 15G), or an anti-OX40 agonistic
antibody tested with cells from two different donors (FIGS. 15H and
15I).
[0446] The abilities of BA002 and BA006 to activate cynomolgus
PBMCs were examined by a similar method. Briefly, primary
cynomolgus monkey PBMCs from donors 12 and 13 were stimulated with
100 ng/mL of staphylococcal enterotoxin A (SEA) superantigen in the
presence of 10 .mu.g/mL of BA002 or BA006, and 10 .mu.g/mL of an
anti-PD-1 antibody or an isotype control antibody for 4 days. The
amounts of IL-2 and IFN.gamma. in the culture supernatants were
measured using AlphaLISA kits.
[0447] As shown in FIG. 16A, BA002 and BA006 enhanced IL-2
secretion from the cynomolgus PBMCs either alone or in combination
with the anti-PD-1 antibody. Similarly, as shown in FIG. 16B, BA002
and BA006 enhanced IFN.gamma. secretion from the cynomolgus PBMCs
either alone or in combination with the anti-PD-1 antibody.
6.4.3 Anti-TIGIT Antibodies Enhance T Cell Memory Recall
[0448] In this example, the functions of BA002 and BA006 were
tested in type I and type II T cell memory recall assays.
Type I T Cell Memory Recall
[0449] In the type I T cell memory recall assay, frozen aliquots of
primary cytomegalovirus (CMV)-reactive HLA-A*02:01 PBMCs from a
human donor were retrieved from liquid nitrogen and immediately
thawed in 37.degree. C. water until floating ice was observed.
Cells were transferred to 9 mL of pre-warmed X-Vivo 15 media and
immediately centrifuged at 1500 rpm, 5 min. Cells were then
re-suspended in 10 mL of pre-warmed R10 media. To count cells and
check the viability, 20 .mu.L of sample was removed and added to
380 .mu.L of viability dye, mixed and read using a Muse apparatus.
Cells were then re-suspended to a 2.times. intermediate
concentration and total volume of 10 mL.
[0450] The primary PBMCs were stimulated with a final concentration
of 1.75 .mu.g/mL of CMV pp65 peptide (NLVPMVATV; SEQ ID NO: 61),
and treated with 10 .mu.g/mL of BA002, BA006, anti-TIGIT reference
antibody #7, or an isotype control antibody. Specifically, 35 .mu.L
of a 1000 .mu.g/mL stock of CMVpp65 peptide was added to the 10 mL
of cells prepared above. The cells were gently mixed by inverting,
and 100 .mu.L of the mixture were pipetted into corresponding
wells. The anti-TIGIT antibodies were similarly prepared into
2.times. intermediate stocks, and 100 .mu.L of each antibody was
added to the wells. The cells at a final density of
2.5.times.10.sup.5 cells/well were incubated in tissue culture
incubator at 37.degree. C. and 5% CO.sub.2 within a humidified
chamber.
[0451] Fresh CMVpp65 peptide and anti-TIGIT antibodies were added
daily for 5 days. Specifically, the cells were centrifuged at 1500
rpm, 2 min, 20 .mu.L of supernatant was removed, and 10 .mu.L of
CMV pp65 peptide and 10 .mu.L of an anti-TIGIT antibody were added
to the cells. The final concentrations of the CMV pp65 peptide and
anti-TIGIT antibody were 1.75 .mu.g/mL and 10 .mu.g/mL,
respectively. IFN.gamma. secretion was assessed daily for six days
by AlphaLISA kit (Perkin Elmer) according to the manufacturer's
protocol.
[0452] As shown in FIG. 17A, BA002 and BA006 both induced
increasing IFN.gamma. secretion over time in the type I memory
recall assay relative to reference antibody #7 or isotype control,
and BA006 induced greater levels of IFN.gamma. than BA002.
[0453] To characterize the expression of TIGIT on memory T cells,
CMV-reactive HLA-A*02:01 PBMCs were stimulated with 1.75 .mu.g/mL
CMV pp65 peptide for 5 days as described above. CD8 effector memory
T cells were enriched by sequentially gating on the FSC-A vs.
SSC-A, FSC-H vs FSC-A, SSC-A vs SSC-H, CD3 vs SSC-A, CD4 vs. CD8,
and CD45RO vs. CD197. CD8 effector memory T cells were identified
as CD8.sup.+ CD4.sup.- CD45RO.sup.+ CD197.sup.-. The expression
levels of TIGIT, CD226, and CD96 on CD8 effector memory T cells
were determined by flow cytometry.
[0454] As shown in FIG. 17B, TIGIT, CD226, and CD96 were all
expressed on the CD8 effector memory T cells. This result suggested
that an anti-TIGIT antibody could have a direct effect on CD8
effector memory T cells.
[0455] To further characterize the function of the anti-TIGIT
antibodies in T cell memory recall, CMV-reactive HLA-A*02:01 PBMCs
were stimulated with 1.75 .mu.g/mL CMV pp65 peptide in the presence
of 10 .mu.g/mL BA002, BA006, anti-TIGIT reference antibody #7,
and/or an anti-PD-1 antibody for 5 days as described above. The
secretion of IFN.gamma. and TNF.alpha. was assessed by AlphaLISA
kits (Perkin Elmer) according to the manufacturer's protocol. In a
similar experiment, CMV-reactive HLA-A*02:01 PBMCs were stimulated
with 1.75 .mu.g/mL CMV pp65 peptide in the presence of 10 .mu.g/mL
BA002, BA006, anti-TIGIT reference antibody #7, and/or an anti-PD-1
antibody for 6 days as described above. CD8 effector memory T cells
were identified as CD8.sup.+ CD45RO.sup.+ CD197.sup.-, and CD4
effector memory T cells were identified as CD4.sup.+ CD45RO.sup.+
CD197.sup.-, and T cell proliferation was assessed by Ki67
expression by flow cytometry.
[0456] As shown in FIGS. 17C and 17D, BA002 and BA006 both enhanced
IFN.gamma. and TNF.alpha. secretion in the type I memory recall
assay, and BA006 was more potent than BA002. Addition of the
anti-PD-1 antibody further increased IFN.gamma. and TNF.alpha.
secretion. BA002 and BA006 also enhanced CD8 effector memory T cell
proliferation in the type I memory recall assay, and BA006 was more
potent than BA002 (FIG. 17E). Addition of the anti-PD-1 antibody
did not substantially increase the CD8 effector memory T cell
proliferation in the BA006 treatment group. The proliferation of
CD4 effector memory T cells was not as substantially affected by
BA002, BA006, or the anti-PD-1 antibody (FIG. 17F).
Type II T Cell Memory Recall
[0457] In the type II T cell memory recall assay, 1 .mu.g/mL of CMV
whole antigen (Astarte Biologics, Cat #1004), known to be primarily
processed and presented on MHC class II molecules (though these
antigens may also be cross-presented on MHC class I molecules),
were used for stimulating CMV-reactive PBMCs. Specifically, 200
.mu.L of a 100 .mu.g/mL stock of CMV whole antigen was added to 10
mL of donor PBMCs in R10 medium. The cells were gently mixed by
inverting, and 100 .mu.L of the mixture was pipetted into
corresponding wells. 10 .mu.g/mL of an anti-PD-1 antibody was added
to some of the wells. The cells were incubated in tissue culture
incubator at 37.degree. C. and 5% CO2 within a humidified chamber
for 4 days at a density of 220,000 cells/well (Donor 11) or 250,000
cells/well (Donor 10). The cell samples were analyzed by
sequentially gating on FSC-A vs. SSC-A, FSC-H vs FSC-A, SSC-A vs
SSC-H, CD3 vs SSC-A, and CD4 vs CD8. The CD4.sup.+ T cell subset
was identified as CD4.sup.+ CD8.sup.-, and the CD8.sup.+ T cell
subset was identified as CD8.sup.+ CD4.sup.-. Within each subset,
naive T cells, effector T cells (T.sub.Eff), effector memory T
cells (T.sub.EM), and central memory T cells (T.sub.CM) were
identified as CD45RO.sup.- CD197.sup.+, CD45RO.sup.- CD197.sup.-,
CD45RO.sup.+ CD197.sup.-, and CD45RO.sup.+ CD197.sup.+,
respectively. Each subset was analyzed for its expression of TIGIT
as detected by an APC-conjugated anti-TIGIT antibody.
[0458] As shown in FIG. 18A, the CMV whole antigen increased the
expression level of TIGIT on CD4.sup.+ T.sub.EFF, T.sub.EM, and
T.sub.CM cells, and the anti-PD-1 antibody further enhanced TIGIT
expression on T.sub.EM and T.sub.CM cells. Increased TIGIT
expression was also observed on CD8.sup.+ T.sub.EFF, T.sub.EM, and
T.sub.CM cells (FIG. 18B), likely due to the cross-presentation of
the antigens on MHC class I molecules.
[0459] To further characterize the function of anti-TIGIT
antibodies in type II T cell memory recall, CMV-reactive PBMCs were
incubated with 1 .mu.g/mL of CMV whole antigen in the presence or
absence of 10 .mu.g/mL of BA002, BA006, anti-TIGIT reference
antibody #7, and/or an anti-PD-1 antibody for 4 days. IFN.gamma.
secretion in the culture medium was analyzed by AlphaLISA kits
(Perkin Elmer) according to the manufacturer's protocol.
[0460] As shown in FIGS. 18C and 18D, BA002 and BA006, when
combined with the anti-PD-1 antibody, both enhanced IFN.gamma.
secretion from the PBMCs, and BA006 was more potent than BA002.
