NZ794974A - Anti-ctla-4 antibodies and methods of use thereof - Google Patents
Anti-ctla-4 antibodies and methods of use thereofInfo
- Publication number
- NZ794974A NZ794974A NZ794974A NZ79497417A NZ794974A NZ 794974 A NZ794974 A NZ 794974A NZ 794974 A NZ794974 A NZ 794974A NZ 79497417 A NZ79497417 A NZ 79497417A NZ 794974 A NZ794974 A NZ 794974A
- Authority
- NZ
- New Zealand
- Prior art keywords
- antibody
- amino acid
- seq
- ctla
- certain embodiments
- Prior art date
Links
Abstract
The instant disclosure provides antibodies that specifically bind to CTLA-4 (e.g., human CTLA-4) and antagonize CTLA-4 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. bodies, and methods of treating a subject using these antibodies.
Description
The instant disclosure provides antibodies that specifically bind to CTLA-4 (e.g., human CTLA-4)
and nize CTLA-4 function. Also provided are ceutical 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.
NZ 794974
ANTI-CTLA-4 ANTIBODIES AND METHODS OF USE THEREOF
1. RELATED ATIONS
This application is a divisional of New Zealand patent application 754106, which
is the national phase entry in New Zealand of PCT international application
shed as
Provisional Application No: 62/431,272, filed December 7, 2016, which is incorporated by
reference herein in its ty.
2. FIELD
The instant disclosure relates to antibodies that specifically bind to CTLA-4 (e.g.,
human CTLA-4) and methods of using the same.
3. BACKGROUND
T-lymphocytes are central to the adaptive immune response to antigen. At least two
signals are required for full tion of naive T-cells (Bretscher 1999, Proc Natl Acad Sci
USA 96:185-90). A first, antigen-specific signal is provided by interaction of the T-cell
receptor (TCR) with MHC/peptide complex on an antigen-presenting cell (APC). A second,
co-stimulatory signal is provided by the interactions between receptors on the T-cell and their
ligands on an antigen presenting cell (APC). Engagement of both TCR/MHC and costimulatory
interactions leads to T-cell activation via a number of intracellular ys,
including calcium-calcineurin and RAS mitogen-activated protein kinase, and subsequent
activation of transcription factors for a number of effector compounds, including cytokines
such as IL-2. These events lead to T-cell proliferation, generation of a CD4+ helper T-cell (TH)
pool, and expansion of activated CD8+ cytotoxic T-cells. Not only is co-stimulation al
for full T-cell activation, its absence during TCR/MHC engagement results in anergy and/or
apoptosis.
[0004] Multiple positive and negative co-stimulatory pathways are involved in T-cell
regulation; however, the most critical are between CD28 on T-cells and B7-1 (CD80) and B7-
2 (CD86) on APCs. CD28 promotes T-cell differentiation into TH1 phenotype cells and
enhances antibody tion by B cells and activation of T-cells. B7-1 and B7-2, expressed
on APCs such as dendritic cells (DC) and B cells, have overlapping but distinct ons. B7-
2 is constitutively expressed and is rapidly lated on APCs coincident with C
engagement (signal 1). B7-1 sion is very low on the resting cell, but is typically induced
after ged T-cell stimulation. These differences suggest that while B7-2 may be important
in initialization of T-cell activation, B7-1 may play a greater role in perpetuating the immune
response.
After T-cell activation, the negative regulatory receptor Cytotoxic T-Lymphocyte
Antigen 4 (CTLA-4) is upregulated on T-cells (Alegre et al., 2001, Nat Rev Immunol 1:220-
8). CTLA-4 is urally homologous to CD28 but binds more tightly to both B7-1 and B7-
2 ligands. CTLA-4 ts the immune response in several ways: it competes with CD28 for
the B7 ligands and thus blocks co-stimulation; it negatively signals to inhibit T-cell activation;
and it can capture CD80 and CD86 from opposing cells by trans-endocytosis, resulting in
impaired costimulation via CD28 (Krummel and Allison, 1995, J Exp Med 182:459-465;
Walunas et al., 1994, Immunity 1:405-413; Qureshi et al., 2011, Science 332:600-603).
Given the critical role of the B7 co-stimulatory pathway in promoting and
maintaining an immune response, therapeutic agents designed to antagonize this pathway are
promising for the treatment of autoimmune diseases and disorders.
4. Y
[0007] The instant disclosure provides antibodies that specifically bind to human CTLA-4
and antagonize CTLA-4 function, e.g., -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 described herein are particularly
useful for increasing T-cell activation in response to an antigen (e.g., a tumor antigen or an
infectious disease antigen) and/or decreasing Treg-mediated immune ssion, and hence
for ng cancer in a subject or for treating or preventing an infectious disease in a subject.
Accordingly, in one aspect the instant disclosure es an isolated antibody
sing a heavy chain variable region comprising complementarity determining s
CDRH1, CDRH2, and CDRH3 and a light chain variable region comprising complementarity
determining regions CDRL1, CDRL2, and CDRL3, n:
(a) CDRH1 comprises the amino acid sequence of SYSMN (SEQ ID NO: 10);
(b) CDRH2 comprises the amino acid sequence of SISSSSSYIYYAXSVKG (SEQ ID NO:
18), wherein X is E or D;
(c) CDRH3 ses the amino acid sequence of VGLXGPFDI (SEQ ID NO: 19), n X
is F or M;
(d) CDRL1 comprises the amino acid sequence of RASQSVSRYLG (SEQ ID NO: 15);
(e) CDRL2 ses the amino acid sequence of GASTRAT (SEQ ID NO: 16); and
(f) CDRL3 comprises the amino acid sequence of QQYGSSPWT (SEQ ID NO: 17),
and wherein the CDRH1, CDRH2, and CDRH3 ces of the antibody are not SEQ ID NOs:
, 11, and 13, respectively.
In certain embodiments, the CDRH2 comprises the amino acid ce of SEQ
ID NO: 11. In certain embodiments, the CDRH2 ses the amino acid sequence of SEQ
ID NO: 12. In certain embodiments, the CDRH3 comprises the amino acid sequence of SEQ
ID NO: 13. In certain ments, the CDRH3 comprises the amino acid sequence of SEQ
ID NO: 14. In certain embodiments, CDRH1, CDRH2, and CDRH3 comprise the CDRH1,
CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 10, 11, and 14; 10, 12,
and 13; or 10, 12, and 14, respectively. In certain embodiments, CDRH1, CDRH2, and CDRH3
comprise the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs:
, 12, and 14, respectively. In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1,
CDRL2, and CDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 10, 11, 14,
, 16, and 17; 10, 12, 13, 15, 16, and 17; or 10, 12, 14, 15, 16, and 17, respectively. In n
embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 se the amino
acid sequences set forth in SEQ ID NOs: 10, 12, 14, 15, 16, and 17, respectively.
In another aspect, the instant disclosure provides an isolated antibody that
specifically binds to human CTLA-4, comprising a heavy chain variable region comprising
mentarity determining s 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 SYSMN (SEQ ID NO: 10);
(b) CDRH2 comprises the amino acid sequence of SISSSSSYIYYAXSVKG (SEQ ID NO:
18), wherein X is E or D;
(c) CDRH3 comprises the amino acid sequence of VGLXGPFDI (SEQ ID NO: 19), wherein X
is F or M;
(d) CDRL1 comprises the amino acid sequence of RASQSVSRYLG (SEQ ID NO: 15);
(e) CDRL2 comprises the amino acid sequence of GASTRAT (SEQ ID NO: 16); and
(f) CDRL3 comprises the amino acid sequence of QQYGSSPWT (SEQ ID NO: 17),
and wherein the CDRH1, CDRH2, and CDRH3 sequences of the antibody are not SEQ ID
NOs: 10, 11, and 13, respectively.
In certain embodiments, the CDRH2 comprises the amino acid sequence of SEQ
ID NO: 11. In certain ments, the CDRH2 comprises the amino acid sequence of SEQ
ID NO: 12. In certain embodiments, the CDRH3 comprises the amino acid sequence of SEQ
ID NO: 13. In certain embodiments, the CDRH3 comprises the amino acid sequence of SEQ
ID NO: 14. In certain embodiments, CDRH1, CDRH2, and CDRH3 se the CDRH1,
CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 10, 11, and 14; 10, 12,
and 13; or 10, 12, and 14, respectively. In n embodiments, CDRH1, CDRH2, and CDRH3
comprise the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs:
, 12, and 14, respectively. In certain ments, CDRH1, CDRH2, CDRH3, CDRL1,
CDRL2, and CDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 10, 11, 14,
, 16, and 17; 10, 12, 13, 15, 16, and 17; or 10, 12, 14, 15, 16, and 17, respectively. In n
embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino
acid sequences set forth in SEQ ID NOs: 10, 12, 14, 15, 16, and 17, respectively.
In another aspect, the instant disclosure provides an ed antibody that
specifically binds to human CTLA-4, comprising a heavy chain le region comprising
complementarity ining s CDRH1, CDRH2, and CDRH3, and a light chain
variable region comprising complementarity determining regions CDRL1, CDRL2, and
CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the
amino acid sequences set forth in SEQ ID NOs: 10, 12, 14, 15, 16, and 17, respectively.
In certain embodiments, the antibody comprises a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the antibody
comprises a heavy chain variable region comprising an amino acid sequence which is at least
75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence selected from
the group consisting of SEQ ID NOs: 2 and 4-8. In certain embodiments, the antibody
comprises a heavy chain variable region comprising an amino acid sequence which is at least
75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence selected from
the group consisting of SEQ ID NOs: 3. In certain embodiments, the heavy chain variable
region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:
2 and 4-8. In certain embodiments, the heavy chain variable region comprises the amino acid
sequence of SEQ ID NO: 8. In certain embodiments, the heavy chain variable region comprises
the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the dy comprises a
heavy chain sing the amino acid sequence of SEQ ID NO: 23. In certain embodiments,
the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24.
In n embodiments, the antibody comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO: 25. In certain embodiments, the antibody ses a heavy chain
comprising the amino acid sequence of SEQ ID NO: 26. In certain embodiments, the antibody
comprises a heavy chain variable region having an amino acid sequence derived from a human
IGHV3-21 germline sequence (e.g., IGHV3-21*01, e.g., having amino acid sequence of SEQ
ID NO: 21).
In certain embodiments, the antibody comprises a light chain variable region
comprising an amino acid sequence which is at least 75%, 80%, 85%, 90%, 95%, 99%, or
100% identical to the amino acid ce of SEQ ID NO: 9. In certain embodiments, the
antibody comprises a light chain variable region comprising the amino acid sequence of SEQ
ID NO: 9. In certain embodiments, the antibody comprises a light chain sing the amino
acid ce of SEQ ID NO: 27. In n embodiments, the antibody comprises a light
chain variable region having an amino acid sequence derived from a human IGKV3-20
germline sequence (e.g., IGKV3-20*01, e.g., having amino acid sequence of SEQ ID NO: 22).
In n embodiments, the instant disclosure provides an isolated antibody that
specifically binds to human CTLA-4, the antibody comprising a heavy chain variable region
having an amino acid sequence d from a human IGHV3-21 germline sequence (e.g.,
IGHV3-21*01, e.g., having amino acid sequence of SEQ ID NO: 21), wherein the heavy chain
variable region comprises the amino acid sequence set forth in SEQ ID NO: 14. In certain
embodiments, the antibody comprises a light chain le region having an amino acid
sequence d from a human IGKV3-20 ne sequence (e.g., IGKV3-20*01, e.g.,
having amino acid sequence of SEQ ID NO: 22).
In another aspect, the t disclosure provides an isolated antibody that
specifically binds to human CTLA-4, comprising a heavy chain variable region comprising an
amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8. In certain
embodiments, the antibody comprises a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 8. In certain embodiments, the antibody comprises a heavy chain
comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-26.
In certain embodiments, the antibody comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO: 23. In certain ments, the antibody comprises a heavy chain
comprising the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the antibody
comprises a heavy chain sing the amino acid sequence of SEQ ID NO: 25. In certain
embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of
SEQ ID NO: 26.
In another aspect, the instant sure provides an isolated antibody that
specifically binds to human CTLA-4, comprising a heavy chain variable region and a light
chain variable region, wherein the heavy chain variable region and the light chain variable
region comprise the amino acid sequences set forth in SEQ ID NOs: 2 and 9; 3 and 9; 4 and 9;
and 9; 6 and 9; 7 and 9; or 8 and 9, respectively. In certain embodiments, the antibody
comprises a heavy chain variable region comprising the amino acid ce of SEQ ID NO:
8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9. In
certain ments, the antibody comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO: 23; and a light chain comprising the amino acid sequence of SEQ ID
NO: 27. In certain embodiments, the antibody ses a heavy chain comprising the amino
acid sequence of SEQ ID NO: 24; and a light chain comprising the amino acid sequence of
SEQ ID NO: 27. In certain embodiments, the antibody ses a heavy chain comprising
the amino acid sequence of SEQ ID NO: 25; and a light chain comprising the amino acid
sequence of SEQ ID NO: 27. In certain embodiments, the antibody comprises a heavy chain
comprising the amino acid sequence of SEQ ID NO: 26; and a light chain comprising the amino
acid ce of SEQ ID NO: 27.
In another aspect, the instant disclosure provides an ed antibody that
specifically binds to human , comprising a heavy chain variable region having an
amino acid sequence derived from a human IGHV3-21*01 germline sequence (e.g., IGHV3-
21*01, e.g., having amino acid sequence of SEQ ID NO: 21); and a light chain variable region
having an amino acid sequence derived from a human IGKV3-20*01 germline sequence (e.g.,
IGKV3-20*01, e.g., having amino acid sequence of SEQ ID NO: 22).
In certain embodiments, the antibody comprises a heavy chain constant region
selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain
ments, the heavy chain constant region is IgG1. In certain embodiments, the heavy
chain constant region is IgG2. In certain embodiments, the antibody comprises a light chain
constant region selected from the group consisting of human Igκ and Igλ.