6.4.4 Anti-TIGIT Antibodies Enhance Antigen-Specific T Cell
Cytotoxicity
[0461] In this example, the effects of BA002 and BA006 on T cell
cytotoxicity were tested. Specifically, primary human T cells
ectopically expressing an NY-ESO-1 TCR were co-cultured with
NY-ESO-1 expressing U251MG tumor cells for 13 days to model T cell
exhaustion. For live imaging, KARPAS 299 cells ectopically
expressing NY-ESO-1 were first incubated with 1 .mu.M CellTrace Far
Red Cell Proliferation Dye (Life Technologies) in PBS for 30
minutes at 37.degree. C. and 5% CO.sub.2 to label the cell bodies.
The labeled cells were resuspended in fresh culture media and
seeded at a density of 15,000 cells per well in a 384-well
microscopy plate. The exhausted T cells were then added at a
density of 30,000 cells per well. BA002, BA006, or a corresponding
isotype control antibody was added to the co-culture at the
concentration of 10 .mu.g/mL in combination with 10 .mu.g/mL of an
anti-PD-1 antibody or an isotype control antibody.
[0462] Live images were collected using an ImageXpress Micro
Confocal High-Content microscope (Molecular Devices) at 37.degree.
C. and 5% CO2 in the Cy5 channel (CellTrace Far Red Cell
Proliferation Dye) every two hours over a course of 24 hours. In
total, for each condition at each time point, eight images
(20.times. magnification) were acquired with an average of 1,211
cells (.+-.88 cells, standard deviation). Image analysis to
quantify the amount of killed KARPAS 299 cells was performed using
MetaXpress analysis software (Molecular Devices).
[0463] As shown in FIG. 19, BA002 and BA006, either alone or in
combination with the anti-PD-1 antibody, enhanced the cytotoxicity
of the T cells against the antigen-expressing tumor cells.
6.4.5 Anti-TIGIT Antibodies Enhance NK Cell Activity
[0464] In this example, the effects of BA002 and BA006 on NK cell
activation were studied.
[0465] Briefly, freshly thawed PBMCs were cultured in RPMI medium
supplemented with 10% fetal bovine serum and 100UI of IL-2 and
IL-15. The cells were treated with 20 .mu.g/mL of BA002, BA006,
reference antibody #1 (human IgG1), reference antibody #1
Fc-enhanced variant (human IgG1 with S239D/A330L/I332E
substitutions in the Fc region), or a corresponding isotype control
antibody for 5 hours. K562 cells were optionally added as target
cells for co-culture at the amount of 10% of the PBMCs.
[0466] To stain the NK cell activation marker CD107a, an
anti-CD107a antibody conjugated with APC (Biolegend) was added to
the cell culture at a 1:400 dilution. Monensin (eBiosciences) was
also added to prevent acidification of endocytic vesicles, thereby
avoiding degradation of CD107a that was re-internalized from the
cell surface. Additionally, Brefeldin A (eBiosciences) was added to
the cell culture to prevent exocytosis of cytokine-containing
vesicles, thereby allowing visualization of cytokine production
following stimulation.
[0467] Following the treatment, the PBMCs were stained for cell
surface markers for 30 min using an anti-CD56 antibody conjugated
with BUV737 (BD Biosciences) and an anti-CD3 antibody conjugated
with BV421 (Biolegend). After washing, the cells were incubated in
BD Cytofix/Cytoperm solution for 20 min at 4.degree. C. for
fixation and permeabilization. The cells were then washed twice and
incubated for 30 min at 4.degree. C. with an anti-IFN.gamma.
antibody conjugated with AlexaFluor700 and an anti-TNF.alpha.
antibody conjugated with PECy7 (BD Biosciences) in BD Perm/Wash
solution. The stained cells were analyzed by flow cytometry.
[0468] As shown in FIG. 20A, the lymphocyte population was
identified by a first plot gating on forward scatter-Area (FSC-A)
versus side scatter Area (SSC-A), and a second plot gating on FSC-A
versus FSC-Height (FSC-H) for selection of single cells. The NK
cells were further identified from the lymphocyte population as
CD3.sup.- CD56.sup.+. The activated NK cells were identified as
CD107a.sup.+.
[0469] As shown in FIG. 20B, the anti-TIGIT antibodies enhanced the
activation marker of CD107a on the NK cells from PBMCs. The
anti-TIGIT antibodies also increased the production of IFN.gamma.
(FIG. 20C) and TNF.alpha. (FIG. 20D) in the NK cells. Similar
effects were observed with the NK cells in PBMCs co-cultured with
K562 target cells (FIGS. 20E-20G). BA006 showed more potent effects
on NK cell activation than BA002. Similarly, the reference antibody
#1 variant comprising S239D/A330L/I332E substitutions in the Fc
region was more potent in NK cell activation than reference
antibody #1 comprising a wild type IgG1 Fc region.
6.5 Example 5: Epitope Mapping
[0470] The epitopes of BA002 and BA006 were studied by
hydrogen-deuterium exchange (HDX) mass spectrometry and antigen
mutagenesis.
6.5.1 Epitope Mapping of Anti-TIGIT Antibody by HDX
[0471] The interaction of TIGIT with the F(ab').sub.2 fragment of
BA002 (BA002-F(ab').sub.2) was evaluated using the methods
described below.
TIGIT Interaction with Anti-Human TIGIT F(ab).sub.2
[0472] 10 .mu.L human TIGIT (6.16 .mu.g) or 20 .mu.L human TIGIT
and F(ab').sub.2 mixture (6.16 .mu.g: 30.8 .mu.g) was incubated
with 110 .mu.L deuterium oxide labeling buffer (50 mM sodium
phosphate, 100 mM sodium chloride at pD 7.4) for 0 sec, 60 sec, 300
sec, 1800 sec, 7200 sec and 14400 sec at 24.degree. C.
Hydrogen/deuterium exchange was quenched by adding 125 .mu.L of 4 M
guanidine hydrochloride, 0.85 M TCEP buffer (final pH 2.5).
Subsequently, the quenched samples were subjected to on column
pepsin/protease XIII digestion and LC-MS analysis as described
below. The mass spectra were recorded in MS only mode.
HDX Data Analysis
[0473] Raw MS data was processed using HDX WorkBench software for
the analysis of H/D exchange MS data. The deuterium levels were
calculated using the average mass difference between the deuterated
peptide and its native form (to). For the calculation of deuterium
incorporation, the mass spectra for a given peptide were combined
across the extracted ion chromatogram peak and the weighted average
m/z was calculated. The mass increase from the mass of the native
peptide (0 minute) to the weighted averaged mass corresponds to the
level of deuterium incorporation.
Pepsin/Protease XIII Digestion and LC-MS
[0474] 5 .mu.g of native or human TIGIT in 120 .mu.L control buffer
(50 mM phosphate, 100 mM sodium chloride at pH 7.4) was denatured
by adding 120 .mu.L of 4 M guanidine hydrochloride, 0.85 M TCEP
buffer (final pH is 2.5) and incubating the mixture for three
minutes at 24.degree. C. The mixture was then subjected to
on-column pepsin/protease XIII digestion using a packed
pepsin/protease XIII (w/w, 1:1) column, and the resultant peptides
was analyzed using an UPLC-MS system comprised of a Waters Acquity
UPLC coupled to a Q Exactive.TM. Hybrid Quadrupole-Orbitrap Mass
Spectrometer (Thermo). The peptides were separated on a 50
mm.times.1 mm C8 column with a 20.5 min gradient from 2-28% solvent
B (0.2% formic acid in acetonitrile). Peptide identification was
performed by searching MS/MS data against the human TIGIT sequence
with Mascot. The mass tolerance for the precursor and product ions
was 10 ppm and 0.05 Da, respectively.
Epitope Binding of Anti-Human TIGIT F(ab')2
[0475] Most of the TIGIT peptides displayed identical or similar
deuterium levels with and without BA002-F(ab').sub.2 present.
Several peptide segments, however, were found to have significantly
decreased deuterium incorporation upon BA002-F(ab').sub.2 binding.
All the residues in this paragraph are numbered according to the
full length TIGIT sequence set forth in SEQ ID NO: 29. Two regions,
consisting of residues 110-125 (YHTYPDGTYTGRIFLE, SEQ ID NO: 31)
and residues 54-57 (VTQV, SEQ ID NO: 32), exhibited substantial
deuterium protection when human TIGIT was bound to BA002-F(ab')2.
An additional region consisting of residues 68-81 (ICNADLGWHISPSF,
SEQ ID NO: 33) also showed deuterium protection when human TIGIT
was bound to BA002-F(ab')2. Thus, these regions correspond to one
or more epitopes, or portions thereof, of BA002 on human TIGIT, as
shown in FIG. 21.
6.5.2 Epitope Mapping of Anti-TIGIT Antibody by Antigen
Mutagenesis
[0476] In this example, the binding of BA006, as well as six
reference antibodies, to human TIGIT and mutant proteins was
characterized by surface plasmon resonance (SPR). Briefly, the
structure of the extracellular domain of human TIGIT was obtained
from the PDB database (reference No. 3UDW) and was isolated from
the structure of a TIGIT-PVR complex. Among the amino acid residues
located within the epitope regions identified from Section 6.5.1,
T34, Q35, I47, N49, L52, H55, P58, H90, T96, T98, R100, and F102
were found to have a side chain facing the PVR-binding surface
(FIG. 22A). These residues were selected for antigen mutagenesis
analysis. The amino acid sequences of the mutated human TIGIT
proteins are provided in Table 3.
[0477] In the SPR experiment, the anti-TIGIT antibodies were
individually captured at a flow rate of 10 .mu.l/min on flow-cells
2, 3 and 4, keeping the flow-cell 1 as reference, on a CM5 chip on
which an anti-human Fab antibody had been immobilized by amine
coupling. The wild-type and mutant TIGIT proteins were
independently run over all the flow-cells at a concentration of 100
nM at 50 .mu.l/min for 90 seconds, followed by a dissociation phase
of 400 seconds. The maximum binding response was measured based on
the sensorgrams, and the percentages of binding of each antibody
relative to the affinity to the wild-type TIGIT protein are shown
in Table 5.
TABLE-US-00005 TABLE 5 Binding of anti-TIGIT antibodies to
wild-type and mutant TIGIT. SEQ ID Ref. Ref. Ref. Ref. Ref. Ref.
TIGIT NO BA006 1 2 3 4 5 6 WT 42 + + + + + + + T34A 43 + + + + + +
+ Q35A 44 - +/- +/- +* + + +/- I47E 45 - + + - + +* - N49A 46 +/- +
+ + + + +/- L52A 47 + - + +* + +/-* - L52E 48 +/- - + +/-* +* - -
H55A 49 + - + +* +* + +* P58A 50 + + + + + + + H90A 51 +/- +/- +/-
+ + + +/- T96A 52 - + + + + + + T96I 53 - + + + + + + T98A 54 + + +
+ + + + R100A 55 + + + + + + + F102A 56 + + +/- + + + + C48Y, 57
+/- + + + + + +* N49S, A50V I56V, 58 + + +/- + + +* +/-* S57A,
P58S, S59V T96I, 59 - + + + + + + T98K +: at least 70% relative to
the binding affinity to wild-type TIGIT protein +/-: less than 70%
and at least 20% relative to the binding affinity to wild-type
TIGIT protein -: less than 20% relative to the binding affinity to
wild-type TIGIT protein *faster dissociation rate observed
[0478] As shown in Table 5, the single mutations of Q35A, 147E,
N49A, H90A, T96A, and T96I reduced the binding of BA006 to human
TIGIT, suggesting that BA006 likely binds to TIGIT via one or more
conformational epitopes comprising Q35, I47, N49, H90, and/or T96
(FIG. 22B). BA006 was not sensitive to the mutations of L52A, H55A,
F102A, and I56V/S57A/P58S/S59V in these experiments, indicating
that BA006 likely did not bind directly to L52, H55, I56, S57, P58,
S59, or F102 of human TIGIT. This set of epitopes is unique and is
not identical to the epitope mapping results of the reference
antibodies.
6.6 Example 6: In Vivo Pharmacology of an Anti-TIGIT Antibody in a
Mouse Model
[0479] As described above, BA002 and BA006 robustly enhanced T cell
activities in vitro. However, these antibodies did not bind to
murine TIGIT protein. In order to study the in vivo functions of
BA002 and BA006 in mouse models, surrogate antibodies that bound to
murine TIGIT were generated. Briefly, the VH and VL regions of a
TIGIT reference antibody were linked to murine heavy chain and
light chain constant regions, respectively. Surrogate antibody
mIgG2a, surrogate antibody mIgG2a-N297Q, surrogate antibody mIgG1,
and surrogate antibody mIgG2 (Fc enhanced) have different Fc
regions, but share the same light chain sequence. It is generally
recognized in the art that mIgG2a is functionally similar to human
IgG1. The amino acid sequences of these antibodies are shown in
Table 6.
TABLE-US-00006 TABLE 6 Amino acid sequences of mouse surrogate
anti-TIGIT antibodies. SEQ Amino Acid ID Description Sequence NO:
Surrogate XVQLVESGGGLTQPG 62 antibody KSLKLSCEASGFTFS VH SFTMHW
VRQSPGKGLEWVAFI RSGSGIVFYADAVRG RFTISR DNAKNLLFLQMNDLK
SEDTAMYYCARRPLG HNTF DSWGQGTLVTVSS, wherein X is glutamate (E) or
pyroglutamate (pE) Surrogate DIVMTQSPSSLAVSP 63 antibody VL
GEKVTMTCKSSQSLY YSGVKENLLAWYQQK PGQSPKLLIYYASIR FTGVPDRFTGSGSGT
DYTLTITSVQAEDMG QYFCQQGINNPLTFG DGTKLEIK Surrogate XVQLVESGGGLTQPG
64 antibody KSLKLSCEASGFTFS mIgG2a full SFTMHWVRQSPGKGL length
EWVAFIRSGSGIVFY heavy chain ADAVRGRFTISRDNA without KNLLFLQMNDLKSED
C-terminal TAMYYCARRPLGHNT lysine FDSWGQGTLVTVSSA (used in the
KTTAPSVYPLAPVCG experiments in DTTGSSVTLGCLVKG Sections 6.6.2,
YFPEPVTLTWNSGSL 6.6.3, SSGVHTFPAVLQSDL and 6.6.4) YTLSSSVTVTSSTWP
SQSITCNVAHPASST KVDKKIEPRGPTIKP CPPCKCPAPNLLGGP SVFIFPPKIKDVLMI
SLSPIVTCVVVDVSE DDPDVQISWFVNNVE VHTAQTQTHREDYNS TLRVVSALPIQHQDW
MSGKEFKCKVNNKDL PAPIERTISKPKGSV RAPQVYVLPPPEEEM TKKQVTLTCMVTDFM
PEDIYVEWTNNGKTE LNYKNTEPVLDSDGS YFMYSKLRVEKKNWV ERNSYSCSVVHEGLH
NHHTTKSFSRTPG, wherein X is glutamate (E) or pyroglutamate (pE)
Surrogate XVQLVESGGGLTQPGK 65 antibody SLKLSCEASGFTFSS mIgG2a full
FTMHWVRQSPGKGLE length WVAFIRSGSGIVFYA heavy chain DAVRGRFTISRDNAK
with C- NLLFLQMNDLKSEDT terminal AMYYCARRPLGHNTF lysine
DSWGQGTLVTVSSAK (used TTAPSVYPLAPVCGD in the TTGSSVTLGCLVKGY
experiments in FPEPVTLTWNSGSLS Section 6.6.1) SGVHTFPAVLQSDLY
TLSSSVTVTSSTWPS QSITCNVAHPASSTK VDKKIEPRGPTIKPC PPCKCPAPNLLGGPS
VFIFPPKIKDVLMIS LSPIVTCVVVDVSED DPDVQISWFVNNVEV HTAQTQTHREDYNST
LRVVSALPIQHQDWM SGKEFKCKVNNKDLP APIERTISKPKGSVR APQVYVLPPPEEEMT
KKQVTLTCMVTDFMP EDIYVEWTNNGKTEL NYKNTEPVLDSDGSY FMYSKLRVEKKNWVE
RNSYSCSVVHEGLHN HHTTKSFSRTPGK, wherein X is glutamate (E) or
pyroglutamate (pE) Surrogate XVQLVESGGGLTQPG 66 antibody
KSLKLSCEASGFTFS mIgG2a-N297Q SFTMHWVRQSPGKGL full EWVAFIRSGSGIVFY
length heavy ADAVRGRFTISRDNA chain with KNLLFLQMNDLKSED C-terminal
TAMYYCARRPLGHNT lysine FDSWGQGTLVTVSSA (used in the KTTAPSVYPLAPVCG
experiments DTTGSSVTLGCLVKG in Section YFPEPVTLTWNSGSL 6.6.1)
SSGVHTFPAVLQSDL YTLSSSVTVTSSTWP SQSITCNVAHPASST KVDKKIEPRGPTIKP
CPPCKCPAPNLLGGP SVFIFPPKIKDVLMI SLSPIVTCVVVDVSE DDPDVQISWFVNNVE
VHTAQTQTHREDYQS TLRVVSALPIQHQDW MSGKEFKCKVNNKDL PAPIERTISKPKGSV
RAPQVYVLPPPEEEM TKKQVTLTCMVTDFM PEDIYVEWTNNGKTE LNYKNTEPVLDSDGS
YFMYSKLRVEKKNWV ERNSYSCSVVHEGLH NHHTTKSFSRTPGK, wherein X is
glutamate (E) or pyroglutamate (pE) Surrogate XVQLVESGGGLTQPG 67
antibody KSLKLSCEASGFTFS mlgGl full SFTMHWVRQSPGKGL length
EWVAFIRSGSGIVFY heavy chain ADAVRGRFTISRDNA with C- KNLLFLQMNDLKSED
terminal TAMYYCARRPLGHNT lysine (used FDSWGQGTLVTVSSA in the
KTTPPSVYPLAPGSA experiments in AQTNSMVTLGCLVKG Section 6.6.1)
YFPEPVTVTWNSGSL SSGVHTFPAVLQSDL YTLSSSVTVPSSTWP SETVTCNVAHPASST
KVDKKIVPRDCGCKP CICTVPEVSSVFIFP PKPKDVLTITLTPKV TCVVVDISKDDPEVQ
FSWFVDDVEVHTAQT QPREEQFNSTFRSVS ELPIMHQDWLNGKEF KCRVNSAAFPAPIEK
TISKTKGRPKAPQVY TIPPPKEQMAKDKVS LTCMITDFFPEDITV EWQWNGQPAENYKNT
QPIMDTDGSYFVYSK LNVQKSNWEAGNTFT CSVLHEGLHNHHTEK SLSHSPGK, wherein X
is glutamate (E) or Pyroglutamate (pE) Surrogate XVQLVESGGGLTQPG 68
antibody KSLKLSCEASGFTFS mIgG2 SFTMHWVRQSPGKGL (Fc enhanced)
EWVAFIRSGSGIVFY full length ADAVRGRFTISRDNA heavy chain
KNLLFLQMNDLKSED without TAMYYCARRPLGHNT C-terminal FDSWGQGTLVTVSSA
lysine (used KTTAPSVYPLAPVCG in the DTTGSSVTLGCLVKG experiments in
YFPEPVTLTWNSGSL Sections 6.6.2, SSGVHTFPAVLQSDL 6.6.3,
YTLSSSVTVTSSTWP and 6.6.4) SQSITCNVAHPASST KVDKKIEPRGPTIKP
CPPCKCPAPNLLGGP DVFIFPPKIKDVLMI SLSPIVTCVVVDVSE DDPDVQISWFVNNVE
VHTAQTQTHREDYNS TLRVVSALPIQHQDW MSGKEFKCKVNNKDL PLPEERTISKPKGSV
RAPQVYVLPPPEEEM TKKQVTLTCMVTDFM PEDIYVEWTNNGKTE LNYKNTEPVLDSDGS
YFMYSKLRVEKKNWV ERNSYSCSVVHEGLH NHHTTKSFSRTPG, wherein X is
glutamate (E) or pyroglutamate (pE) Surrogate DIVMTQSPSSLAVSP 69
antibody IgK GEKVTMTCKSSQSLY full length YSGVKENLLAWYQQK light
chain PGQSPKLLIYYASIR FTGVPDRFTGSGSGT DYTLTITSVQAEDMG
QYFCQQGINNPLTFG DGTKLEIKRADAAPT VSIFPPSSEQLTSGG ASVVCFLNNFYPKDI