In certain ments, the antibody comprises an IgG1 heavy chain constant
region. In certain embodiments, the antibody comprises a heavy chain constant region
comprising the amino acid sequence of SEQ ID NO: 28. In certain embodiment, the amino
acid sequence of the IgG1 heavy chain constant region comprises S239D/I332E mutations,
numbered according to the EU numbering system. In certain embodiments, the antibody
comprises a heavy chain constant region comprising the amino acid ce of SEQ ID NO:
29. In certain embodiments, the amino acid sequence of the IgG1 heavy chain constant region
comprises S239D/A330L/I332E mutations, numbered according to the EU numbering system.
In n embodiments, the antibody comprises a heavy chain constant region sing the
amino acid sequence of SEQ ID NO: 30. In certain ments, the amino acid ce of
the IgG1 heavy chain constant region comprises L235V/F243L/R292P/Y300L/P396L
mutations, numbered according to the EU numbering system. In certain embodiments, the
dy comprises a heavy chain constant region comprising the amino acid sequence of SEQ
ID NO: 31. In certain embodiments, the IgG1 heavy chain constant region is afucosylated IgG1.
In certain embodiments, the antibody comprises a human IgG heavy chain constant
region that is a variant of a wild type human IgG heavy chain constant region, wherein the
variant human IgG heavy chain constant region binds to FcγRIIIA with a higher affinity than
the wild type human IgG heavy chain nt region binds to FcγRIIIA. In certain
embodiments, the variant human IgG heavy chain constant region is a variant human IgG1
heavy chain constant region.
[0022] In certain embodiments, the antibody comprises a light chain constant region
selected from the group consisting of human Igκ and Igλ. In certain embodiments, the antibody
comprises an Igκ light chain constant region. In certain embodiments, the antibody comprises
a light chain constant region comprising the amino acid sequence of SEQ ID NO: 32.
In another aspect, the instant disclosure provides an isolated antibody that cross-
competes for binding to human CTLA-4 with an dy described herein. In another aspect,
the instant disclosure es an isolated antibody that cross-competes for g to human
CTLA-4 with an antibody comprising the heavy and light chain variable region amino acid
sequences set forth in SEQ ID NOs: 8 and 9, respectively.
In another aspect, the t disclosure provides an isolated antibody that binds to
the same epitope on human CTLA-4 as an antibody described . In another aspect, the
instant disclosure provides an isolated antibody that binds to the same epitope on human
CTLA-4 as an antibody comprising the heavy and light chain variable region amino acid
sequences set forth in SEQ ID NOs: 8 and 9, respectively.
In another aspect, the instant disclosure provides an isolated antibody that binds,
e.g., specifically binds, to an epitope of human CTLA-4. In certain embodiments, the antibody
binds to an epitope located within a region of human CTLA-4 consisting of an amino acid
ce selected from the group ting of SEQ ID NOs: 34-39. In n embodiments,
the antibody binds to an epitope located within a region of human CTLA-4 consisting of the
amino acid sequence of SEQ ID NO: 37. In certain embodiments, the antibody binds to an
epitope located within a region of human CTLA-4 consisting of the amino acid sequence of
SEQ ID NO: 36. In n embodiments, the antibody binds to an epitope located within a
region of human CTLA-4 consisting of the amino acid ce of SEQ ID NO: 35. In n
embodiments, the antibody binds to an epitope located within a region of human CTLA-4
consisting of the amino acid sequence of SEQ ID NO: 34. In certain embodiments, the antibody
binds to an epitope located within a region of human CTLA-4 ting of the amino acid
sequence of SEQ ID NO: 38. In certain embodiments, the antibody binds to an epitope located
within a region of human CTLA-4 consisting of the amino acid sequence of SEQ ID NO: 39.
In another aspect, the instant disclosure provides an antibody or isolated antibody
that specifically binds to the same epitope of human CTLA-4 as any antibody of the present
invention. In certain embodiments, the antibody binds to an epitope located within a region of
human CTLA-4 consisting of an amino acid sequence selected from the group ting of
SEQ ID NOs: 34-39. In certain embodiments, the antibody binds to an epitope located within
a region of human CTLA-4 consisting of the amino acid sequence of SEQ ID NO: 37. In
certain embodiments, the antibody binds to an epitope located within a region of human CTLA-
4 consisting of the amino acid sequence of SEQ ID NO: 36. In certain embodiments, the
antibody binds to an epitope located within a region of human CTLA-4 consisting of the amino
acid ce of SEQ ID NO: 35. In certain embodiments, the antibody binds to an epitope
located within a region of human CTLA-4 consisting of the amino acid sequence of SEQ ID
NO: 34. In certain embodiments, the antibody binds to an epitope located within a region of
human CTLA-4 consisting of the amino acid ce of SEQ ID NO: 38. In certain
embodiments, the antibody binds to an epitope located within a region of human CTLA-4
consisting of the amino acid sequence of SEQ ID NO: 39.
In another aspect, the t disclosure provides an antibody that, when bound to a
human CTLA-4 protein or fragment thereof, e.g., comprising the amino acid sequence of
residues 37-162 of SEQ ID NO: 33, reduces en/deuterium exchange in a region
consisting of the amino acid sequence set forth in SEQ ID NO: 34 relative to
hydrogen/deuterium ge in the region consisting of the amino acid sequence set forth in
SEQ ID NO: 34 in the absence of the antibody, as determined by a hydrogen/deuterium assay.
In another aspect, the instant sure provides an antibody that, when bound to a human
CTLA-4 protein or fragment thereof, e.g., comprising the amino acid sequence of residues 37-
162 of SEQ ID NO: 33, reduces hydrogen/deuterium exchange in a region ting of the
amino acid sequence set forth in SEQ ID NO: 35 relative to hydrogen/deuterium exchange in
the region ting of the amino acid ce set forth in SEQ ID NO: 35 in the absence of
the dy, as determined by a hydrogen/deuterium assay. In another aspect, the instant
disclosure provides an antibody that, when bound to a human CTLA-4 protein or fragment
thereof, e.g., comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33,
reduces en/deuterium exchange in a region consisting of the amino acid sequence set
forth in SEQ ID NO: 36 relative to hydrogen/deuterium exchange in the region consisting of
the amino acid sequence set forth in SEQ ID NO: 36 in the absence of the antibody, as
determined by a hydrogen/deuterium assay. In another aspect, the instant disclosure provides
an antibody that, when bound to a human CTLA-4 protein or fragment thereof, e.g., comprising
the amino acid ce of residues 37-162 of SEQ ID NO: 33, reduces en/deuterium
exchange in a region ting of the amino acid sequence set forth in SEQ ID NO: 37 relative
to hydrogen/deuterium exchange in the region consisting of the amino acid sequence set forth
in SEQ ID NO: 37 in the absence of the antibody, as determined by a hydrogen/deuterium
assay. In another aspect, the instant disclosure provides an antibody that, when bound to a
human CTLA-4 protein or fragment thereof, e.g., comprising the amino acid sequence of
residues 37-162 of SEQ ID NO: 33, reduces hydrogen/deuterium exchange in a region
consisting of the amino acid sequence set forth in SEQ ID NO: 38 relative to
hydrogen/deuterium exchange in the region consisting of the amino acid sequence set forth in
SEQ ID NO: 38 in the absence of the antibody, as determined by a hydrogen/deuterium assay.
In another aspect, the instant disclosure provides an antibody that, when bound to a human
CTLA-4 protein or fragment thereof, e.g., comprising the amino acid sequence of residues 37-
162 of SEQ ID NO: 33, reduces hydrogen/deuterium ge in a region ting of the
amino acid sequence set forth in SEQ ID NO: 39 relative to hydrogen/deuterium exchange in
the region consisting of the amino acid sequence set forth in SEQ ID NO: 39 in the absence of
the antibody, as ined by a hydrogen/deuterium assay.
[0028] In another aspect, the instant disclosure provides an antibody or isolated dy
that specifically binds to the same epitope of human CTLA-4 as any antibody of the present
invention. In certain embodiments, the antibody, when bound to a human CTLA-4 protein or
fragment thereof, e.g., comprising the amino acid sequence of residues 37-162 of SEQ ID NO:
33, reduces en/deuterium exchange in a region consisting of the amino acid sequence
set forth in SEQ ID NO: 34 ve to hydrogen/deuterium exchange in the region consisting
of the amino acid sequence set forth in SEQ ID NO: 34 in the absence of the antibody, as
determined by a hydrogen/deuterium assay. In n embodiments, the antibody, when bound
to a human CTLA-4 protein or fragment thereof, e.g., comprising the amino acid ce of
residues 37-162 of SEQ ID NO: 33, s hydrogen/deuterium exchange in a region
consisting of the amino acid sequence set forth in SEQ ID NO: 35 relative to
hydrogen/deuterium exchange in the region consisting of the amino acid sequence set forth in
SEQ ID NO: 35 in the absence of the antibody, as determined by a hydrogen/deuterium assay.
In certain embodiments, the antibody, when bound to a human CTLA-4 protein or fragment
thereof, e.g., comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33,
reduces hydrogen/deuterium exchange in a region consisting of the amino acid sequence set
forth in SEQ ID NO: 36 relative to hydrogen/deuterium exchange in the region ting of
the amino acid sequence set forth in SEQ ID NO: 36 in the e of the antibody, as
determined by a hydrogen/deuterium assay. In certain embodiments, the antibody, when bound
to a human CTLA-4 protein or fragment f, e.g., comprising the amino acid sequence of
residues 37-162 of SEQ ID NO: 33, reduces hydrogen/deuterium exchange in a region
consisting of the amino acid sequence set forth in SEQ ID NO: 37 relative to
hydrogen/deuterium exchange in the region consisting of the amino acid sequence set forth in
SEQ ID NO: 37 in the absence of the antibody, as determined by a hydrogen/deuterium assay.
In certain embodiments, the antibody, when bound to a human CTLA-4 protein or nt
f, e.g., comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33,
reduces hydrogen/deuterium exchange in a region consisting of the amino acid sequence set
forth in SEQ ID NO: 38 relative to hydrogen/deuterium exchange in the region consisting of
the amino acid sequence set forth in SEQ ID NO: 38 in the absence of the antibody, as
determined by a hydrogen/deuterium assay. In certain embodiments, the antibody, when bound
to a human CTLA-4 protein or fragment thereof, e.g., comprising the amino acid ce of
residues 37-162 of SEQ ID NO: 33, reduces hydrogen/deuterium exchange in a region
ting of the amino acid sequence set forth in SEQ ID NO: 39 relative to
hydrogen/deuterium exchange in the region consisting of the amino acid sequence set forth in
SEQ ID NO: 39 in the absence of the antibody, as determined by a hydrogen/deuterium assay.
In certain embodiments, the antibody is a human dy. In certain embodiment,
the antibody is a bispecific antibody.
In certain embodiments, the antibody is antagonistic to human CTLA-4. In certain
embodiments, the dy deactivates, reduces, or inhibits an activity of human CTLA-4. In
certain embodiments, the antibody ts g of human CTLA-4 to human CD80 or
human CD86. In certain embodiments, the antibody induces IL-2 production by peripheral
blood mononuclear cells (PBMCs) stimulated with staphylococcal enterotoxin A (SEA).
In certain embodiments, the antibody is conjugated to a xic agent, cytostatic
agent, toxin, radionuclide, or able label.
[0032] In certain embodiments, the N-terminal amino acid residue of the heavy chain
variable region and/or the light chain variable region of the antibody has been ted to
pyroglutamate.
In one embodiment, the present invention relates to an antibody of the present
invention for use as a medicament.
In one embodiment, the present invention relates to use of an dy of the present
invention for preparing pharmaceutical compositions or medicaments for immunotherapy. In
certain embodiments, the immunotherapy is for sing T-cell activation in response to an
antigen in a subject, optionally for treating cancer, or treating or preventing infectious diseases.
[0035] In one embodiment, the present invention relates to an antibody of the t
invention for use as a diagnostic.
In one ment, the present invention relates to the use of an antibody of the
present invention for in vitro detection of human CTLA-4 in a biological sample.
In another aspect, the instant disclosure provides a pharmaceutical composition
comprising an anti-CTLA-4 antibody described herein and a pharmaceutically acceptable
carrier or excipient.
In another aspect, the instant disclosure provides an isolated polynucleotide
encoding a heavy and/or light chain of an antibody described herein. In another , the
instant disclosure es a vector comprising the polynucleotide. In r aspect, the
instant disclosure provides a recombinant host cell comprising the polynucleotide or the vector.
In another aspect, the instant disclosure provides a method of producing an dy that binds
to human CTLA-4, the method sing culturing the host cell so that the polynucleotide is
expressed and the dy is produced.
In another aspect, the instant disclosure provides a method of sing T-cell
activation in response to an antigen in a t, the method comprising administering to the
subject an effective amount of an anti-CTLA-4 antibody or pharmaceutical composition
described 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 anti-
CTLA-4 antibody or pharmaceutical composition described herein.
[0040] In certain ments, the subject has cancer. In certain embodiments, the subject
has a metastatic or locally advanced tumor (e.g., solid . In certain embodiments, the
cancer is treated in accordance with a method described herein as a first cancer therapy after
diagnosis of the metastatic or y advanced tumor (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2,
3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis). In certain ments,
the cancer is treated in accordance with a method bed herein as the first cancer therapy
after diagnosis of tumor progression (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12
weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis of tumor progression) that has occurred
despite previous treatment of the tumor with a different cancer therapy, optionally wherein the
method described herein is provided as the second cancer therapy administered. In certain
embodiments, the cancer is treated in accordance with a method described herein as the first
cancer therapy after diagnosis of toxicity of a different cancer therapy (e.g., within 1, 2, 3, 4,
, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis of
toxicity of the ent cancer therapy), optionally n the method described herein is
provided as the second cancer therapy administered. In certain embodiments, the cancer treated
in accordance with the methods described herein is a metastatic or locally advanced cancer
(e.g., solid tumor) for which no standard therapy is available. In other embodiments, the cancer
treated in accordance with the methods described herein is a metastatic or locally advanced
cancer (e.g., solid tumor) for which a standard therapy has failed (i.e., the cancer has progressed
after the rd therapy). In certain embodiments, a therapy fails if the cancer is refractory
to the therapy. In n embodiments, a therapy fails if the cancer relapses after responding,
fully or partially, to the therapy. In certain ments, metastatic or locally advanced cancer
(e.g., solid tumor) has been confirmed ogically or cytologically.
In certain embodiments, the cancer expresses PD-L1. In certain embodiments, the
percentage of tumor cells in a sample of the cancer that exhibit detectable membrane expression
(e.g., partial or complete membrane sion) of PD-L1 is at least 1% (e.g., at least 2%, 3%,
4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%). In
certain embodiments, the percentage of tumor cells in a sample of the cancer that exhibit
detectable membrane expression (e.g., partial or complete membrane sion) of PD-L1 is
at least 1%. In certain embodiments, the percentage of tumor cells in a sample of the cancer
that exhibit detectable membrane expression (e.g., partial or te membrane sion)
of PD-L1 is at least 5%. In n embodiments, the percentage of tumor cells in a sample of
the cancer that exhibit detectable ne expression (e.g., partial or complete membrane
expression) of PD-L1 is at least 25%. In certain embodiments, the percentage of tumor cells
in a sample of the cancer that exhibit detectable membrane expression (e.g., partial or complete
membrane sion) of PD-L1 is at least 50%.
In certain embodiments, the atic or locally advanced tumor ses PD-L1.
In certain embodiments, the percentage of tumor cells in a sample of the metastatic or locally
advanced tumor that exhibit detectable membrane expression (e.g., partial or complete
membrane expression) of PD-L1 is at least 1% (e.g., at least 2%, 3%, 4%, 5%, 10%, 15%, 20%,
%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%). In certain embodiments, the
percentage of tumor cells in a sample of the metastatic or locally advanced tumor that exhibit
detectable membrane expression (e.g., l or complete membrane expression) of PD-L1 is
at least 1%. In certain embodiments, the percentage of tumor cells in a sample of the metastatic
or locally advanced tumor that t detectable membrane expression (e.g., l or
te membrane expression) of PD-L1 is at least 5%. In certain embodiments, the
tage of tumor cells in a sample of the metastatic or y advanced tumor that exhibit
detectable membrane expression (e.g., partial or complete membrane expression) of PD-L1 is
at least 25%. In certain embodiments, the percentage of tumor cells in a sample of the
metastatic or locally advanced tumor that exhibit detectable membrane sion (e.g., partial
or complete membrane expression) of PD-L1 is at least 50%.
In certain embodiments, the cancer is a al cancer. In certain ments,
the cancer is a metastatic or locally advanced cancer (e.g., solid tumor). In certain
embodiments, the metastatic or locally advanced cancer (e.g., solid tumor) is a metastatic or
locally advanced, unresectable squamous cell carcinoma, adenosquamous carcinoma, or
adenocarcinoma of the . In certain embodiments, no standard therapy is available for the
cancer (e.g., cervical cancer) or metastatic or locally advanced tumor (e.g., solid tumor). In
certain embodiments, the cancer (e.g., cervical cancer) or metastatic or locally advanced tumor
(e.g., solid tumor) is refractory to a standard therapy. In certain embodiments, the cancer (e.g.,
cervical cancer) or metastatic or locally advanced tumor (e.g., solid tumor) has ed after a
standard therapy. In certain embodiments, the standard therapy comprises a platinumcontaining
chemotherapy. In certain embodiments, the standard y is a platinumcontaining
t. In certain embodiments, the cancer (e.g., cervical cancer) is a metastatic
or locally advanced, unresectable squamous cell carcinoma, adenosquamous carcinoma, or
adenocarcinoma of the cervix that has relapsed after a platinum-containing doublet
administered for treatment of advanced rent, unresectable, or metastatic) disease. In
certain embodiments, the cancer (e.g., cervical cancer) or metastatic or y advanced tumor
is HPV positive. In certain embodiments, the cancer or metastatic or locally advanced solid
tumor is head and neck cancer, melanoma, renal cell carcinoma, urothelial oma, or
endometrial carcinoma. In certain embodiments, the cancer (e.g., cervical cancer) or metastatic
or locally advanced solid tumor is associated with microsatellite instability.
In certain embodiments, the subject has cervical cancer (e.g., a metastatic or locally
advanced, unresectable squamous cell carcinoma, adenosquamous carcinoma, or
adenocarcinoma of the cervix), and the method comprises administering to the t an
effective amount of an anti-CTLA-4 antibody described herein, e.g., 84.H3 (IgG1
S239D/A330L/I332E), or pharmaceutical composition sing such anti-CTLA-4 antibody
as a first cancer therapy after diagnosis of the cervical cancer (e.g., within 1, 2, 3, 4, 5, or 6
days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis), optionally
wherein the anti-CTLA-4 antibody described herein, e.g., AGEN1884.H3 (IgG1
S239D/A330L/I332E), or pharmaceutical composition comprising such anti-CTLA-4 antibody
is administered at the dosage and frequency selected from the group consisting of 0.3 mg/kg
every four weeks, 1 mg/kg every four weeks, 3 mg/kg every four weeks, 0.3 mg/kg every six
weeks, 1 mg/kg every six weeks, 3 mg/kg every six weeks, 0.3 mg/kg every twelve weeks, 1
mg/kg every twelve weeks, and 3 mg/kg every twelve weeks. In certain embodiments, the
subject has cervical cancer (e.g., a metastatic or locally advanced, unresectable squamous cell
carcinoma, adenosquamous carcinoma, or adenocarcinoma of the cervix), and the method
comprises administering to the subject an effective amount of a therapeutic combination
comprising an anti-CTLA-4 antibody described , e.g., AGEN1884.H3 (IgG1
S239D/A330L/I332E), or pharmaceutical composition comprising such anti-CTLA-4
antibody, and pembrolizumab as a first cancer therapy after diagnosis of the cervical cancer
(e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months
after sis), optionally wherein the anti-CTLA-4 antibody described , e.g.,
AGEN1884.H3 (IgG1 S239D/A330L/I332E), or pharmaceutical composition comprising such
anti-CTLA-4 antibody, is administered at the dosage and frequency selected from the group
consisting of 0.3 mg/kg every four weeks, 1 mg/kg every four weeks, 3 mg/kg every four
weeks, 0.3 mg/kg every six weeks, 1 mg/kg every six weeks, 3 mg/kg every six weeks, 0.3
mg/kg every twelve weeks, 1 mg/kg every twelve weeks, and 3 mg/kg every twelve weeks,
and pembrolizumab is administered at 200 mg every three weeks.
In n embodiments, the cancer is a non-small cell lung cancer ). In
certain embodiments, the NSCLC is a Stage IV NSCLC. In certain embodiments, the
percentage of tumor cells in a sample of the NSCLC that exhibit detectable membrane
expression (e.g., l or complete membrane expression) of PD-L1 is at least 1% (e.g., at
least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%,
or 90%). In certain embodiments, the percentage of tumor cells in a sample of the NSCLC that
exhibit detectable membrane expression (e.g., partial or complete membrane expression) of
PD-L1 is at least 1%. In certain embodiments, the percentage of tumor cells in a sample of the
NSCLC that exhibit detectable membrane expression (e.g., partial or te ne
expression) of PD-L1 is at least 5%. In certain embodiments, the tage of tumor cells in
a sample of the NSCLC that exhibit able membrane expression (e.g., partial or te
membrane expression) of PD-L1 is at least 25%. In certain embodiments, the percentage of
tumor cells in a sample of the NSCLC that exhibit detectable membrane expression (e.g., partial
or complete membrane sion) of PD-L1 is at least 50%. In certain embodiments, the
NSCLC has no EGFR or ALK genomic tumor aberrations. In certain embodiments, the
metastatic or y advanced NSCLC has no EGFR sensitizing mutation (e.g., mutation that
is amenable to treatment with a ne kinase inhibitor including erlotinib, gefitinib, or
afatanib) or ALK translocation. In certain ments, the subject has received no prior
systemic chemotherapy treatment for NSCLC.
In certain embodiments, the metastatic or locally advanced solid tumor is a
metastatic or locally advanced non-small cell lung cancer (NSCLC). In certain embodiments,
the metastatic or y advanced solid tumor is a metastatic non-small cell lung cancer
(NSCLC). In n embodiments, the metastatic or locally advanced solid tumor is a Stage
IV, metastatic or locally advanced NSCLC. In certain embodiments, the metastatic or locally
advanced solid tumor is a Stage IV, metastatic NSCLC. In n embodiments, the
percentage of tumor cells in a sample of the metastatic or locally advanced NSCLC that exhibit
detectable membrane expression (e.g., partial or complete membrane expression) of PD-L1 is
at least 1% (e.g., at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
60%, 70%, 80%, or 90%). In n embodiments, the percentage of tumor cells in a sample
of the metastatic or locally advanced NSCLC that exhibit detectable membrane expression
(e.g., partial or complete membrane sion) of PD-L1 is at least 1%. In certain
embodiments, the percentage of tumor cells in a sample of the metastatic or locally advanced
NSCLC that exhibit detectable membrane expression (e.g., partial or te ne
expression) of PD-L1 is at least 5%. In certain embodiments, the tage of tumor cells in
a sample of the metastatic or locally advanced NSCLC that exhibit detectable membrane
expression (e.g., partial or complete membrane expression) of PD-L1 is at least 25%. In certain
embodiments, the percentage of tumor cells in a sample of the metastatic or locally advanced
NSCLC that exhibit detectable membrane expression (e.g., partial or complete membrane
expression) of PD-L1 is at least 50%. In certain ments, the metastatic or locally
advanced NSCLC has no EGFR or ALK c tumor aberrations. In certain embodiments,
the subject has received no prior systemic chemotherapy ent for metastatic or locally
ed NSCLC.
In certain embodiments, the subject has NSCLC (e.g., Stage IV, metastatic, or
locally advanced NSCLC), optionally wherein the percentage of tumor cells in a sample of the
NSCLC that exhibit detectable expression (e.g., membrane expression, partial or complete
membrane expression) of PD-L1 is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%,
%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%, and the method ses administering to
the subject an effective amount of an anti-CTLA-4 antibody described herein, e.g.,
AGEN1884.H3 (IgG1 S239D/A330L/I332E), or pharmaceutical composition comprising such
anti-CTLA-4 antibody, as a first cancer therapy after diagnosis of the cervical cancer (e.g.,
within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after
diagnosis), optionally wherein the anti-CTLA-4 antibody described herein, e.g.,
AGEN1884.H3 (IgG1 S239D/A330L/I332E), or pharmaceutical composition comprising such
anti-CTLA-4 antibody, is administered at the dosage and frequency ed from the group
consisting of 0.3 mg/kg every four weeks, 1 mg/kg every four weeks, 3 mg/kg every four
weeks, 0.3 mg/kg every six weeks, 1 mg/kg every six weeks, 3 mg/kg every six weeks, 0.3
mg/kg every twelve weeks, 1 mg/kg every twelve weeks, and 3 mg/kg every twelve weeks. In
certain embodiments, the subject has NSCLC (e.g., Stage IV, metastatic, or y advanced
NSCLC), optionally n the percentage of tumor cells in a sample of the NSCLC that
exhibit detectable expression (e.g., membrane sion, partial or complete membrane
expression) of PD-L1 is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 60%, 70%, 80%, or 90%, and the method comprises administering to the subject a
therapeutic combination comprising an anti-CTLA-4 antibody described herein, e.g.,
AGEN1884.H3 (IgG1 S239D/A330L/I332E), or pharmaceutical composition comprising such
anti-CTLA-4 antibody, and pembrolizumab as a first cancer y after diagnosis of the
cervical cancer (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4,
6, 8, or 12 months after diagnosis), ally wherein the anti-CTLA-4 antibody described
herein, e.g., AGEN1884.H3 (IgG1 A330L/I332E), or pharmaceutical composition
comprising such anti-CTLA-4 antibody, is administered at the dosage and frequency selected
from the group consisting of 0.3 mg/kg every four weeks, 1 mg/kg every four weeks, 3 mg/kg
every four weeks, 0.3 mg/kg every six weeks, 1 mg/kg every six weeks, 3 mg/kg every six
weeks, 0.3 mg/kg every twelve weeks, 1 mg/kg every twelve weeks, and 3 mg/kg every twelve
weeks, and pembrolizumab is administered at 200 mg every three weeks.
In certain embodiments, the cancer is a cutaneous squamous-cell carcinoma
(cSCC). In certain embodiments, the metastatic or y advanced solid tumor is a Stage IV
cutaneous squamous-cell carcinoma (cSCC). In certain embodiments, the cSCC is diagnosed
ogically or cytologically according to the eighth edition of the American Joint Committee
on Cancer g manual. In certain embodiments, the cSCC is not curable with radiation
y. In certain embodiments, the subject has cSCC (e.g., Stage IV cSCC), and the method
comprises administering to the subject an effective amount of an anti-CTLA-4 antibody
described herein, e.g., AGEN1884.H3 (IgG1 S239D/A330L/I332E), or pharmaceutical
composition comprising such anti-CTLA-4 antibody, as a first cancer y after diagnosis
of the cSCC (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8,
or 12 months after diagnosis), optionally wherein the anti-CTLA-4 antibody described herein,
e.g., AGEN1884.H3 (IgG1 S239D/A330L/I332E), or pharmaceutical composition comprising
such anti-CTLA-4 antibody, is administered at the dosage and frequency selected from the
group consisting of 0.3 mg/kg every four weeks, 1 mg/kg every four weeks, 3 mg/kg every
four weeks, 0.3 mg/kg every six weeks, 1 mg/kg every six weeks, 3 mg/kg every six weeks,
0.3 mg/kg every twelve weeks, 1 mg/kg every twelve weeks, and 3 mg/kg every twelve weeks.
In certain embodiments, the t has cSCC (e.g., Stage IV cSCC), and the method comprises
administering to the subject an effective amount of a therapeutic combination comprising an
anti-CTLA-4 antibody described herein, e.g., 84.H3 (IgG1 S239D/A330L/I332E), or
pharmaceutical composition comprising such anti-CTLA-4 antibody, and lizumab as a
first cancer y after diagnosis of the cSCC (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4,
6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis), optionally wherein the anti-
CTLA-4 antibody bed herein, e.g., AGEN1884.H3 (IgG1 S239D/A330L/I332E), or
pharmaceutical composition comprising such anti-CTLA-4 antibody, is administered at the
dosage and frequency ed from the group consisting of 0.3 mg/kg every four weeks, 1
mg/kg every four weeks, 3 mg/kg every four weeks, 0.3 mg/kg every six weeks, 1 mg/kg every
six weeks, 3 mg/kg every six weeks, 0.3 mg/kg every twelve weeks, 1 mg/kg every twelve
weeks, and 3 mg/kg every twelve weeks, and pembrolizumab is stered at 200 mg every
three weeks.