NVKWKIDGSERQNGV LNSWTDQDSKDSTYS MSSTLTLTKDEYERH NSYTCEATHKTSTSP
IVKSFNRNEC
6.6.1 Anti-TIGIT Antibodies Inhibited Tumor Growth in an Early
Intervention Model
[0480] The mouse surrogate antibodies were tested in an early
intervention mouse model. Specifically, Balb/c mice (Jackson Labs
#000651) 6-8 weeks of age were first acclimated for two weeks and
were shaved and tagged. CT26 mouse colorectal carcinoma cells
(ATCC.RTM. CRL-2638.TM.) were expanded in tissue culture in RPMI
medium supplemented with 10% heat-inactivated FBS and normocin for
1 week. The mice were injected subcutaneously with 1.times.10.sup.5
CT26 cells suspended in 100 .mu.L of PBS. The implanted tumor cells
were allowed to establish for 7 days to reach the size of
approximately 35-40 mm.sup.3. The mice were then randomized and
treated with 200 .mu.g of surrogate antibody mIgG2a, surrogate
antibody mIgG2a-N297Q, surrogate antibody mIgG1, or an isotype
control antibody (mIgG2a) twice a week via intraperitoneal
administration. For comparison, 200 .mu.g of an anti-PD-1 antibody
was administered to the mice intraperitoneally twice a week. The
tumor volumes were measured biweekly by caliper, and were
calculated as length.times.width.sup.2.times.0.5.
[0481] As shown in FIGS. 23A-23F, surrogate antibody mIgG2a led to
a complete response in one out of five mice and substantially
suppressed tumor growth in three out of five mice. The anti-PD-1
antibody also reduced the rate of tumor growth. By contrast,
surrogate antibody mIgG2a-N297Q and surrogate antibody mIgG1 had
little effect on tumor growth. This result corroborated the in
vitro observation that the effector function of Fc enhanced the
ability of an anti-TIGIT antibody to activate T cell immunity.
[0482] Next tested were combination treatments of an anti-TIGIT
antibody and an anti-PD-1 antibody in the early intervention model.
Mice harboring CT26 tumors were generated as described above, and
were treated with 200 .mu.g of surrogate antibody mIgG2a, surrogate
antibody mIgG2a-N297Q, surrogate antibody mIgG1, or an isotype
control antibody (mIgG2a) in combination with 200 .mu.g of the
anti-PD-1 antibody twice a week via intraperitoneal
administration.
[0483] As shown in FIGS. 24A-24G, the combination treatment of
surrogate antibody mIgG2a and the anti-PD-1 antibody led to a
complete response in three out of five mice. By contrast, the
combination of surrogate antibody mIgG2a-N297Q and the anti-PD-1
antibody had little effect on tumor growth relative to the
anti-PD-1 antibody alone.
6.6.2 Anti-TIGIT Antibodies Inhibit Tumor Growth in a Late
Intervention Model
[0484] The anti-TIGIT antibodies and combinations were also
examined in a late intervention mouse model. Specifically, Balb/c
mice (Jackson Labs #000651) 6-8 weeks of age were first acclimated
for two weeks and were shaved and tagged. CT26 mouse colorectal
carcinoma cells (ATCC.RTM. CRL-2638.TM.) were expanded in tissue
culture in RPMI medium supplemented with 10% heat-inactivated FBS
and normocin for 1 week. The mice were injected with
5.times.10.sup.4 CT26 cells in 100 .mu.L of PBS subcutaneously. The
implanted tumor cells were allowed to establish for 12 days, when
the mean tumor size was 85 mm.sup.3. The mice without detectable
tumors or with tumor volumes greater than 300 mm.sup.3 were
excluded from the study. On days 12, 16, and 20 post-tumor
implantation, the mice were injected intraperitoneally with 100
.mu.g of surrogate antibody mIgG2a or surrogate antibody mIgG2a (Fc
enhanced), or the respective isotype control antibody. The tumor
volumes were measured biweekly by caliper, and were calculated as
length.times.width.sup.2.times.0.5.
[0485] As shown in FIGS. 25A-25E, surrogate antibody mIgG2a and
surrogate antibody mIgG2a (Fc enhanced) both reduced tumor growth,
and surrogate antibody mIgG2a (Fc enhanced) was more potent than
surrogate antibody mIgG2a.
[0486] The effect of combining an anti-TIGIT antibody with another
checkpoint targeting molecule on tumor suppression was also tested
in the late intervention model. Specifically, the mice were
inoculated as described above, and the mean tumor size 12 days
after inoculation was 70-120 mm.sup.3. The mice were treated with
100 .mu.g of surrogate antibody mIgG2a (Fc enhanced), 100 .mu.g of
an anti-PD-1 antibody or an anti-CTLA-4 antibody, or a combination
thereof on days 12, 16, and 20 post-tumor implantation. Tumor
growth was monitored bi-weekly using a digital caliper.
[0487] As shown in FIGS. 26A and 26B, combinations of surrogate
antibody mIgG2a (Fc enhanced) with the anti-PD-1 antibody or the
anti-CTLA-4 antibody substantially reduced tumor growth in this
animal model.
6.6.3 Anti-TIGIT Antibodies Promote Infiltration of CD8.sup.+ T
Cells into Tumors
[0488] The inhibition of tumor growth by the anti-TIGIT antibodies
could be due to activation of effector T cells or suppression of
regulatory T cells (Tregs). To understand the mechanism of this
regulation, BALB/c mice were inoculated with 5.times.10.sup.4 CT26
cells subcutaneously. When the tumors reached approximately 50-80
mm.sup.3 after 12-14 days, the mice were randomized and treated
with a single dose of 100 .mu.g of surrogate antibody mIgG2a,
surrogate antibody mIgG2a (Fc enhanced), or the respective isotype
control antibody via intraperitoneal administration. An anti-GITR
antibody in the mIgG2a format ("DTA-1 (mIgG2a)") that was known to
deplete Tregs was used as a positive control. The mice were
sacrificed at 0, 24, 72, or 120 hours post-treatment for collection
of tumor and tumor-draining lymph node (TDLN) samples (FIG.
27A).
[0489] As shown in FIGS. 27B-27F, administration of surrogate
antibody mIgG2a or surrogate antibody mIgG2a (Fc enhanced) did not
substantially affect the amount of intratumoral FoxP3.sup.+ Tregs,
intratumoral CD4.sup.+ non-Tregs, or TDLN FoxP3.sup.+ Tregs, but
significantly increased the amount of intratumoral CD8.sup.+ T
cells. Thus, while not wishing to be bound by theory, it was
hypothesized that surrogate antibody mIgG2a or surrogate antibody
mIgG2a (Fc enhanced) inhibited tumor growth by promoting
infiltration of CD8.sup.+ T cells in the tumors.
6.6.4 Anti-TIGIT Antibodies Activate Effector T Cells in an
Fc.gamma.RIV-Dependent Manner
[0490] As described above, surrogate antibody mIgG2a (Fc enhanced)
was more potent than surrogate antibody mIgG2a in tumor
suppression, suggesting that the ability of the Fc to bind to
Fc.gamma. receptors might play a role in the function of anti-TIGIT
antibodies. Murine Fc.gamma.RIV was known as a primary receptor of
mIgG2a, and the Fc region of surrogate antibody mIgG2a (Fc
enhanced) was known to bind to murine Fc.gamma.RIV with a higher
affinity than the Fc region of surrogate antibody mIgG2a. Thus, the
ability of surrogate antibody mIgG2a (Fc enhanced) to enhance
Fc.gamma.RIV signaling was examined. Briefly, CHO cells engineered
to express murine TIGIT were cultured in RPMI 1640 medium
supplemented with 10% FBS, 10 mM HEPES, 1.times.