In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition
described herein is administered intravenously. In certain embodiments, the anti-CTLA-4
antibody or pharmaceutical composition described herein is administered intravenously at 0.01
mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg, optionally
at an interval of once every two weeks. In certain embodiments, the anti-CTLA-4 antibody or
pharmaceutical composition described herein is administered intravenously at 0.01 mg/kg, 0.03
mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg, ally at an al
of once every three weeks. In certain embodiments, the anti-CTLA-4 antibody or
pharmaceutical composition described herein is administered intravenously at 0.01 mg/kg, 0.03
mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg, optionally at an interval
of once every four weeks. In certain embodiments, the anti-CTLA-4 antibody or
pharmaceutical composition described herein is administered intravenously at 0.01 mg/kg, 0.03
mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg, optionally at an interval
of once every six weeks. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical
composition described herein is administered intravenously at 0.01 mg/kg, 0.03 mg/kg, 0.1
mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg, ally at an interval of once
every twelve weeks. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical
composition described herein is administered intratumorally. In certain embodiments, the anti-
CTLA-4 antibody or pharmaceutical composition described herein is stered
intratumorally at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, or 3 mg/kg,
optionally at an interval of once every three weeks. In certain embodiments, the TLA-4
antibody or pharmaceutical composition described herein is administered intratumorally at
0.03 mg/kg, 0.1 mg/kg, or 0.3 mg/kg, optionally at an interval of once every three weeks. In
certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described
herein is administered intratumorally at a dose that is up to 5-fold, 10-fold, 20-fold, 30-fold,
40-fold, 50-fold, 60-fold, d, 80-fold, 90-fold, 100-fold, or 200-fold lower than a dose
given by systemic administration. In certain embodiments, the anti-CTLA-4 antibody or
pharmaceutical composition described herein is stered intratumorally at a dose that is
up to 10-fold lower than a dose given by systemic administration. In certain embodiments, the
anti-CTLA-4 dy or pharmaceutical ition bed herein is administered
intratumorally at a dose that is up to 100-fold lower than a dose given by systemic
administration. In certain ments, the anti-CTLA-4 antibody or ceutical
composition described herein is administered (e.g., intratumorally or systemically) as a
erapy. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical
composition bed herein is administered intratumorally and the method further comprises
administering an additional therapeutic agent to the subject. In n embodiments, the
additional therapeutic agent is administered systemically. In certain embodiments, the subject
has a solid tumor and the additional therapeutic agent is an anti-PD-1 antibody. In certain
embodiments, the D-1 antibody is pembrolizumab or nivolumab. In certain
embodiments, the pembrolizumab is administered at a dose of 200 mg every three weeks. In
certain embodiments, the subject has head and neck squamous cell carcinoma and the
additional therapeutic agent is an anti-EGFR antibody. In certain embodiments, the anti-EGFR
antibody is cetuximab. In certain embodiments, the subject has HER2+ breast cancer and the
additional therapeutic agent is an anti-HER2 antibody. In certain embodiments, the anti-HER2
antibody is trastuzumab. In certain embodiments, these methods further comprise
administering a chemotherapeutic agent to the subject. In certain embodiments, the
chemotherapeutic agent is administered systemically. In certain embodiments, the
herapeutic agent is gemcitabine. In n embodiments, the anti-CTLA-4 antibody or
pharmaceutical composition described herein is administered intratumorally and the subject
has an ed or metastatic solid tumor. In certain ments, the anti-CTLA-4 antibody
or pharmaceutical composition described herein is administered intratumorally and the subject
has head and neck cancer (e.g., relapsed/refractory head and neck us cell carcinoma).
In n embodiments, the anti-CTLA-4 dy or pharmaceutical composition described
herein is administered intratumorally and the subject has breast cancer (e.g., relapsed/refractory
HER2+ breast cancer). In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical
composition described herein is delivered to a tumor draining lymph node. In certain
embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is
delivered via a localized administration (e.g., subcutaneous administration). In certain
embodiments, the anti-CTLA-4 dy or pharmaceutical composition described herein is
delivered via a localized stration (e.g., subcutaneous administration) at 0.01 mg/kg, 0.03
mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, or 3 mg/kg. In certain embodiments, the anti-CTLA-
4 antibody or pharmaceutical composition described herein is delivered via a localized
administration (e.g., subcutaneous administration) at a dose that is up to 5-fold, 10-fold, 20-
fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, d, 90-fold, 100-fold, or 200-fold lower
than a dose given by systemic administration. In certain embodiments, the TLA-4
antibody or pharmaceutical composition described herein is delivered via a localized
administration (e.g., subcutaneous administration) at a dose that is up to 10-fold lower than a
dose given by systemic administration. In certain embodiments, the anti-CTLA-4 antibody or
pharmaceutical composition described herein is delivered via a localized administration (e.g.,
subcutaneous administration) at a dose that is up to 100-fold lower than a dose given by
systemic administration. In certain ments, the anti-CTLA-4 dy or ceutical
composition described herein is delivered via a localized stration (e.g., subcutaneous
administration) and the method further ses stering an additional therapeutic agent
to the subject. In certain embodiments, the additional therapeutic agent is a vaccine. In certain
embodiments, the vaccine comprises a heat shock protein e complex (HSPPC)
comprising a heat shock protein complexed with an antigenic peptide. In one embodiment, the
heat shock n is gp96 protein and is complexed with a tumor-associated antigenic peptide,
wherein the HSPPC is derived from a tumor ed from a subject. In certain embodiments,
the heat shock protein is selected from the group consisting of hsc70, hsp70, hsp90, hsp110,
grp170, gp96, iculin, a mutant thereof, and combinations of two or more thereof. In
certain embodiments, the heat shock protein is hsc70. In certain embodiments, the heat shock
protein is hsp70. In certain embodiments, the antigenic peptide is synthetic. In certain
embodiments, the t has cancer. In certain embodiments, the subject has an infectious
disease. 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 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 nist anti-PD-L2 antibody, an antagonist anti-CTLA-
4 antibody, an antagonist anti-TIM-3 antibody, an antagonist anti-LAG-3 antibody, an
antagonist anti-CEACAM1 antibody, an agonist anti-GITR antibody, an agonist anti-OX40
antibody, and an agonist anti-CD137 antibody, an agonist R3 antibody, an agonist anti-
TNFSF14 antibody, and an agonist anti-CD27 antibody. In n ments, the
additional therapeutic agent is radiotherapy. In certain ments, the additional therapeutic
agent is an inhibitor of indoleamine-2,3-dioxygenase (IDO). Suitable IDO inhibitors include,
without limitation, epacadostat, 7, indoximod, and NLG919. In certain embodiments,
the additional therapeutic agent is a vaccine. In certain embodiments, the vaccine comprises a
heat shock protein peptide complex (HSPPC) sing a heat shock protein complexed with
an antigenic peptide. In one embodiment, the heat shock protein is gp96 protein and is
complexed with a tumor-associated antigenic e, wherein the HSPPC is derived from a
tumor obtained from a subject.
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 anti-CTLA-4 antibody or pharmaceutical composition described . In another
aspect, the t disclosure provides a method of preventing an infectious disease in a subject,
the method sing administering to the subject an ive amount of an anti-CTLA-4
antibody or pharmaceutical ition described herein.
[0051] In one embodiment, the present invention relates to an antibody of the present
invention, a polynucleotide of the invention, a vector of the ion, and/or a recombinant
host cell of the invention, for use as a medicament.
In one ment, the present invention relates to an antibody of the present
invention, a polynucleotide of the invention, a vector of the invention, and/or a recombinant
host cell of the invention, for use as a diagnostic.
In one embodiment, the present invention relates to the use of an antibody of the
present invention, a polynucleotide of the invention, a vector of the ion, and/or a
recombinant host cell of the invention, for the in vitro detection of human CTLA-4 in a
biological sample.
In one aspect, provided herein is a pharmaceutical composition comprising an anti-
CTLA-4 antibody described herein and a ceutically acceptable carrier or excipient, for
use as a medicament.
In one aspect, provided herein is a pharmaceutical composition comprising an anti-
CTLA-4 antibody described herein and a pharmaceutically acceptable r or excipient, for
use as a diagnostic.
In one aspect, ed herein is a ceutical composition comprising an anti-
CTLA-4 antibody described herein, a polynucleotide of the invention, a vector of the invention,
and/or a recombinant host cell of the invention, and a pharmaceutically acceptable carrier or
excipient. In one aspect, the pharmaceutical composition is for use as a medicament and/or
diagnostic.
In one aspect, the present invention relates to an antibody, polynucleotide, vector,
recombinant host cell, and/or pharmaceutical composition of the t ion, for use in
a method for increasing T-cell activation in response to an antigen.
[0058] In one aspect, the present invention relates to an antibody, polynucleotide, vector,
recombinant host cell, and/or pharmaceutical composition of the present invention, for use in
a method for increasing T-cell activation in response to an antigen in a subject.
In one aspect, the present invention relates to an antibody, polynucleotide, vector,
recombinant host cell, and/or pharmaceutical composition of the present invention, for use in
a method for sing T-cell activation in response to an n in a subject comprising
administering to the subject an effective amount of an dy, polynucleotide, vector,
recombinant host cell, and/or pharmaceutical composition of the invention.
In one aspect, the present invention relates to an antibody, polynucleotide, vector,
recombinant host cell, and/or pharmaceutical composition of the present invention, for use in
a method for the treatment of .
In one aspect, the present invention relates to an antibody, polynucleotide, vector,
recombinant host cell, and/or pharmaceutical composition of the present invention, for use in
a method for the treatment of cancer in a subject.
In one aspect, the present invention relates to an antibody, polynucleotide, vector,
recombinant host cell, and/or pharmaceutical composition of the present invention, for use in
a method for the ent of cancer in a subject comprising stering to the subject an
effective amount of an antibody, polynucleotide, vector, recombinant host cell, and/or
pharmaceutical composition of the invention.
In one aspect, the present ion relates to (a) an antibody, polynucleotide,
vector, recombinant host cell, and/or pharmaceutical composition of the t invention and
(b) an additional therapeutic agent, preferably an anti-PD-1 antibody, for use as a medicament.
In one , the t invention relates to (a) an antibody, cleotide,
vector, recombinant host cell, and/or pharmaceutical composition of the t invention and
(b) an additional therapeutic agent, preferably an anti-PD-1 antibody, for use in a method for
the treatment of cancer. In a preferred embodiment, the cancer is a solid tumor. In another
red embodiment, the antibody, polynucleotide, vector, recombinant host cell, and/or
pharmaceutical composition of the present invention is administered intratumorally to the
subject, preferably administered intratumorally to the subject at 0.01 mg/kg, 0.03 mg/kg, 0.1
mg/kg, 0.3 mg/kg, 1 mg/kg, or 3 mg/kg, optionally at an interval of once every three weeks.
In one aspect, the present invention relates to a pharmaceutical composition, kit or
kit-of-parts comprising (a) an antibody, polynucleotide, vector, recombinant host cell, and/or
pharmaceutical composition of the present invention and (b) an additional therapeutic agent,
preferably an anti-PD-1 antibody.
[0066] In one aspect, the present invention relates to (a) an antibody, polynucleotide,
vector, recombinant host cell, and/or pharmaceutical composition of the t invention and
(b) an anti-EGFR antibody, and optionally (c) a chemotherapeutic agent, for use as a
medicament.
In one aspect, the present invention s to (a) an antibody, polynucleotide,
vector, recombinant host cell, and/or pharmaceutical composition of the t invention and
(b) an GFR dy, and optionally (c) a chemotherapeutic agent, for use in a method
for the treatment of cancer. In a preferred embodiment, the cancer is head and neck squamous
cell carcinoma. In another preferred embodiment, the antibody, polynucleotide, vector,
recombinant host cell, and/or pharmaceutical composition of the present invention is
administered intratumorally to the subject, preferably administered intratumorally to the
subject at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, or 3 mg/kg, optionally at
an al of once every three weeks.
In one aspect, the present invention relates to a pharmaceutical composition, kit or
kit-of-parts comprising (a) an antibody, polynucleotide, vector, recombinant host cell, and/or
ceutical composition of the present ion and (b) an anti-EGFR antibody, and
optionally (c) a chemotherapeutic agent.
In one aspect, the present invention relates to (a) an antibody, polynucleotide,
vector, recombinant host cell, and/or pharmaceutical composition of the present invention and
(b) an anti-HER2 antibody, and ally (c) a chemotherapeutic agent, for use as a
ment.
In one aspect, the present invention relates to (a) an antibody, cleotide,
vector, recombinant host cell, and/or ceutical composition of the present invention and
(b) an anti-HER2 antibody, and optionally (c) a chemotherapeutic agent, for use in a method
for the treatment of HER2+ breast cancer. In another preferred embodiment, the antibody,
polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition of the
present ion is administered umorally to the subject, preferably administered
umorally to the t at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, or 3
mg/kg, optionally at an interval of once every three weeks.
[0071] In one aspect, the present invention relates to a ceutical composition, kit or
kit-of-parts comprising (a) an antibody, polynucleotide, vector, recombinant host cell, and/or
pharmaceutical composition of the present invention and (b) an anti-HER2 antibody, and
optionally (c) a chemotherapeutic agent.
In one aspect, the present invention relates to an antibody, polynucleotide, vector,
recombinant host cell, and/or pharmaceutical composition of the present invention, for use in
a method for the treatment of cancer, wherein the antibody, polynucleotide, vector,
recombinant host cell, and/or pharmaceutical composition of the present invention is
administered intratumorally to the subject, preferably administered intratumorally to the
subject at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, or 3 mg/kg, optionally at
an interval of once every three weeks.
In one , the present invention relates to an antibody, polynucleotide, vector,
recombinant host cell, and/or pharmaceutical ition of the present invention, for use in
a method for the treatment of cancer, wherein the antibody, polynucleotide, vector,
recombinant host cell, and/or pharmaceutical composition of the present invention is
administered subcutaneously or intravenously to the subject, preferably administered
intravenously to the subject at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3
mg/kg, 6 mg/kg, or 10 mg/kg, optionally at an al of once every three weeks.
In one aspect, the present invention relates to (a) an antibody, polynucleotide,
vector, recombinant host cell, and/or pharmaceutical composition of the present invention and
(b) an additional therapeutic agent, for use as a medicament. In a preferred ment, the
additional eutic agent is a chemotherapeutic agent or a checkpoint targeting agent or an
inhibitor of indoleamine-2,3-dioxygenase (IDO) or a vaccine.
In one aspect, the present invention relates to (a) an antibody, polynucleotide,
vector, recombinant host cell, and/or pharmaceutical ition of the present invention and
(b) an additional therapeutic agent, for use in a method for the treatment of cancer. In a
preferred embodiment, the additional therapeutic agent is a chemotherapeutic agent or a
checkpoint targeting agent or an inhibitor of indoleamine-2,3-dioxygenase (IDO) or a vaccine.
In one aspect, the t invention relates to a pharmaceutical composition, kit or
kit-of-parts comprising (a) an antibody, polynucleotide, vector, recombinant host cell, and/or
pharmaceutical ition of the present invention and (b) an additional therapeutic agent.
In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic agent or a
checkpoint targeting agent or an inhibitor of indoleamine-2,3-dioxygenase (IDO) or a vaccine.
In one aspect, the present invention relates to an antibody, polynucleotide, vector,
recombinant host cell, and/or pharmaceutical ition of the present ion, for use in
a method for the treatment of cancer, and/or for use in a method for increasing T-cell activation
in se to an antigen, wherein the antibody, polynucleotide, , recombinant host cell,
and/or pharmaceutical composition of the present invention is delivered to a tumor draining
lymph node.
[0078] In one aspect, the t invention relates to the use of an antibody, polynucleotide,
vector, recombinant host cell, and/or pharmaceutical composition of the present invention in a
method for the ent of cancer, and/or in a method for increasing T-cell activation in
response to an antigen in a subject, wherein the antibody, polynucleotide, vector, recombinant
host cell, and/or pharmaceutical composition of the present invention is red to a tumor
draining lymph node.
In one aspect, the present invention relates to the use of an antibody, polynucleotide,
vector, recombinant host cell, and/or pharmaceutical composition of the present ion, for
preparing medicaments for therapy, for example, for increasing T-cell activation in
response to an antigen in a subject, treating cancer, or treating or ting infectious diseases.
[0080] In one aspect, the present invention relates to the use of an dy, polynucleotide,
vector, recombinant host cell, and/or pharmaceutical composition of the t ion, for
preparing medicaments for immunotherapy, for example, for increasing T-cell activation in
response to an n in a subject, treating cancer, or treating or preventing infectious diseases,
wherein the antibody, cleotide, vector, recombinant host cell, and/or pharmaceutical
composition of the present invention is delivered to a tumor draining lymph node.
In one aspect, the present invention relates to the use of (a) an antibody,
polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition of the
present invention and (b) an anti-HER2 antibody, and optionally (c) a chemotherapeutic agent,
to prepare a medicament for immunotherapy, for example, for increasing T-cell activation in
response to an antigen in a subject, treating cancer, or treating or preventing infectious diseases.
In one aspect, the present invention relates to the use of (a) an antibody,
polynucleotide, , recombinant host cell, and/or pharmaceutical composition of the
present invention and (b) an anti-HER2 antibody, and optionally (c) a chemotherapeutic agent,
to prepare a medicament for immunotherapy, for example, for increasing T-cell activation in
response to an antigen in a t, treating cancer, or treating or preventing infectious diseases,
wherein the antibody, polynucleotide, vector, recombinant host cell, and/or pharmaceutical
composition of the present invention is red to a tumor draining lymph node.
. BRIEF DESCRIPTION OF THE GS
[0083] Figures 1A, 1B, 1C, 1D, 1E, 1F, and 1G are flow cytometry histograms showing
the binding of anti-CTLA-4 dies or an IgG1 isotype control antibody to Jurkat cells
engineered to express human CTLA-4 on the cell surface. The anti-CTLA-4 antibodies tested
are: AGEN1884.H1.1 (IgG1), AGEN1884.H1.2 (IgG1), AGEN1884.H1.3 (IgG1),
84.H2.1 (IgG1), AGEN1884.H2.2 (IgG1), 84.H2.3 (IgG1), and
AGEN1884.H3 (IgG1).
Figure 2 is a graph g IL-2 production of primary human PBMCs following
incubation under sub-optimal stimulation with the Staphylococcal Enterotoxin A (SEA)
superantigen in the e or presence of the anti-CTLA-4 antibody AGEN1884.H3 (IgG1)
or an isotype control antibody (IgG1). Replicates of eight were performed for each group and
the mean values of the eight replicates are indicated with a black bar.
Figure 3 is a graph showing results from an ILluciferase reporter assay
demonstrating that de of CTLA-4 leads to T cell activation. Fold response of luciferase
sion, a surrogate marker for IL-2 gene activation, is plotted over a range of antibody
concentrations for AGEN1884.H3 (IgG1) or an isotype control antibody (IgG1).
[0086] Figure 4 is a graph showing results from a reporter assay where simultaneous
engagement of AGEN1884.H3 (IgG1) to target cells (via CTLA-4 binding) and effector cells
(via FcγRIIIA binding) rs expression of luciferase by the effector cell line. Luciferase
activity is a surrogate marker for FcγRIIIA signaling. Fold response of RLU values is plotted
against a range of antibody concentrations for AGEN1884.H3 (IgG1) and an isotype control
antibody (IgG1).
Figures 5A, 5B, 5C, and 5D are flow try histograms showing CTLA
expressing Jurkat cells incubated with the anti-CTLA-4 antibody 84.H3 (IgG1),
AGEN1884.H3 (IgG1 S239D/I332E), 84.H3 (IgG1 S239D/A330L/I332E), or
AGEN1884.H3 (IgG1 L235V/F243L/R292P/Y300L/P396L), or an e control antibody.
Antibody binding was detected using a fluorochrome-conjugated ary antibody.
s 6A and 6B are graphs showing ng of binding n human CTLA-
4 and its s, CD80 and CD86, respectively, by AGEN1884.H3 (IgG1
S239D/A330L/I332E). Jurkat cells engineered to constitutively express human CTLA-4 were
incubated with anti-CTLA-4 antibody AGEN1884.H3 (IgG1-S239D/A330E/I332E), a
reference anti-CTLA-4 antibody, or an isotype control dy (IgG1), and then stained with
the indicated fluorescently labeled ligand. Ligand binding was then assessed by flow
cytometry.
[0089] Figures 7A, 7B, and 7C are graphs showing IL-2 production of primary human
PBMCs cultured under sub-optimal stimulation with the SEA superantigen in the absence or
presence of an isotype control antibody (IgG1) or an anti-CTLA-4 antibody. Figures 7A and
7B are graphs showing IL-2 production stimulated by either a single dose or a dose titration of
the isotype control antibody (IgG1) or the anti-CTLA-4 dies AGEN1884.H3 (IgG1),
AGEN1884.H3 (IgG1 S239D/I332E), AGEN1884.H3 (IgG1 S239D/A330L/I332E), and
AGEN1884.H3 (IgG1 L235V/F243L/R292P/Y300L/P396L). In the study shown in Figure 7B,
in addition to the isotype control antibody (IgG1) or the anti-CTLA-4 antibody, the cells in each
sample were also incubated with an IgG4 S228P isotype control antibody. Figure 7C is a graph
showing IL-2 production ated by a dose titration of the isotype control antibody (IgG1)
or the anti-CTLA-4 antibodies 84 , AGEN1884 (IgG1 S239D/I332E),
AGEN1884 (IgG1 S239D/A330L/I332E), and afucosylated AGEN1884 (IgG1).
Figure 8A is an immunoblot analysis for phosphorylated ZAP70 (Y493) in human
PBMCs following stimulation with 50 ng/ml of SEA superantigen and 10 µg/ml of isotype
l dy (IgG1) or the anti-CTLA-4 antibodies AGEN1884.H3 (IgG1), AGEN1884.H3
(IgG1 S239D/A330L/I332E), or AGEN1884.H3 (IgG1 N297A). Figure 8B is a chart showing
normalized densitometric analysis of the data shown in Figure 8A.
Figures 9A, 9B, 9C, and 9D are graphs showing antitumor efficacy and
intratumoral regulatory T cell (Treg) depletion induced by Fc variants of murine anti-CTLA-4
antibody 9D9. Figure 9A shows tumor growth in CT26 mice following single-dose treatment
with murine anti-CTLA-4 antibody 9D9 (mIgG2a), an Fc-silent variant of anti-CTLA-4
antibody 9D9 (mIgG2a-N297A), an Fc variant of anti-CTLA-4 antibody 9D9 (mIgG2a-
S239D/A330L/I332E), or an isotype l antibody (mIgG2a). The upper panel shows
median tumor volume over time for each treatment group. The remaining panels show tumor
volume over time for individual s in each treatment group. Figure 9B shows the effect
of anti-CTLA-4 antibody ent on T cell tions from tumor infiltrates collected from
mice treated with single doses of anti-CTLA-4 dy 9D9 (mIgG2a), anti-CTLA-4 antibody
9D9 a-N297A), anti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E), or
isotype control antibody (mIgG2a). Tumor infiltrates were harvested and analyzed by flow
cytometry at indicated time points after injection with dy. Cell populations analyzed
include: FoxP3+ Tregs (upper left panel), CD45+ leukocytes (upper right panel), and CD4+
non-Tregs (lower left panel). The lower right panel shows the ratio of CD8+ T cells to Tregs
observed in tumor rates. Figure 9C shows FoxP3+ Treg populations over time in raining
lymph nodes ted from mice treated as described for Figure 9B. Figure 9D
shows fold-change in splenic FoxP3+ Tregs at 72 hours after treatment as described for Figure
Figure 10 is a series of graphs showing antitumor efficacy of murine anti-CTLA-4
antibodies when combined with tumor vaccine derived from a HPV+ tumor (viral antigens
E6/E7). Shown are tumor volume over time for individual mice receiving no treatment, isotype
control antibody (mIgG2a), anti-CTLA-4 antibody 9D9 (mIgG2a), or an Fc variant of anti-
CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E). Graphs in the top row show results
for animals that were stered the indicated antibody treatment only. Graphs in the bottom
row show results for animals that were administered the indicated antibody treatment in
combination with tumor vaccine.
[0092] Figures 11A and 11B are graphs g gene expression and CpG methylation of
human T cell populations. CD4+ CD25+/- FOXP3- non-regulatory T cells (Teff) and CD4+
CD25+ FOXP3+ regulatory T cells (Treg) were isolated from peripheral blood of healthy
donors, expanded, and activated. Figure 11A shows FOXP3, intracellular , and
membrane CTLA-4 levels in each activated T cell population, as determined by flow
cytometry. Figure 11B shows the level of CpG methylation in CpG regions within the FOXP3
(top panel) and CTLA4 (bottom panel) loci in naïve T cells, activated effector T cells, and
activated regulatory T cells, each from the same donor.
Figures 12A and 12B are graphs showing time courses of antibody dependent
cellular cytotoxicity (ADCC) of human CTLA-4+ target cells after incubation with anti-CTLA-
4 antibody 84.H3 (IgG1) or Fc variants thereof. NK-92 cells (FcγRIIIA V158-
expressing) were co-cultured with + target cells that were incubated with different Fc
variants of anti-CTLA-4 antibodies or an IgG1 isotype control (10 µg/ml). High content
microscopy of caspase 3/7 activation was then used to fy ADCC activity. Figure 12A
shows ADCC activity in Jurkat cells engineered to express CTLA-4 on the cell surface, when
incubated with AGEN1884.H3 , AGEN1884.H3 (IgG1 N297A), AGEN1884.H3 (IgG1
A330L/I332E), AGEN1884.H3 (IgG1 S267E/L328F), afucosylated 84.H3
(IgG1), or an isotype control antibody (IgG1). Figure 12B shows ADCC activity in primary
human activated effector T cells (left panel) or tory T cells (right panel) when incubated
with these antibodies.
Figures 13A, 13B, 13C, and 13D are graphs showing the effects of anti-CTLA-4
antibody variants on T cell function when administered alone or in combination with an anti-
PD-1 antibody. Human PBMCs were isolated from two donors and incubated under
stimulatory culture conditions with anti-CTLA-4 antibody AGEN1884.H3 (IgG1), an Fc
variant anti-CTLA-4 antibody AGEN1884.H3 (IgG1 S239D/A330L/I332E), or an e
control antibody (IgG1), in combination with a reference anti-PD-1 antibody or an isotype
control antibody (IgG4), as indicated. For each treatment ion listed, a dosage titration
was used for the first-listed antibody, and a fixed concentration (5 μg/ml) was used for the
second-listed antibody. This experiment was performed twice, for a total of two replicates per
donor. The levels of IL-2 production induced by each antibody combination on PBMCs
collected from the first donor are shown in Figure 13A (replicate 1) and Figure 13B (replicate
2). The levels of IL-2 production induced by each antibody combination on PBMCs collected
from the second donor are shown in Figure 13C (replicate 1) and Figure 13D cate 2).
Figures 14A, 14B, and 14C are a series of sequence alignments. Figure 14A is a
ce alignment for human CTLA-4 (SEQ ID NO: 33), cynomolgus monkey CTLA-4 (SEQ
ID NO: 40), mouse CTLA-4 (SEQ ID NO: 41), and rat CTLA-4 (SEQ ID NO: 42). Dots
represent residues identical to corresponding human residues. An “*” (asterisk) indicates
positions which have a , fully conserved residue. A “:” (colon) indicates conservation
between groups of strongly r properties. A “.” (period) tes conservation between
groups of weakly r properties. Figures 14B and 14C are sequence alignments for human
CTLA-4 (residues 1-144 and residues 145-223 of SEQ ID NO: 33, respectively), cynomolgus
monkey CTLA-4 (residues 1-144 and residues 145-223 of SEQ ID NO: 40, respectively),
human CD28 (residues 1-127 and residues 0 of SEQ ID NO: 43, respectively), human
ICOS (residues 1-124 and residues 125-199 of SEQ ID NO: 44, respectively), human BTLA
(residues 1-125 and residues 126-289 of SEQ ID NO: 45, respectively), and human PD-1
(residues 1-143 and residues 144-288 of SEQ ID NO: 46, respectively). The two s
showing strong se in deuterium uptake when human CTLA-4 was bound to AGEN1884-
Fab are underlined in Figures 14A-14C: residues 80-82 (QVT, SEQ ID NO: 39) and residues
135-149 (YPPPYYLGIGNGTQI, SEQ ID NO: 37), numbered according to SEQ ID NO: 33.