Pen/Strep-Glutamine, and 1 .mu.g/mL puromycin. The cells were
resuspended in fresh culture medium at 2.4.times.10.sup.6 cells/mL,
and 25 .mu.L of the cells were added to each well of a white
96-well assay plate. A dilution series of surrogate antibody
mIgG2a, or its isotype control antibody, or surrogate antibody
mIgG2a (Fc enhanced) or its isotype control antibody were prepared
in culture medium, and 25 .mu.l of the antibody was added to the
cells. Effector T cells (Jurkat cells) stably expressing murine
Fc.gamma.RIV and having a firefly luciferase reporter under the
control of a nuclear factor of activated T-cells (NFAT)-responsive
promoter (ADCC V variant, Promega) were thawed and resuspended at
6.times.10.sup.6 cells/mL in RPMI 1640 supplemented with 4% FBS,
and 25 .mu.l of the effector cells were added to each well. The
co-culture was incubated at 37 C, 5% CO2 for 20 hours. 75 .mu.l of
Bio-Glo Luciferase assay reagent was added to each well, and
luminescence values were measured with a plate reader (Envision)
after 5-10 minutes of incubation at room temperature.
[0491] As shown in FIG. 28A, surrogate antibody mIgG2a (Fc
enhanced) induced a stronger NFAT activity in the effector T cells
than surrogate antibody mIgG2a, indicating that surrogate antibody
mIgG2a (Fc enhanced) had greater effector function.
[0492] To further elucidate the function of Fc.gamma.RIV in T cell
activation mediated by anti-TIGIT antibodies, C57BL/6 mice were
pretreated with 100 .mu.s of an anti-Fc.gamma.RIV antibody
(Biolegend, Catalog #149502) or vehicle control by intraperitoneal
injection. After 30 minutes, the mice were injected
intraperitoneally with 100 .mu.g of the SEB superantigen together
with 100 of surrogate antibody mIgG2a, an anti-CTLA-4 antibody
(mIgG2a), or an isotype control antibody. T cells were isolated
from the peripheral blood after 3 days, and SEB-specific
(V.beta.8.sup.+) CD4.sup.+ or CD8.sup.+ effector T cells
(CD44.sup.+ CD62L.sup.-) were quantified by flow cytometry for
proliferation (% Ki67 positive).
[0493] As shown in FIGS. 28B and 28C, the anti-Fc.gamma.RIV
antibody significantly reduced the proliferation of
antigen-specific CD4.sup.+ and CD8.sup.+ effector T cells mediated
by surrogate antibody mIgG2a or the anti-CTLA-4 antibody. Thus,
Fc.gamma.R co-engagement enhanced T cell co-stimulation mediated by
the anti-TIGIT and anti-CTLA-4 antibodies in this murine model of T
cell priming.
[0494] The 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 will become apparent to
those skilled in the art from the foregoing description and
accompanying figures. Such modifications are intended to fall
within the scope of the appended claims.
[0495] All references (e.g., publications or patents or patent
applications) cited herein are incorporated herein by reference in
their entireties and for all purposes to the same extent as if each
individual reference (e.g., publication or patent or patent
application) was specifically and individually indicated to be
incorporated by reference in its entirety for all purposes.
[0496] Other embodiments are within the following claims.
Sequence CWU 1
1
7015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Ser Tyr Gly Ile Ser1 527PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Gly
Tyr Thr Phe Ala Ser Tyr1 5317PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 3Gly Ile Thr Pro Phe Phe Asn
Arg Val Asp Val Ala Glu Lys Phe Gln1 5 10 15Gly46PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Thr
Pro Phe Phe Asn Arg1 5513PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 5Asp Leu Arg Arg Gly Gly Val
Gly Asp Ala Phe Asp Ile1 5 10614PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 6Thr Gly Thr Ser Ser Asp
Val Gly Ser His Asn Tyr Val Ser1 5 1077PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 7Glu
Val Ser Tyr Arg Pro Ser1 5810PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 8Ser Ser Tyr Thr Pro Ser Ser
Ala Thr Val1 5 109122PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMOD_RES(1)..(1)Glu or
pyro-Glu 9Xaa Val Gln Leu Val Gln Ser Gly Ala Glu Val Glu Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Ala Ser Tyr 20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Gly Ile Thr Pro Phe Phe Asn Arg Val Asp
Val Ala Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr
Ser Thr Asn Thr Val Tyr65 70 75 80Ile Glu Leu Ser Ser Leu Thr Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu Arg Arg Gly
Gly Val Gly Asp Ala Phe Asp Ile Trp 100 105 110Gly Arg Gly Thr Leu
Val Thr Val Ser Ser 115 12010110PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptideMOD_RES(1)..(1)Gln or
pyro-Glu 10Xaa Ser Ala Leu Thr Gln Pro Arg Ser Val Ser Gly Ser Pro
Gly Gln1 5 10 15Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val
Gly Ser His 20 25 30Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys
Ala Pro Gln Leu 35 40 45Met Ile Tyr Glu Val Ser Tyr Arg Pro Ser Glu
Ile Ser Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Ile Ser Gly Leu65 70 75 80Gln Pro Glu Asp Glu Ala Asp Tyr
Tyr Cys Ser Ser Tyr Thr Pro Ser 85 90 95Ser Ala Thr Val Phe Gly Ala
Gly Thr Lys Leu Thr Val Leu 100 105 11011451PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMOD_RES(1)..(1)Glu or pyro-Glu 11Xaa Val Gln Leu Val Gln
Ser Gly Ala Glu Val Glu Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Tyr 20 25 30Gly Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile
Thr Pro Phe Phe Asn Arg Val Asp Val Ala Glu Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Val Tyr65 70 75
80Ile Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Leu Arg Arg Gly Gly Val Gly Asp Ala Phe Asp Ile
Trp 100 105 110Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 45012451PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMOD_RES(1)..(1)Glu or
pyro-Glu 12Xaa Val Gln Leu Val Gln Ser Gly Ala Glu Val Glu Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Ala Ser Tyr 20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Gly Ile Thr Pro Phe Phe Asn Arg Val Asp
Val Ala Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr
Ser Thr Asn Thr Val Tyr65 70 75 80Ile Glu Leu Ser Ser Leu Thr Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu Arg Arg Gly
Gly Val Gly Asp Ala Phe Asp Ile Trp 100 105 110Gly Arg Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235 240Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265
270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala
Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390
395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly 45013451PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMOD_RES(1)..(1)Glu or pyro-Glu 13Xaa Val Gln Leu Val Gln
Ser Gly Ala Glu Val Glu Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Tyr 20 25 30Gly Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile
Thr Pro Phe Phe Asn Arg Val Asp Val Ala Glu Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Val Tyr65 70 75
80Ile Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Leu Arg Arg Gly Gly Val Gly Asp Ala Phe Asp Ile
Trp 100 105 110Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Phe Phe225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 45014451PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMOD_RES(1)..(1)Glu or
pyro-Glu 14Xaa Val Gln Leu Val Gln Ser Gly Ala Glu Val Glu Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Ala Ser Tyr 20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Gly Ile Thr Pro Phe Phe Asn Arg Val Asp
Val Ala Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr
Ser Thr Asn Thr Val Tyr65 70 75 80Ile Glu Leu Ser Ser Leu Thr Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu Arg Arg Gly
Gly Val Gly Asp Ala Phe Asp Ile Trp 100 105 110Gly Arg Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235 240Gly Gly Pro Asp
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265
270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro 325 330 335Glu Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390
395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly 45015451PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMOD_RES(1)..(1)Glu or pyro-Glu 15Xaa Val Gln Leu Val Gln
Ser Gly Ala Glu Val Glu Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Tyr 20 25 30Gly Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile
Thr Pro Phe Phe Asn Arg Val Asp Val Ala Glu Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Val Tyr65 70 75
80Ile Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Leu Arg Arg Gly Gly Val Gly Asp Ala Phe Asp Ile
Trp 100 105 110Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe
Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215
220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu225 230 235 240Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Leu Pro 325 330
335Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser
Pro Gly 45016451PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMOD_RES(1)..(1)Glu or pyro-Glu 16Xaa
Val Gln Leu Val Gln Ser Gly Ala Glu Val Glu Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Tyr
20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Gly Ile Thr Pro Phe Phe Asn Arg Val Asp Val Ala Glu
Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn
Thr Val Tyr65 70 75 80Ile Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu Arg Arg Gly Gly Val Gly
Asp Ala Phe Asp Ile Trp 100 105 110Gly Arg Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Val225 230 235 240Gly Gly Pro Ser Val Phe Leu
Leu Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val
His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr 290 295
300Leu Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Leu
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410
415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 435 440 445Ser Pro Gly 45017451PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMOD_RES(1)..(1)Glu or pyro-Glu 17Xaa Val Gln Leu Val Gln
Ser Gly Ala Glu Val Glu Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Tyr 20 25 30Gly Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile
Thr Pro Phe Phe Asn Arg Val Asp Val Ala Glu Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Val Tyr65 70 75
80Ile Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Leu Arg Arg Gly Gly Val Gly Asp Ala Phe Asp Ile
Trp 100 105 110Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Glu 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Phe Pro Ala Pro
325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 45018448PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMOD_RES(1)..(1)Glu or
pyro-Glu 18Xaa Val Gln Leu Val Gln Ser Gly Ala Glu Val Glu Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Ala Ser Tyr 20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Gly Ile Thr Pro Phe Phe Asn Arg Val Asp
Val Ala Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr
Ser Thr Asn Thr Val Tyr65 70 75 80Ile Glu Leu Ser Ser Leu Thr Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu Arg Arg Gly
Gly Val Gly Asp Ala Phe Asp Ile Trp 100 105 110Gly Arg Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265
270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu Gly 435 440 44519329PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
19Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155
160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
32520329PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 20Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Ala Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235
240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro Gly 32521329PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 21Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70