6. DETAILED DESCRIPTION
The instant sure provides antibodies that specifically bind to CTLA-4 (e.g.,
human CTLA-4) and antagonize CTLA-4 function, e.g., CTLAmediated immune
suppression. Also provided are pharmaceutical compositions comprising these antibodies,
nucleic acids encoding these dies, expression vectors and host cells for making these
antibodies, and s of treating a subject using these antibodies. The dies described
herein are particularly useful for increasing T cell tion in response to an n (e.g., a
tumor n or an infectious disease antigen), and hence 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 dies 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.
The skilled worker will appreciate that a glutamate (E) or glutamine (Q) amino acid
residue at the N-terminus of a heavy chain variable region and/or a light chain variable region
of any one of the antibodies described herein (e.g., an anti-CTLA4 antibody) can, under certain
conditions, spontaneously convert to pyroglutamate by post-translational cyclization of the free
amino group to form a lactam. Accordingly, in certain embodiments of each and every one of
the methods, uses, pharmaceutical compositions, or kits described herein, the N-terminal amino
acid e of one or more heavy chain variable s and/or light chain variable regions of
the antibody has been converted to pyroglutamate (e.g., as a result of post-translational
cyclization of the free amino group of the N-terminal E or Q residue).
6.1 Definitions
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 ed meaning of the recited value or range.
As used herein, the term “CTLA-4” refers to cytotoxic hocyte-associated
protein 4. As used herein, the term “human ” refers to a human CTLA-4 protein
encoded by a wild type human CTLA-4 gene, e.g., GenBank™ accession number
NM_005214.4 or NM_001037631.2. An exemplary immature amino acid sequence of human
CTLA-4 is provided as SEQ ID NO: 33.
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 or VL regions. Examples of antibodies e monoclonal
dies, recombinantly ed antibodies, monospecific dies, multispecific
dies (including bispecific antibodies), human antibodies, humanized antibodies,
chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising
two heavy chain and two light chain molecules, an dy 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, conjugate antibodies,
antibody-drug conjugates, single domain antibodies, monovalent antibodies, single chain
antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’)2
fragments, ide-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., IgG1,
IgG2, IgG3, IgG4, IgA1 or IgA2), or any subclass (e.g., IgG2a or IgG2b) of globulin
molecule. In certain embodiments, dies described herein are IgG antibodies, or a class
(e.g., human IgG1 or IgG4) or ss thereof. In a specific embodiment, the antibody is a
humanized monoclonal antibody. In another specific embodiment, the antibody is a human
monoclonal antibody.
As used herein, the terms “VH region” and “VL region” refer to single antibody
heavy and light chain variable s, respectively, comprising FR (Framework Regions) 1,
2, 3 and 4 and CDR (Complementarity Determining s) 1, 2 and 3 (see Kabat et al.,
(1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242,
Bethesda), which is herein orated by reference in its entirety).
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 logical
interest. (1991), by Chothia et al., J. Mol. Biol. 196:901-917 (1987), and by MacCallum et al.,
J. Mol. Biol. 2-745 (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 Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein
of immunological interest. (1991). In n embodiments, the term “CDR” is a CDR as
defined by Chothia et al., J. Mol. Biol. 196:901-917 (1987). In certain ments, 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 is of Antibody Variable s,” in Antibody
Engineering, mann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin
(2001).
As used herein, the term “framework (FR) amino acid es” refers to those
amino acids in the framework region of an globulin 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 Chothia definition of
CDRs).
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 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 icity of a particular antibody for its particular antigen. The ility in
sequence is concentrated in those regions called complementarity determining regions (CDRs)
while the more highly conserved regions in the variable domain are called ork regions
(FR). Without wishing to be bound by any particular ism 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 le region comprises rodent or murine CDRs
and human framework regions (FRs). In particular embodiments, the variable region is a
primate (e.g., non-human e) variable region. In certain ments, the variable region
comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions
(FRs).
[00105] The terms “VL” and “VL domain” are used interchangeably to refer to the light
chain variable region of an antibody.
The terms “VH” and “VH domain” are used interchangeably to refer to the heavy
chain variable region of an antibody.
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
le generally has a more conserved amino acid sequence relative to an immunoglobulin
variable domain.
As used herein, the term “heavy chain” when used in reference to an antibody can
refer to any distinct type, e.g., alpha (α), delta (δ), n (ε), gamma (γ), and mu (µ), based
on the amino acid sequence of the nt domain, which give rise to IgA, IgD, IgE, IgG, and
IgM classes of dies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and
IgG4.
As used herein, the term “light chain” when used in reference to an antibody can
refer to any distinct type, e.g., kappa (κ) or lambda (λ) based on the amino acid sequence of the
constant s. Light chain amino acid sequences are well known in the art.
[00110] As used herein, the term “EU numbering system” refers to the EU numbering
convention for the constant regions of an antibody, as described in n, G.M. et al., Proc.
Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of ns of Immunological
Interest, U.S. Dept. Health and Human es, 5th edition, 1991, each of which is herein
incorporated by reference in its entirety.
[00111] “Binding affinity” generally refers to the strength of the sum total of valent
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 s 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 (KD). Affinity can be measured and/or expressed in a
number of ways known in the art, including, but not limited to, equilibrium dissociation
constant (KD), and equilibrium association constant (KA). The K D is calculated from the
quotient of koff/kon, s KA is calculated from the quotient of kon/koff. k on refers to the
association rate nt of, e.g., an antibody to an antigen, and koff refers to the dissociation
rate constant of, e.g., an antibody to an antigen. The kon and koff can be determined by
techniques known to one of ordinary skill in the art, such as BIAcore® or KinExA. As used
herein, a “lower affinity” refers to a larger KD.
As used herein, the terms “specifically binds,” “specifically recognizes,”
“immunospecifically ” 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
x) as such binding is understood by one skilled in the art. For e, a le that
specifically binds to an antigen can bind to other peptides or polypeptides, lly with lower
affinity as determined by, e.g., immunoassays, BIAcore®, KinExA 3000 instrument (Sapidyne
Instruments, Boise, ID), or other assays known in the art. In a ic embodiment, molecules
that specifically bind to an antigen bind to the antigen with a KA that is at least 2 logs (i.e.,
factors of 10), 2.5 logs, 3 logs, 4 logs or r than the KA when the molecules bind nonspecifically
to another antigen.
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 CTLA-4 do not cross react with other non-
CTLA-4 ns. In a specific embodiment, provided herein is an antibody that binds to
CTLA-4 (e.g., human CTLA-4) with higher affinity than to another unrelated antigen. In
certain embodiments, provided herein is an antibody that binds to CTLA-4 (e.g., human CTLA-
4) 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 ed by, e.g., a
radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In a specific
embodiment, the extent of binding of an anti-CTLA-4 antibody described herein to an
unrelated, non-CTLA-4 protein is less than 10%, 15%, or 20% of the binding of the antibody
to CTLA-4 protein as measured by, e.g., a mmunoassay.
As used herein, the term sylation” or “afucosylated” in the context of an Fc
refers to a substantial lack of a fucose covalently attached, directly or indirectly, to residue 297
of the human IgG1 Fc region, numbered according to the EU numbering system, or the
corresponding e in non-IgG1 or non-human IgG1 immunoglobulins. Thus, in a
composition comprising a plurality of afucosylated antibodies, at least 70% of the antibodies
will not be fucosylated, directly or indirectly (e.g., via intervening sugars) at residue 297 of the
Fc region of the antibodies, and in some embodiments at least 80%, 85%, 90%, 95%, or 99%
will not be fucosylated, ly or indirectly, at residue 297 of the Fc .
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 uous 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, en/deuterium
exchange coupled with mass spectrometry (e.g., liquid tography 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 mational
epitopes), and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray
llography, crystallization may be accomplished using any of the known methods in the
art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350;
son A (1990) Eur J Biochem 189: 1-23; Chayen NE (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). dy:antigen crystals may be studied using well known X-
ray diffraction techniques and may be refined using er software such as X-PLOR (Yale
University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985)
volumes 114 & 115, eds f HW et al.,; U.S. 2004/0014194), and BUSTER (Bricogne G
(1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; ne G (1997) Meth l
276A: 361-423, ed Carter CW; 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 ties.
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 BC
& Wells JA (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 ques. 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 US Patent 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 ined using e 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.
[00116] As used , the term “an epitope located within a region of human CTLA-4”
consisting of a particular amino acid sequence or a set of amino acid residues refers to an
epitope comprising one or more of the amino acid residues of the specified region, wherein the
specified region includes the first specified amino acid residue and the last specified amino
acid e of the region of human CTLA-4. In certain embodiments, the epitope comprises
each one of the amino acid residues located within the specified region. In certain
embodiments, one or more additional amino acid residues of human CTLA-4 outside the
specified region bind to an antibody together with an epitope located within the specified
region.
[00117] As used herein, the terms “T cell or” and “TCR” are used interchangeably
and refer to full length heterodimeric αβ or γδ 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 ning le
regions of TCRs attached by a flexible linker, TCR chains linked by an engineered ide
bond, monospecific TCRs, multi-specific TCRs (including bispecific TCRs), TCR fusions,
human TCRs, humanized TCRs, ic 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 es a first portion from a TCR of
a first species and a second portion from a TCR of a second species).
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.
As used , the term “peptide-MHC complex” refers to an MHC le
(MHC class I or MHC class II) with a peptide bound in the art-recognized peptide binding
pocket of the MHC.
As used herein, the term “treat,” “treating,” and “treatment” refer to therapeutic or
preventative measures bed herein. The s of “treatment” employ administration
of an antibody to a subject having a disease or disorder, or predisposed to having such a disease
or er, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more
symptoms of the disease or er or recurring disease or disorder, or in order to prolong the
survival of a subject beyond that expected in the absence of such treatment.
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 .
[00122] As used herein with respect to the response of a cancer to a therapy, the terms
“refractory” and “resistant” have their art-recognized meaning and are used interchangeably.
As used herein, the term “subject” includes any human or non-human animal. In
one ment, the subject is a human or non-human mammal. In one embodiment, the
subject is a human.
The determination of “percent identity” between two sequences (e.g., amino acid
sequences or nucleic acid sequences) can be accomplished using a mathematical thm. A
specific, non-limiting example of a mathematical algorithm utilized for the comparison of two
sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, ed
as in Karlin S & Altschul SF (1993) PNAS 90: 877, each of which is herein incorporated
by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST
ms of Altschul SF 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, ngth=12 to obtain nucleotide
ces 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 SF 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 t 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 ) 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 ces, a PAM120 weight residue table, a gap length penalty of 12,
and a gap penalty of 4 can be used.
The percent identity between two sequences can be determined using techniques
similar to those described above, with or without allowing gaps. In ating percent identity,
typically only exact matches are counted.
6.2 Anti-CTLA-4 Antibodies
[00126] In one aspect the t disclosure provides antibodies that specifically bind to
CTLA-4 (e.g., human CTLA-4) and antagonize CTLA-4 function. The amino acid sequences
of ary dies are set forth in Tables 1-4 herein.
Table 1. Amino acid ces of exemplary anti-CTLA-4 antibodies.*
SEQ Description Amino acid sequence
ID NO:
1 AGEN1884 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
NWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGL
MGPFDIWGQGTMVTVSS
2 AGEN1884_M102F VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
NWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF
GPFDIWGQGTMVTVSS
3 AGEN1884_M113L VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
NWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGL
MGPFDIWGQGTLVTVSS
4 AGEN1884_D62E VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGL
MGPFDIWGQGTMVTVSS
AGEN1884_M102F_M11 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
3L VH NWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF
GPFDIWGQGTLVTVSS
6 AGEN1884_D62E_M102 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
F VH PGKGLEWVSSISSSSSYIYYAESVKGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF
GPFDIWGQGTMVTVSS
7 AGEN1884_D62E_M113 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
L VH NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGL
MGPFDIWGQGTLVTVSS
8 AGEN1884_D62E_M102 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
F_M113L VH NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRF
AKNSLYLQMNSLRAEDTAVYYCARVGLF
GPFDIWGQGTLVTVSS
9 84 VL SPGTLSLSPGERATLSCRASQSVSRYLGW
YQQKPGQAPRLLIYGASTRATGIPDRFSGSGSGTD
FTLTITRLEPEDFAVYYCQQYGSSPWTFGQGTKVE
CDRH1 SYSMN
11 CDRH2 SISSSSSYIYYADSVKG
12 CDRH2 SISSSSSYIYYAESVKG
13 CDRH3 VGLMGPFDI
14 CDRH3 VGLFGPFDI
CDRL1 SRYLG
16 CDRL2 GASTRAT
17 CDRL3 QQYGSSPWT
18 CDRH2 consensus SISSSSSYIYYAXSVKG, wherein:
sequence X is E or D
SEQ Description Amino acid sequence
ID NO:
19 CDRH3 consensus VGLXGPFDI, wherein:
sequence X is F or M
VH consensus sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
NWVRQAPGKGLEWVSSISSSSSYIYYAX1SVKGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGL
IWGQGTX3VTVSS, wherein:
X1 is E or D,
X2 is F or M, and
X3 is L or M.
23 AGEN1884.H3 (IgG1) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
heavy chain NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF
GPFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTS
GCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
24 84.H3 (IgG1 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
S239D/I332E) heavy NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRF
chain TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF
GPFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTS
GCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPD
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAPEEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS
SEQ Description Amino acid sequence
ID NO:
AGEN1884.H3 (IgG1 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
S239D/A330L/I332E) NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRF
heavy chain TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF
GPFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPD
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPLPEEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS
26 AGEN1884.H3 (IgG1 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
L235V/F243L/R292P/Y3 NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRF
00L/P396L) heavy chain TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF
GQGTLVTVSSASTKGPSVFPLAPSSKSTS
GCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKRVEPKSCDKTHTCPPCPAPELVGGPS
VFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS
47 AGEN1884.H3 (IgG1 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
N297A) heavy chain PGKGLEWVSSISSSSSYIYYAESVKGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF
GPFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
SGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYASTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
SEQ Description Amino acid sequence
ID NO:
48 84.H3 (IgG1 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
S267E/L328F) heavy NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRF
chain TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF
GQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
27 AGEN1884.H3 light EIVLTQSPGTLSLSPGERATLSCRASQSVSRYLGW
chain YQQKPGQAPRLLIYGASTRATGIPDRFSGSGSGTD
FTLTITRLEPEDFAVYYCQQYGSSPWTFGQGTKVE
IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP
WKVDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
FNRGEC
28 IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT
PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
LDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPG
29 IgG1 S239D/I332E ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPEEKTISKAKGQPREPQVYTLPPSRE
EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPG
SEQ Description Amino acid sequence
ID NO:
IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
S239D/A330L/I332E PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
LGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRE
EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPG
31 IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
F243L/R292P/Y3 PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
00L/P396L LGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPG
32 Light chain constant RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR
region EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
NRGEC
* CDRs are defined according to the Kabat numbering system.
Table 2. Heavy chain CDR amino acid sequences of exemplary anti-CTLA-4 dies.*
VH (SEQ ID NO:) CDRH1 CDRH2 CDRH3
(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:)
AGEN1884 VH (1) SYSMN (10) SISSSSSYIYYADSV VGLMGPFDI (13)
KG (11)
84_M102F VH (2) SYSMN (10) SISSSSSYIYYADSV VGLFGPFDI (14)
KG (11)
AGEN1884_M113L VH (3) SYSMN (10) SISSSSSYIYYADSV VGLMGPFDI (13)
KG (11)
AGEN1884_D62E VH (4) SYSMN (10) SISSSSSYIYYAESV VGLMGPFDI (13)
KG (12)
AGEN1884_M102F_M113 SYSMN (10) SISSSSSYIYYADSV FDI (14)
L VH (5) KG (11)
AGEN1884_D62E_M102F SYSMN (10) SISSSSSYIYYAESV VGLFGPFDI (14)
VH (6) KG (12)
AGEN1884_D62E_M113L SYSMN (10) SISSSSSYIYYAESV VGLMGPFDI (13)
VH (7) KG (12)
AGEN1884_D62E_M102F SYSMN (10) SISSSSSYIYYAESV VGLFGPFDI (14)
_M113L VH (8) KG (12)
*Defined according to the Kabat numbering system.
Table 3. Light chain CDR amino acid sequences of exemplary anti-CTLA-4 antibodies.*
VL CDRL1 CDRL2 CDRL3
(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:)
AGEN1884 VL (9) SRYLG (15) GASTRAT (16) QQYGSSPWT (17)
*Defined according to the Kabat numbering system.
Table 4. Exemplary anti-CTLA-4 antibodies.
Antibody Heavy chain le region SEQ Light chain SEQ ID
ID NO: variable region NO:
AGEN1884 AGEN1884 VH 1 AGEN1884 VL 9
AGEN1884.H1.1 AGEN1884_M102F VH 2 AGEN1884 VL 9
AGEN1884.H1.2 84_M113L VH 3 AGEN1884 VL 9
AGEN1884.H1.3 AGEN1884_D62E VH 4 AGEN1884 VL 9
AGEN1884.H2.1 AGEN1884_M102F_M113L VH 5 AGEN1884 VL 9
84.H2.2 AGEN1884_D62E_M102F VH 6 AGEN1884 VL 9
AGEN1884.H2.3 AGEN1884_D62E_M113L VH 7 AGEN1884 VL 9
AGEN1884.H3 AGEN1884_D62E_M102F_M113L 8 AGEN1884 VL 9
Table 5. Closest germline genes.
SEQ ID NO: Closest germline gene Amino acid ce
21 IGHV3-21*01 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYS
MNWVRQAPGKGLEWVSSISSSSSYIYYADSVK
GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCA
22 IGKV3-20*01 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYL
AWYQQKPGQAPRLLIYGASSRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYCQQYGSSP
Table 6. Exemplary sequences of CTLA-4 and family s.
SEQ Description Amino acid Sequence
ID NO:
33 Human CTLA-4 immature MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFC
protein (P16410) KAMHVAQPAVVLASSRGIASFVCEYASPGKATEV
RVTVLRQADSQVTEVCAATYMMGNELTFLDDSI
CTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYP
PPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSS
GLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPP
TEPECEKQFQPYFIPIN
34 CTLA-4 epitope YLGI
CTLA-4 epitope LGI
36 CTLA-4 epitope YLGIGNGTQI
37 CTLA-4 epitope YPPPYYLGIGNGTQI
SEQ Description Amino acid Sequence
ID NO:
38 CTLA-4 epitope MYPPPYY
39 CTLA-4 epitope QVT
40 MACFA CTLA-4 MACLGFQRHKARLNLATRTRPYTLLFSLLFIPVFS
(G7PL88) QPAVVLANSRGIASFVCEYASPGKATE
VRVTVLRQADSQVTEVCAATYMMGNELTFLDDS
ICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMY
GIGNGTQIYVIDPEPCPDSDFLLWILAAVS
SGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMP
PTEPECEKQFQPYFIPIN
41 Mouse CTLA-4 (P09793) MACLGLRRYKAQLQLPSRTWPFVALLTLLFIPVFS
EAIQVTQPSVVLASSHGVASFPCEYSPSHNTDEVR
VTVLRQTNDQMTEVCATTFTEKNTVGFLDYPFCS
RVNLTIQGLRAVDTGLYLCKVELMYPPP
YFVGMGNGTQIYVIDPEPCPDSDFLLWILVAVSLG
LFFYSFLVSAVSLSKMLKKRSPLTTGVYVKMPPT
EPECEKQFQPYFIPIN
42 Rat CTLA-4 (Q62859) MACLGLQRYKTHLQLPSRTWPFGVLLSLLFIPIFSE
AIQVTQPSVVLASSHGVASFPCEYASSHNTDEVR
TNDQVTEVCATTFTVKNTLGFLDDPFCS
GTFNESRVNLTIQGLRAADTGLYFCKVELMYPPP
YFVGMGNGTQIYVIDPEPCPDSDFLLWILAAVSSG
LFFYSFLVTAVSLNRTLKKRSPLTTGVYVKMPPTE
PECEKQFQPYFIPIN
43 Human CD28 (P10747) MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDN
AVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVV
YGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQN
LYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVK
GKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLV
TVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRK
HYQPYAPPRDFAAYRS
44 Human ICOS (Q9Y6W8) MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHN
GGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTK
GSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHA
NYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQL
KFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVH
DPNGEYMFMRAVNTAKKSRLTDVTL
45 Human BTLA (Q7Z6A9) MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESC
KRQSE
TWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEP
VLPNDNGSYRCSANFQSNLIESHSTTLYVTDVKSA
SERPSKDEMASRPWLLYRLLPLGGLPLLITTCFCL
FCCLRRHQGKQNELSDTAGREINLVDAHLKSEQT
EASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEV
YSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKE
APTEYASICVRS
SEQ Description Amino acid Sequence
ID NO:
46 Human PD-1 6) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPW
NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNW
YRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLP
NGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIK
ESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV
LLGSLVLLVWVLAVICSRAARGTIGARR
EDPSAVPVFSVDYGELDFQWREKTPEPP
VPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRS
EDGHCSWPL
In certain embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a VH domain
sing 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 one of VH domains set forth in
Table 1. In certain embodiments, the antibody comprises the CDRH2 of one of the VH
domains set forth in Table 1. In certain embodiments, the antibody comprises the CDRH3 of
one of the VH domains set forth in Table 1.
In certain embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), 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 one of VL domains set forth in
Table 1. In certain embodiments, the antibody comprises the CDRL2 of one of the VL domains
set forth in Table 1. In certain embodiments, the antibody comprises the CDRL3 of one of the
VL domains set forth in Table 1.
In certain ments, the CDRs of an antibody can be determined according to
Kabat et al., J. Biol. Chem. 252, 616 (1977) and Kabat et al., ces of protein of
immunological interest (1991), each of which is herein incorporated by reference in its entirety.
In certain embodiments, the CDRs of an antibody can be determined according to
the Chothia ing scheme, which refers to the location of immunoglobulin structural loops
(see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; ikani 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. Patent 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 a 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 a 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).
In certain embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human ), the antibody comprising the Chothia VH
CDRs of a VH disclosed in Table 1 herein. In certain embodiments, the instant disclosure
provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), the
antibody comprising the Chothia VL CDRs of a VL disclosed in Table 1 . In certain
embodiments, the instant disclosure provides an isolated antibody that specifically binds to
CTLA-4 (e.g., human ), the antibody comprising the Chothia VH CDRs and Chothia
VL CDRs of an antibody disclosed in Table 1 herein. In certain ments, antibodies that
specifically bind to CTLA-4 (e.g., human CTLA-4) comprise one or more CDRs, in which the
Chothia and Kabat CDRs have the same amino acid sequence. In certain embodiments, the
instant disclosure es an isolated antibody that specifically binds to CTLA-4 (e.g., human
CTLA-4) and comprises combinations of Kabat CDRs and Chothia CDRs.
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: 2, each of which is herein
incorporated by reference in its entirety.
[00133] In n embodiments, the instant disclosure provides antibodies that specifically
bind to CTLA-4 (e.g., human CTLA-4) and comprise CDRs of an antibody disclosed in Table
1 herein, as ined by the IMGT numbering system, for example, as described in Lefranc
M-P (1999) supra and c M-P et al., (1999) supra.
In certain embodiments, the CDRs of an dy can be ined according to
the AbM numbering scheme, which refers to AbM hypervariable regions, which represent a
compromise n 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 t sure
provides antibodies that specifically bind to CTLA-4 (e.g., human CTLA-4) and comprise
CDRs of an antibody disclosed in Table 1 herein as determined by the AbM numbering scheme.
In certain embodiments, the instant disclosure es an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), 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: 2, 4, 5, 6, 7, or 8, and a light chain variable
region comprising the CDRL1, CDRL2, and CDRL3 region amino acid ces of a VL
domain set forth in SEQ ID NO: 9, wherein each CDR is defined in accordance with the
MacCallum definition, the Kabat definition, the Chothia definition, the combination of the
Kabat definition and the Chothia definition, the IMGT numbering system, or the AbM
definition of CDR.
In certain embodiments, the instant disclosure provides an isolated antibody that
ically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising:
(a) a CDRH1 ses the amino acid sequence of SYSMN (SEQ ID NO: 10); and/or
(b) a CDRH2 comprises the amino acid ce of SISSSSSYIYYAXSVKG (SEQ ID
NO: 18), wherein X is E or D; and/or
(c) a CDRH3 comprises the amino acid sequence of VGLXGPFDI (SEQ ID NO: 19),
wherein X is F or M; and/or
(d) CDRL1 comprises the amino acid sequence of RASQSVSRYLG (SEQ ID NO: 15);
and/or
(e) CDRL2 ses the amino acid sequence of GASTRAT (SEQ ID NO: 16); and/or
(f) CDRL3 comprises the amino acid sequence of QQYGSSPWT (SEQ ID NO: 17),
and wherein the CDRH1, CDRH2, and CDRH3 sequences of the antibody are not SEQ ID
NOs: 10, 11, and 13, tively.
In certain embodiments, CDRH2 comprises the amino acid sequence of SEQ ID
NO: 11. In certain embodiments, CDRH2 comprises the amino acid sequence of SEQ ID NO:
12. In certain embodiments, CDRH3 comprises the amino acid sequence of SEQ ID NO: 13.
In n embodiments, CDRH3 comprises the amino acid sequence of SEQ ID NO: 14.
] In certain embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), wherein the antibody comprises a VH
domain sing the CDRH1, CDRH2, and CDRH3 amino acid ces set forth in SEQ
ID NOs: 10, 11, and 14; 10, 12, and 13; or 10, 12, and 14, respectively. In certain embodiments,
the instant disclosure es an isolated antibody that specifically binds to CTLA-4 (e.g.,
human CTLA-4), wherein the antibody comprises a VH domain comprising the CDRH1,
CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 10, 12, and 14,
respectively.
In n embodiments, the t disclosure provides an ed antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), wherein the antibody comprises a heavy
chain variable region comprising CDRH1, CDRH2, and CDRH3 s, 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: 10, 11, 14, 15, 16, and 17; 10, 12, 13, 15, 16, and 17; or 10, 12, 14,
, 16, and 17, respectively. In n embodiments, the instant disclosure provides an isolated
dy that specifically binds to CTLA-4 (e.g., human CTLA-4), n the antibody
comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions,
and a light chain le region sing CDRL1, CDRL2, and CDRL3 regions, n
the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid
sequences set forth in SEQ ID NOs: 10, 12, 14, 15, 16, and 17, respectively.
In certain embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), comprising a heavy chain variable region
comprising an amino acid sequence of SEQ ID NO: 20. In certain embodiments, the instant
disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-
4), comprising a heavy chain le region comprising an amino acid sequence that is at least
75%, 80%, 85%, 90%, 95%, 99%, 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: 2, 4, 5, 6, 7,
or 8. In certain embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), comprising a heavy chain variable region
comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, 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: 3. In certain embodiments, the antibody comprises a
heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, 4, 5,
6, 7, or 8. In certain embodiments, the antibody comprises a heavy chain variable region having
the amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the antibody
comprises a heavy chain variable region having the amino acid ce set forth in SEQ ID
NO: 3. In certain embodiments, the antibody comprises a heavy chain variable region having
the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the antibody
comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID
NO: 5. In n embodiments, the antibody comprises a heavy chain variable region having
the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the antibody
comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID
NO: 7. In n embodiments, the antibody comprises a heavy chain variable region having
the amino acid sequence set forth in SEQ ID NO: 8.