75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 100 105 110Pro Ala Pro Glu Phe Phe Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr
Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly 32522329PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
22Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly
Gly Pro Asp Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155
160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
32523329PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 23Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Leu
Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235
240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro Gly 32524329PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 24Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Leu Val Gly Gly Pro Ser Val Phe Leu
Leu Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Pro Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Leu Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Leu
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly 32525329PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
25Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val
Asp Val Glu His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155
160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
32526326PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 26Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu Phe
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120
125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235
240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu
Gly 32527216PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMOD_RES(1)..(1)Gln or pyro-Glu 27Xaa
Ser Ala Leu Thr Gln Pro Arg Ser Val Ser Gly Ser Pro Gly Gln1 5 10
15Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser His
20 25 30Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Gln
Leu 35 40 45Met Ile Tyr Glu Val Ser Tyr Arg Pro Ser Glu Ile Ser Asn
Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile
Ser Gly Leu65 70 75 80Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser
Ser Tyr Thr Pro Ser 85 90 95Ser Ala Thr Val Phe Gly Ala Gly Thr Lys
Leu Thr Val Leu Gly Gln 100 105 110Pro Lys Ala Ala Pro Ser Val Thr
Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125Leu Gln Ala Asn Lys Ala
Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140Pro Gly Ala Val
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys145 150 155 160Ala
Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170
175Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
Glu Lys 195 200 205Thr Val Ala Pro Thr Glu Cys Ser 210
21528106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 28Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser1 5 10 15Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp 20 25 30Phe Tyr Pro Gly Ala Val Thr Val Ala
Trp Lys Ala Asp Ser Ser Pro 35 40 45Val Lys Ala Gly Val Glu Thr Thr
Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60Lys Tyr Ala Ala Ser Ser Tyr
Leu Ser Leu Thr Pro Glu Gln Trp Lys65 70 75 80Ser His Arg Ser Tyr
Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95Glu Lys Thr Val
Ala Pro Thr Glu Cys Ser 100 10529244PRTHomo sapiens 29Met Arg Trp
Cys Leu Leu Leu Ile Trp Ala Gln Gly Leu Arg Gln Ala1 5 10 15Pro Leu
Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn 20 25 30Ile
Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser 35 40
45Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln
50 55 60Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro
Ser65 70 75 80Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu
Thr Leu Gln 85 90 95Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys
Ile Tyr His Thr 100 105 110Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile
Phe Leu Glu Val Leu Glu 115 120 125Ser Ser Val Ala Glu His Gly Ala
Arg Phe Gln Ile Pro Leu Leu Gly 130 135 140Ala Met Ala Ala Thr Leu
Val Val Ile Cys Thr Ala Val Ile Val Val145 150 155 160Val Ala Leu
Thr Arg Lys Lys Lys Ala Leu Arg Ile His Ser Val Glu
165 170 175Gly Asp Leu Arg Arg Lys Ser Ala Gly Gln Glu Glu Trp Ser
Pro Ser 180 185 190Ala Pro Ser Pro Pro Gly Ser Cys Val Gln Ala Glu
Ala Ala Pro Ala 195 200 205Gly Leu Cys Gly Glu Gln Arg Gly Glu Asp
Cys Ala Glu Leu His Asp 210 215 220Tyr Phe Asn Val Leu Ser Tyr Arg
Ser Leu Gly Asn Cys Ser Phe Phe225 230 235 240Thr Glu Thr
Gly30117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 30Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn
Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile Leu Gln Cys His Leu
Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Gln Val Asn Trp Glu Gln Gln
Asp Gln Leu Leu Ala Ile Cys 35 40 45Asn Ala Asp Leu Gly Trp His Ile
Ser Pro Ser Phe Lys Asp Arg Val 50 55 60Ala Pro Gly Pro Gly Leu Gly
Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75 80Asp Thr Gly Glu Tyr
Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95Tyr Thr Gly Arg
Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110His Gly
Ala Arg Phe 1153116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 31Tyr His Thr Tyr Pro Asp Gly Thr Tyr
Thr Gly Arg Ile Phe Leu Glu1 5 10 15324PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 32Val
Thr Gln Val13314PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 33Ile Cys Asn Ala Asp Leu Gly Trp His
Ile Ser Pro Ser Phe1 5 103498PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 34Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile
Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg3598PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 35Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro
Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg36118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 36Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn
Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile Leu Gln Cys His Leu
Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Gln Val Asn Trp Glu Gln Gln
Asp Gln Leu Leu Ala Ile Cys 35 40 45Ser Ala Asp Leu Gly Trp His Ile
Ser Pro Ser Phe Lys Asp Arg Val 50 55 60Ala Pro Gly Pro Gly Leu Gly
Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75 80Asp Thr Gly Glu Tyr
Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95Tyr Thr Gly Arg
Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110His Gly
Ala Arg Phe Gln 1153799PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 37Gln Ser Ala Leu Thr Gln
Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30Asn Tyr Val Ser
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45Met Ile Tyr
Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75
80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser
85 90 95Ser Thr Leu3899PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 38Gln Ser Ala Leu Thr Gln
Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30Asn Leu Val Ser
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45Met Ile Tyr
Glu Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75
80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Ser
85 90 95Ser Thr Phe3999PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 39Gln Ser Ala Leu Thr Gln
Pro Arg Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15Ser Val Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30Asn Tyr Val Ser
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45Met Ile Tyr
Asp Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75
80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Ser
85 90 95Tyr Thr Phe40223PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 40Met Met Thr Gly Thr Ile
Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile
Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Gln Val
Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45Asn Ala Asp
Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val 50 55 60Ala Pro
Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75
80Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr
85 90 95Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala
Glu 100 105 110His Gly Ala Arg Phe Gln Ile Pro Leu Leu Gly Ala Met
Ala Ala Thr 115 120 125Leu Val Val Ile Cys Thr Ala Val Ile Val Val
Val Ala Leu Thr Arg 130 135 140Lys Lys Lys Ala Leu Arg Ile His Ser
Val Glu Gly Asp Leu Arg Arg145 150 155 160Lys Ser Ala Gly Gln Glu
Glu Trp Ser Pro Ser Ala Pro Ser Pro Pro 165 170 175Gly Ser Cys Val
Gln Ala Glu Ala Ala Pro Ala Gly Leu Cys Gly Glu 180 185 190Gln Arg
Gly Glu Asp Cys Ala Glu Leu His Asp Tyr Phe Asn Val Leu 195 200
205Ser Tyr Arg Ser Leu Gly Asn Cys Ser Phe Phe Thr Glu Thr Gly 210
215 22041120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 41Met Met Thr Gly Thr Ile Glu Thr
Thr Gly Asn Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile Leu Gln
Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Gln Val Asn Trp
Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45Asn Ala Asp Leu Gly
Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val 50 55 60Ala Pro Gly Pro
Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75 80Asp Thr
Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95Tyr
Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105
110His Gly Ala Arg Phe Gln Ile Pro 115 12042118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
42Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys1
5 10 15Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala
Gln 20 25 30Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala
Ile Cys 35 40 45Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys
Asp Arg Val 50 55 60Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser
Leu Thr Val Asn65 70 75 80Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His
Thr Tyr Pro Asp Gly Thr 85 90 95Tyr Thr Gly Arg Ile Phe Leu Glu Val
Leu Glu Ser Ser Val Ala Glu 100 105 110His Gly Ala Arg Phe Gln
11543118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 43Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn
Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile Leu Gln Cys His Leu
Ser Ser Thr Thr Ala Gln 20 25 30Val Ala Gln Val Asn Trp Glu Gln Gln
Asp Gln Leu Leu Ala Ile Cys 35 40 45Asn Ala Asp Leu Gly Trp His Ile
Ser Pro Ser Phe Lys Asp Arg Val 50 55 60Ala Pro Gly Pro Gly Leu Gly
Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75 80Asp Thr Gly Glu Tyr
Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95Tyr Thr Gly Arg
Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110His Gly
Ala Arg Phe Gln 11544118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 44Met Met Thr Gly Thr Ile
Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile
Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Ala Val
Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45Asn Ala Asp
Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val 50 55 60Ala Pro
Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75
80Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr
85 90 95Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala
Glu 100 105 110His Gly Ala Arg Phe Gln 11545118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
45Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys1
5 10 15Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala
Gln 20 25 30Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala
Glu Cys 35 40 45Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys
Asp Arg Val 50 55 60Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser
Leu Thr Val Asn65 70 75 80Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His
Thr Tyr Pro Asp Gly Thr 85 90 95Tyr Thr Gly Arg Ile Phe Leu Glu Val
Leu Glu Ser Ser Val Ala Glu 100 105 110His Gly Ala Arg Phe Gln
11546118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 46Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn
Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile Leu Gln Cys His Leu
Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Gln Val Asn Trp Glu Gln Gln
Asp Gln Leu Leu Ala Ile Cys 35 40 45Ala Ala Asp Leu Gly Trp His Ile
Ser Pro Ser Phe Lys Asp Arg Val 50 55 60Ala Pro Gly Pro Gly Leu Gly
Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75 80Asp Thr Gly Glu Tyr
Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95Tyr Thr Gly Arg
Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110His Gly
Ala Arg Phe Gln 11547118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 47Met Met Thr Gly Thr Ile
Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile
Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Gln Val
Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45Asn Ala Asp
Ala Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val 50 55 60Ala Pro
Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75
80Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr
85 90 95Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala
Glu 100 105 110His Gly Ala Arg Phe Gln 11548118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
48Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys1
5 10 15Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala
Gln 20 25 30Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala
Ile Cys 35 40 45Asn Ala Asp Glu Gly Trp His Ile Ser Pro Ser Phe Lys
Asp Arg Val 50 55 60Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser
Leu Thr Val Asn65 70 75 80Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His
Thr Tyr Pro Asp Gly Thr 85 90 95Tyr Thr Gly Arg Ile Phe Leu Glu Val
Leu Glu Ser Ser Val Ala Glu 100 105 110His Gly Ala Arg Phe Gln
11549118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 49Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn
Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile Leu Gln Cys His Leu
Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Gln Val Asn Trp Glu Gln Gln
Asp Gln Leu Leu Ala Ile Cys 35 40 45Asn Ala Asp Leu Gly Trp Ala Ile
Ser Pro Ser Phe Lys Asp Arg Val 50 55 60Ala Pro Gly Pro Gly Leu Gly
Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75 80Asp Thr Gly Glu Tyr
Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95Tyr Thr Gly Arg
Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110His Gly
Ala Arg Phe Gln 11550118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 50Met Met Thr Gly Thr Ile
Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile
Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Gln Val
Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45Asn Ala Asp
Leu Gly Trp His Ile Ser Ala Ser Phe Lys Asp Arg Val 50 55 60Ala Pro
Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75
80Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr
85 90 95Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala
Glu 100 105 110His Gly Ala Arg Phe Gln
11551118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 51Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn
Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile Leu Gln Cys His Leu
Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Gln Val Asn Trp Glu Gln Gln
Asp Gln Leu Leu Ala Ile Cys 35 40 45Asn Ala Asp Leu Gly Trp His Ile
Ser Pro Ser Phe Lys Asp Arg Val 50 55 60Ala Pro Gly Pro Gly Leu Gly
Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75 80Asp Thr Gly Glu Tyr
Phe Cys Ile Tyr Ala Thr Tyr Pro Asp Gly Thr 85 90 95Tyr Thr Gly Arg
Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110His Gly
Ala Arg Phe Gln 11552118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 52Met Met Thr Gly Thr Ile
Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile
Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Gln Val
Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45Asn Ala Asp
Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val 50 55 60Ala Pro
Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75
80Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Ala
85 90 95Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala
Glu 100 105 110His Gly Ala Arg Phe Gln 11553118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
53Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys1
5 10 15Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala
Gln 20 25 30Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala
Ile Cys 35 40 45Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys
Asp Arg Val 50 55 60Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser
Leu Thr Val Asn65 70 75 80Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His
Thr Tyr Pro Asp Gly Ile 85 90 95Tyr Thr Gly Arg Ile Phe Leu Glu Val
Leu Glu Ser Ser Val Ala Glu 100 105 110His Gly Ala Arg Phe Gln
11554118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 54Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn
Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile Leu Gln Cys His Leu
Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Gln Val Asn Trp Glu Gln Gln
Asp Gln Leu Leu Ala Ile Cys 35 40 45Asn Ala Asp Leu Gly Trp His Ile
Ser Pro Ser Phe Lys Asp Arg Val 50 55 60Ala Pro Gly Pro Gly Leu Gly
Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75 80Asp Thr Gly Glu Tyr
Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95Tyr Ala Gly Arg
Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110His Gly
Ala Arg Phe Gln 11555118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 55Met Met Thr Gly Thr Ile
Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile
Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Gln Val
Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45Asn Ala Asp
Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val 50 55 60Ala Pro
Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75
80Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr
85 90 95Tyr Thr Gly Ala Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala
Glu 100 105 110His Gly Ala Arg Phe Gln 11556118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
56Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys1
5 10 15Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala
Gln 20 25 30Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala
Ile Cys 35 40 45Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys
Asp Arg Val 50 55 60Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser
Leu Thr Val Asn65 70 75 80Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His
Thr Tyr Pro Asp Gly Thr 85 90 95Tyr Thr Gly Arg Ile Ala Leu Glu Val
Leu Glu Ser Ser Val Ala Glu 100 105 110His Gly Ala Arg Phe Gln
11557118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 57Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn
Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile Leu Gln Cys His Leu
Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Gln Val Asn Trp Glu Gln Gln
Asp Gln Leu Leu Ala Ile Tyr 35 40 45Ser Val Asp Leu Gly Trp His Ile
Ser Pro Ser Phe Lys Asp Arg Val 50 55 60Ala Pro Gly Pro Gly Leu Gly
Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75 80Asp Thr Gly Glu Tyr
Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95Tyr Thr Gly Arg
Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110His Gly
Ala Arg Phe Gln 11558118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 58Met Met Thr Gly Thr Ile
Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys1 5 10 15Gly Gly Ser Ile Ile
Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30Val Thr Gln Val
Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45Asn Ala Asp
Leu Gly Trp His Val Ala Ser Val Phe Lys Asp Arg Val 50 55 60Ala Pro
Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn65 70 75
80Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr
85 90 95Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala
Glu 100 105 110His Gly Ala Arg Phe Gln 11559118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
59Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys1
5 10 15Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala
Gln 20 25 30Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala
Ile Cys 35 40 45Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys
Asp Arg Val 50 55 60Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser
Leu Thr Val Asn65 70 75 80Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His
Thr Tyr Pro Asp Gly Ile 85 90 95Tyr Lys Gly Arg Ile Phe Leu Glu Val
Leu Glu Ser Ser Val Ala Glu 100 105 110His Gly Ala Arg Phe Gln
1156099PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 60Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser
Gly Ser Pro Gly Gln1 5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser
Ser Asp Val Gly Ser Tyr 20 25 30Asn Leu Val Ser Trp Tyr Gln Gln His
Pro Gly Lys Ala Pro Lys Leu 35 40 45Met Ile Tyr Glu Gly Ser Lys Arg
Pro Ser Gly Val Ser Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn
Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu
Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95Ser Thr
Leu619PRTCytomegalovirus 61Asn Leu Val Pro Met Val Ala Thr Val1
562119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMOD_RES(1)..(1)Glu or pyro-Glu 62Xaa Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Thr Gln Pro Gly Lys1 5 10 15Ser Leu
Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Thr
Met His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Phe Ile Arg Ser Gly Ser Gly Ile Val Phe Tyr Ala Asp Ala Val
50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Leu
Phe65 70 75 80Leu Gln Met Asn Asp Leu Lys Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Ala Arg Arg Pro Leu Gly His Asn Thr Phe Asp Ser
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11563113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 63Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu
Ala Val Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Lys Ser Ser
Gln Ser Leu Tyr Tyr Ser 20 25 30Gly Val Lys Glu Asn Leu Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ser Pro Lys Leu Leu Ile Tyr Tyr
Ala Ser Ile Arg Phe Thr Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser
Gly Ser Gly Thr Asp Tyr Thr Leu Thr65 70 75 80Ile Thr Ser Val Gln
Ala Glu Asp Met Gly Gln Tyr Phe Cys Gln Gln 85 90 95Gly Ile Asn Asn
Pro Leu Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile 100 105
110Lys64448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMOD_RES(1)..(1)Glu or pyro-Glu 64Xaa Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Thr Gln Pro Gly Lys1 5 10 15Ser Leu
Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Thr
Met His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Phe Ile Arg Ser Gly Ser Gly Ile Val Phe Tyr Ala Asp Ala Val
50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Leu
Phe65 70 75 80Leu Gln Met Asn Asp Leu Lys Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Ala Arg Arg Pro Leu Gly His Asn Thr Phe Asp Ser
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Lys Thr
Thr Ala Pro Ser Val Tyr 115 120 125Pro Leu Ala Pro Val Cys Gly Asp
Thr Thr Gly Ser Ser Val Thr Leu 130 135 140Gly Cys Leu Val Lys Gly
Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp145 150 155 160Asn Ser Gly
Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser 180 185
190Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser
195 200 205Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr
Ile Lys 210 215 220Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu
Leu Gly Gly Pro225 230 235 240Ser Val Phe Ile Phe Pro Pro Lys Ile
Lys Asp Val Leu Met Ile Ser 245 250 255Leu Ser Pro Ile Val Thr Cys
Val Val Val Asp Val Ser Glu Asp Asp 260 265 270Pro Asp Val Gln Ile
Ser Trp Phe Val Asn Asn Val Glu Val His Thr 275 280 285Ala Gln Thr
Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val 290 295 300Val
Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu305 310
315 320Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu
Arg 325 330 335Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln
Val Tyr Val 340 345 350Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys
Gln Val Thr Leu Thr 355 360 365Cys Met Val Thr Asp Phe Met Pro Glu
Asp Ile Tyr Val Glu Trp Thr 370 375 380Asn Asn Gly Lys Thr Glu Leu
Asn Tyr Lys Asn Thr Glu Pro Val Leu385 390 395 400Asp Ser Asp Gly
Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys 405 410 415Lys Asn
Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu 420 425
430Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly
435 440 44565449PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMOD_RES(1)..(1)Glu or pyro-Glu 65Xaa
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Thr Gln Pro Gly Lys1 5 10
15Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Ser Phe
20 25 30Thr Met His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Phe Ile Arg Ser Gly Ser Gly Ile Val Phe Tyr Ala Asp
Ala Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Leu Leu Phe65 70 75 80Leu Gln Met Asn Asp Leu Lys Ser Glu Asp Thr
Ala Met Tyr Tyr Cys 85 90 95Ala Arg Arg Pro Leu Gly His Asn Thr Phe
Asp Ser Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala
Lys Thr Thr Ala Pro Ser Val Tyr 115 120 125Pro Leu Ala Pro Val Cys
Gly Asp Thr Thr Gly Ser Ser Val Thr Leu 130 135 140Gly Cys Leu Val
Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp145 150 155 160Asn
Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser
180 185 190Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro
Ala Ser 195 200 205Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly
Pro Thr Ile Lys 210 215 220Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro
Asn Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Ile Phe Pro Pro
Lys Ile Lys Asp Val Leu Met Ile Ser 245 250 255Leu Ser Pro Ile Val
Thr Cys Val Val Val Asp Val Ser Glu Asp Asp 260 265 270Pro Asp Val
Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr 275 280 285Ala
Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val 290 295
300Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys
Glu305 310 315 320Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala
Pro Ile Glu Arg 325 330 335Thr Ile Ser Lys Pro Lys Gly Ser Val Arg
Ala Pro Gln Val Tyr Val 340 345 350Leu Pro Pro Pro Glu Glu Glu Met
Thr Lys Lys Gln Val Thr Leu Thr 355 360 365Cys Met Val Thr Asp Phe
Met Pro Glu Asp Ile Tyr Val Glu Trp Thr 370 375 380Asn Asn Gly Lys
Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu385 390 395 400Asp
Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys 405 410
415Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu
420 425 430Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr
Pro Gly 435 440 445Lys66449PRTArtificial SequenceDescription of
Artificial Sequence
Synthetic polypeptideMOD_RES(1)..(1)Glu or pyro-Glu 66Xaa Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Thr Gln Pro Gly Lys1 5 10 15Ser Leu
Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Thr
Met His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Phe Ile Arg Ser Gly Ser Gly Ile Val Phe Tyr Ala Asp Ala Val
50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Leu
Phe65 70 75 80Leu Gln Met Asn Asp Leu Lys Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Ala Arg Arg Pro Leu Gly His Asn Thr Phe Asp Ser
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Lys Thr
Thr Ala Pro Ser Val Tyr 115 120 125Pro Leu Ala Pro Val Cys Gly Asp
Thr Thr Gly Ser Ser Val Thr Leu 130 135 140Gly Cys Leu Val Lys Gly
Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp145 150 155 160Asn Ser Gly
Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser 180 185
190Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser
195 200 205Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr
Ile Lys 210 215 220Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu
Leu Gly Gly Pro225 230 235 240Ser Val Phe Ile Phe Pro Pro Lys Ile
Lys Asp Val Leu Met Ile Ser 245 250 255Leu Ser Pro Ile Val Thr Cys
Val Val Val Asp Val Ser Glu Asp Asp 260 265 270Pro Asp Val Gln Ile
Ser Trp Phe Val Asn Asn Val Glu Val His Thr 275 280 285Ala Gln Thr
Gln Thr His Arg Glu Asp Tyr Gln Ser Thr Leu Arg Val 290 295 300Val
Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu305 310
315 320Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu
Arg 325 330 335Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln
Val Tyr Val 340 345 350Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys
Gln Val Thr Leu Thr 355 360 365Cys Met Val Thr Asp Phe Met Pro Glu
Asp Ile Tyr Val Glu Trp Thr 370 375 380Asn Asn Gly Lys Thr Glu Leu
Asn Tyr Lys Asn Thr Glu Pro Val Leu385 390 395 400Asp Ser Asp Gly
Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys 405 410 415Lys Asn
Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu 420 425
430Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly
435 440 445Lys67443PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptideMOD_RES(1)..(1)Glu or pyro-Glu 67Xaa
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Thr Gln Pro Gly Lys1 5 10
15Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Ser Phe
20 25 30Thr Met His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Phe Ile Arg Ser Gly Ser Gly Ile Val Phe Tyr Ala Asp
Ala Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Leu Leu Phe65 70 75 80Leu Gln Met Asn Asp Leu Lys Ser Glu Asp Thr
Ala Met Tyr Tyr Cys 85 90 95Ala Arg Arg Pro Leu Gly His Asn Thr Phe
Asp Ser Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala
Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125Pro Leu Ala Pro Gly Ser
Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140Gly Cys Leu Val
Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp145 150 155 160Asn
Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser
180 185 190Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro
Ala Ser 195 200 205Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp
Cys Gly Cys Lys 210 215 220Pro Cys Ile Cys Thr Val Pro Glu Val Ser
Ser Val Phe Ile Phe Pro225 230 235 240Pro Lys Pro Lys Asp Val Leu
Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255Cys Val Val Val Asp
Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270Trp Phe Val
Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285Glu
Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295
300Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val
Asn305 310 315 320Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys 325 330 335Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr
Ile Pro Pro Pro Lys Glu 340 345 350Gln Met Ala Lys Asp Lys Val Ser
Leu Thr Cys Met Ile Thr Asp Phe 355 360 365Phe Pro Glu Asp Ile Thr
Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380Glu Asn Tyr Lys
Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr385 390 395 400Phe
Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410
415Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His
420 425 430Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435
44068448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMOD_RES(1)..(1)Glu or pyro-Glu 68Xaa Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Thr Gln Pro Gly Lys1 5 10 15Ser Leu
Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Thr
Met His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Phe Ile Arg Ser Gly Ser Gly Ile Val Phe Tyr Ala Asp Ala Val
50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Leu
Phe65 70 75 80Leu Gln Met Asn Asp Leu Lys Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Ala Arg Arg Pro Leu Gly His Asn Thr Phe Asp Ser
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Lys Thr
Thr Ala Pro Ser Val Tyr 115 120 125Pro Leu Ala Pro Val Cys Gly Asp
Thr Thr Gly Ser Ser Val Thr Leu 130 135 140Gly Cys Leu Val Lys Gly
Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp145 150 155 160Asn Ser Gly
Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser 180 185
190Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser
195 200 205Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr
Ile Lys 210 215 220Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu
Leu Gly Gly Pro225 230 235 240Asp Val Phe Ile Phe Pro Pro Lys Ile
Lys Asp Val Leu Met Ile Ser 245 250 255Leu Ser Pro Ile Val Thr Cys
Val Val Val Asp Val Ser Glu Asp Asp 260 265 270Pro Asp Val Gln Ile
Ser Trp Phe Val Asn Asn Val Glu Val His Thr 275 280 285Ala Gln Thr
Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val 290 295 300Val
Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu305 310
315 320Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Leu Pro Glu Glu
Arg 325 330 335Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln
Val Tyr Val 340 345 350Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys
Gln Val Thr Leu Thr 355 360 365Cys Met Val Thr Asp Phe Met Pro Glu
Asp Ile Tyr Val Glu Trp Thr 370 375 380Asn Asn Gly Lys Thr Glu Leu
Asn Tyr Lys Asn Thr Glu Pro Val Leu385 390 395 400Asp Ser Asp Gly
Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys 405 410 415Lys Asn
Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu 420 425
430Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly
435 440 44569220PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 69Asp Ile Val Met Thr Gln Ser Pro
Ser Ser Leu Ala Val Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys
Lys Ser Ser Gln Ser Leu Tyr Tyr Ser 20 25 30Gly Val Lys Glu Asn Leu
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ser Pro Lys Leu Leu
Ile Tyr Tyr Ala Ser Ile Arg Phe Thr Gly Val 50 55 60Pro Asp Arg Phe
Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr65 70 75 80Ile Thr
Ser Val Gln Ala Glu Asp Met Gly Gln Tyr Phe Cys Gln Gln 85 90 95Gly
Ile Asn Asn Pro Leu Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile 100 105
110Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser
115 120 125Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu
Asn Asn 130 135 140Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile
Asp Gly Ser Glu145 150 155 160Arg Gln Asn Gly Val Leu Asn Ser Trp
Thr Asp Gln Asp Ser Lys Asp 165 170 175Ser Thr Tyr Ser Met Ser Ser
Thr Leu Thr Leu Thr Lys Asp Glu Tyr 180 185 190Glu Arg His Asn Ser
Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr 195 200 205Ser Pro Ile
Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 22070245PRTMacaca
fascicularis 70Met Arg Trp Cys Leu Phe Leu Ile Trp Ala Gln Gly Leu
Arg Gln Ala1 5 10 15Pro Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu
Thr Thr Gly Asn 20 25 30Ile Ser Ala Lys Lys Gly Gly Ser Val Ile Leu
Gln Cys His Leu Ser 35 40 45Ser Thr Met Ala Gln Val Thr Gln Val Asn
Trp Glu Gln His Asp His 50 55 60Ser Leu Leu Ala Ile Arg Asn Ala Glu
Leu Gly Trp His Ile Tyr Pro65 70 75 80Ala Phe Lys Asp Arg Val Ala
Pro Gly Pro Gly Leu Gly Leu Thr Leu 85 90 95Gln Ser Leu Thr Met Asn
Asp Thr Gly Glu Tyr Phe Cys Thr Tyr His 100 105 110Thr Tyr Pro Asp
Gly Thr Tyr Arg Gly Arg Ile Phe Leu Glu Val Leu 115 120 125Glu Ser
Ser Val Ala Glu His Ser Ala Arg Phe Gln Ile Pro Leu Leu 130 135
140Gly Ala Met Ala Met Met Leu Val Val Ile Cys Ile Ala Val Ile
Val145 150 155 160Val Val Val Leu Ala Arg Lys Lys Lys Ser Leu Arg
Ile His Ser Val 165 170 175Glu Ser Gly Leu Gln Arg Lys Ser Thr Gly
Gln Glu Glu Gln Ile Pro 180 185 190Ser Ala Pro Ser Pro Pro Gly Ser
Cys Val Gln Ala Glu Ala Ala Pro 195 200 205Ala Gly Leu Cys Gly Glu
Gln Gln Gly Asp Asp Cys Ala Glu Leu His 210 215 220Asp Tyr Phe Asn
Val Leu Ser Tyr Arg Ser Leu Gly Ser Cys Ser Phe225 230 235 240Phe
Thr Glu Thr Gly 245
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