In certain embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), comprising a light chain le region
comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, 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 antibody comprises a
light chain le region having the amino acid sequence set forth in SEQ ID NO: 9.
[00142] In certain embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), comprising a heavy chain variable region
comprising an amino acid sequence of SEQ ID NO: 20, and a light chain variable region
sing an amino acid sequence of SEQ ID NO: 9. In certain embodiments, the instant
disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-
4), comprising a heavy chain variable region comprising an amino acid sequence that is at least
75%, 80%, 85%, 90%, 95%, 99%, 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: 2, 4, 5, 6, 7,
or 8, and a light chain variable region comprising an amino acid sequence that is at least 75%,
80%, 85%, 90%, 95%, 99%, 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 ce set forth in SEQ ID NO: 9. In n
embodiments, the instant disclosure provides an isolated antibody that specifically binds to
CTLA-4 (e.g., human CTLA-4), comprising a heavy chain le region comprising an
amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% (e.g., at least
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) cal to the amino acid sequence
set forth in SEQ ID NO: 3, and a light chain variable region comprising an amino acid sequence
that is at least 75%, 80%, 85%, 90%, 95%, 99%, 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 ce set forth in SEQ ID NO:
9. In certain embodiments, the antibody ses a heavy chain variable region and light
chain variable region having the amino acid sequences set forth in SEQ ID NO: 2 and 9; 4 and
9; 5 and 9; 6 and 9; 7 and 9; or 8 and 9, respectively. In certain embodiments, the antibody
comprises a heavy chain variable region and light chain variable region having the amino acid
sequences set forth in SEQ ID NO: 2 and 9, respectively. In certain ments, the antibody
comprises a heavy chain variable region and light chain variable region having the amino acid
sequences set forth in SEQ ID NO: 3 and 9, respectively. In certain embodiments, the antibody
comprises a heavy chain variable region and light chain variable region having the amino acid
sequences set forth in SEQ ID NO: 4 and 9, respectively. In certain embodiments, the antibody
comprises a heavy chain variable region and light chain variable region having the amino acid
sequences set forth in SEQ ID NO: 5 and 9, respectively. In certain embodiments, the dy
comprises a heavy chain variable region and light chain variable region having the amino acid
sequences set forth in SEQ ID NO: 6 and 9, respectively. In certain embodiments, the antibody
comprises a heavy chain variable region and light chain variable region having the amino acid
sequences set forth in SEQ ID NO: 7 and 9, respectively. In n embodiments, the antibody
comprises a heavy chain le region and light chain variable region having the amino acid
sequences set forth in SEQ ID NO: 8 and 9, respectively.
] In certain embodiments, the t disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), sing a heavy chain le region
having an amino acid sequence d from a human 21 germline sequence (e.g.,
IGHV3-21*01, e.g., having the amino acid sequence of SEQ ID NO: 21). 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 IGHV3-21 germline sequence (e.g.,
IGHV3-21*01, e.g., having the amino acid sequence of SEQ ID NO: 21). In one embodiment,
framework 1, framework 2, framework 3, CDRH1, and CDRH2 are all derived from a human
IGHV3-21 germline sequence (e.g., IGHV3-21*01, e.g., having the amino acid sequence of
SEQ ID NO: 21).
In certain embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), sing a light chain variable region
having an amino acid sequence derived from a human germline sequence selected from the
group consisting of IGKV3-20 (e.g., IGKV3-20*01, e.g., having the amino acid sequence of
SEQ ID NO: 22). One or more s 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 IGKV3-20 (e.g., IGKV3-
*01, e.g., having the amino acid ce of SEQ ID NO: 22). In one embodiment,
ork 1, ork 2, framework 3, CDRL1, and CDRL2 are all derived from a human
germline sequence selected from the group consisting of IGKV3-20 (e.g., IGKV3-20*01, e.g.,
having the amino acid sequence of SEQ ID NO: 22).
In certain embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), comprising a heavy chain variable region
having an amino acid sequence derived from a human IGHV3-21 germline sequence (e.g.,
IGHV3-21*01, e.g., having the amino acid ce of SEQ ID NO: 21), and a light chain
variable region having an amino acid sequence derived from a human germline sequence
selected from the group consisting of IGKV3-20 (e.g., IGKV3-20*01, e.g., having the amino
acid sequence of SEQ ID NO: 22).
[00146] In n embodiments, the instant disclosure provides an isolated antibody that
cross-competes for binding to CTLA-4 (e.g., human CTLA-4) with an dy comprising the
heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 2 and 9;
4 and 9; 5 and 9; 6 and 9; 7 and 9; or 8 and 9, respectively. In certain embodiments, the instant
disclosure provides an isolated antibody that cross-competes for binding to CTLA-4 (e.g.,
human CTLA-4) with an antibody comprising the heavy and light chain variable region amino
acid sequences set forth in SEQ ID NOs: 3 and 9, respectively.
] In certain embodiments, the instant disclosure provides an isolated antibody that
binds to the same or an overlapping epitope of CTLA-4 (e.g., an epitope of human CTLA-4)
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: 2 and 9; 4 and 9; 5 and 9; 6 and 9; 7
and 9; or 8 and 9, respectively. In certain embodiments, the instant disclosure provides an
isolated antibody that binds to the same or an overlapping epitope of CTLA-4 (e.g., an epitope
of human CTLA-4) as an antibody described herein, e.g., an dy comprising the heavy
and light chain variable region amino acid sequences set forth in SEQ ID NOs: 3 and 9,
respectively. In certain embodiments, the e of an antibody can be determined by, e.g.,
NMR spectroscopy, surface plasmon resonance (BIAcore®), X-ray diffraction crystallography
studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g.,
liquid chromatography electrospray mass spectrometry), array-based peptide scanning
assays, and/or mutagenesis mapping (e.g., site-directed nesis g). For X-ray
crystallography, crystallization may be accomplished using any of the known methods in the
art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350;
McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 274;
McPherson A (1976) J Biol Chem 251: 6300-6303, all of which are herein incorporated by
reference in their ties). Antibody:antigen crystals may be studied using well known X-
ray diffraction techniques and may be refined using er software such as X-PLOR (Yale
University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985)
volumes 114 & 115, eds f HW 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 l 276A: 361-423, ed Carter CW; 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
gham BC & Wells JA (1989) supra for a description of nesis techniques,
including alanine scanning nesis 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 es of CTLA-4 (e.g., human CTLA-
4) 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 g icity for an e. 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 CTLA-4 (e.g., human CTLA-4). Numerous
types of competitive binding assays are known, for example: solid phase direct or indirect
radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich
ition assay (see Stahli C et al., (1983) Methods Enzymol 9: 242-253); solid phase direct
biotin-avidin EIA (see Kirkland TN 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 , Cold Spring Harbor Press); solid phase direct label RIA
using I-125 label (see Morel GA et al., (1988) Mol Immunol 25(1): 7-15); solid phase direct
biotin-avidin EIA (see Cheung RC et al., (1990) Virology 176: 546-52); and direct labeled RIA
(see hauer G et al., (1990) Scand J Immunol 32: 77-82), all of which are herein
incorporated by reference in their ties. Typically, such an assay involves the use of
purified antigen (e.g., CTLA-4 such as human CTLA-4) bound to a solid surface or cells
bearing either of these, an led test immunoglobulin and a d 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%, , 65-70%, 70-75% or more. A competition binding assay can be configured
in a large number of different s using either labeled antigen or labeled antibody. In a
common n 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 s 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.
In certain embodiments, the t disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a heavy chain
sing the amino acid ce set forth in SEQ ID NO: 23, 24, 25, or 26. In n
embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set
forth in SEQ ID NO: 23. In certain ments, the antibody comprises a heavy chain
comprising the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the
antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID
NO: 25. In certain embodiments, the antibody comprises a heavy chain comprising the amino
acid sequence set forth in SEQ ID NO: 26.
[00149] In certain embodiments, the instant disclosure provides an ed antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a light chain
comprising the amino acid ce set forth in SEQ ID NO: 27.
In certain embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a heavy chain
comprising the amino acid ce of SEQ ID NO: 23 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 CTLA-4 (e.g., human CTLA-4), the antibody
comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 24 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 ically binds to CTLA-4 (e.g., human
CTLA-4), the antibody comprising a heavy chain comprising the amino acid sequence of SEQ
ID NO: 25 and a light chain sing the amino acid sequence of SEQ ID NO: 27. In certain
embodiments, the instant disclosure provides an isolated antibody that specifically binds to
CTLA-4 (e.g., human CTLA-4), the antibody comprising a heavy chain comprising the amino
acid sequence of SEQ ID NO: 26 and a light chain comprising the amino acid sequence of SEQ
ID NO: 27.
Any Ig nt region can be used in the antibodies described herein. In certain
ments, the Ig region is a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin
molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a
and IgG2b) of immunoglobulin molecule.
In certain embodiments, the instant disclosure provides an ed antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a heavy chain
constant region comprising the amino acid sequence of SEQ ID NO: 28, 29, 30, or 31. In
certain embodiments, the instant disclosure provides an ed antibody that specifically
binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a light chain constant region
sing the amino acid sequence of SEQ ID NO: 32.
In n embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a heavy chain
nt region, e.g., an IgG1 constant region, or fragment thereof comprising a mutation
selected from the group consisting of: S239D, I332E, and a combination thereof, numbered
according to the EU numbering system. In certain embodiments, the dy comprises an
IgG1 heavy chain nt region comprising 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: 29.
In certain embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a heavy chain
constant region, e.g., an IgG1 constant region, or fragment thereof comprising a on
selected from the group consisting of: S239D, A330L, I332E, and combinations thereof,
numbered according to the EU numbering system. In certain embodiments, the antibody
comprises an IgG1 heavy chain constant region comprising S239D, A330L, and I332E
mutations, numbered according to the EU numbering system. In certain embodiments, the
antibody comprises a heavy chain nt region comprising the amino acid sequence of SEQ
ID NO: 30.
[00155] In certain ments, the instant sure provides an ed antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a heavy chain
constant region, e.g., an IgG1 constant region, or fragment thereof comprising a mutation
selected from the group consisting of: L235V, F243L, R292P, Y300L, P396L, and
combinations thereof, numbered ing to the EU numbering system. In certain
embodiments, the dy comprises an IgG1 heavy chain constant region comprising L235V,
F243L, R292P, Y300L, and P396L mutations, numbered ing to the EU numbering
system. In certain ments, the antibody comprises a heavy chain constant region
comprising the amino acid sequence of SEQ ID NO: 31.
In certain ments, the IgG regions of the antibodies described herein have an
increased affinity for IA, e.g., as compared with an antibody with a wild-type Fc region,
e.g., an IgG1 Fc. Sequence alterations that result in increased affinity for FcγRIIIA are known
in the art, for example, in Kellner et al., Methods 65: 105-113 , Lazar et al., Proc Natl
Acad Sci 103: 010 (2006), Shields et al., J Biol Chem. 276(9):6591-6604 (2001), each
of which is herein incorporated by reference in its entirety. In n embodiments, the
antibody comprises a heavy chain constant region, e.g., an IgG1 nt region, or nt
thereof comprising a mutation selected from the group consisting of: G236A, S239D, F243L,
T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and
P396L, and combinations thereof, numbered according to the EU numbering system. In certain
embodiments, the antibody comprises a heavy chain constant region, e.g., an IgG1 constant
region, or fragment thereof comprising S239D, numbered according to the EU numbering
system. In certain embodiments, the antibody comprises a heavy chain constant region, e.g.,
an IgG1 constant region, or fragment f comprising T256A, numbered according to the
EU numbering system. In certain embodiments, the antibody comprises a heavy chain constant
, e.g., an IgG1 constant region, or fragment thereof comprising K290A, numbered
according to the EU numbering system. In certain embodiments, the antibody ses a
heavy chain constant region, e.g., an IgG1 constant region, or fragment f comprising
S298A, numbered according to the EU numbering system. In n embodiments, the
dy comprises a heavy chain constant region, e.g., an IgG1 constant region, or fragment
thereof comprising I332E, numbered according to the EU numbering system. In certain
embodiments, the antibody comprises a heavy chain nt , e.g., an IgG1 constant
region, or fragment thereof comprising E333A, numbered according to the EU numbering
system. In certain embodiments, the antibody comprises a heavy chain constant region, e.g.,
an IgG1 constant region, or fragment thereof comprising K334A, numbered according to the
EU numbering system. In certain embodiments, the antibody comprises a heavy chain constant
region, e.g., an IgG1 constant region, or nt thereof comprising A339T, numbered
according to the EU numbering system. In certain embodiments, the antibody comprises a
heavy chain constant region, e.g., an IgG1 constant region, or fragment thereof comprising
S239D and I332E, numbered according to the EU numbering system. In certain embodiments,
the antibody comprises a heavy chain constant region, e.g., an IgG1 constant region, or
fragment f sing S239D, A330L, and I332E, numbered according to the EU
ing system. In certain ments, the dy comprises a heavy chain constant
region, e.g., an IgG1 constant region, or fragment thereof comprising S298A, E333A, and
K334A, numbered according to the EU numbering system. In certain embodiments, the
antibody comprises a heavy chain constant region, e.g., an IgG1 constant region, or fragment
thereof comprising G236A, S239D, and I332E, numbered according to the EU ing
system. In certain embodiments, the antibody ses a heavy chain constant region, e.g.,
an IgG1 constant region, or fragment thereof comprising F243L, R292P, Y300L, V305I, and
P396L, numbered according to the EU ing system.
In certain embodiments, the antibodies described herein exhibit dy-dependent
cellular cytotoxicity (ADCC) activity. In certain embodiments, the antibodies described herein
te natural killer cell mediated cell depletion. In certain embodiments, the dies
described herein are used for treating tumor infiltrated with natural killer cells. In certain
ments, the dies described herein exhibit antibody-dependent cellular
phagocytosis (ADCP) activity. In certain embodiments, the antibodies described herein initiate
macrophage mediated cell depletion. In certain embodiments, the antibodies described herein
are used for treating tumor infiltrated with macrophages. In certain embodiments, the
antibodies described herein selectively deplete intratumoral regulatory T cells.
[00158] In certain embodiments, an antibody described herein is an table antibody that
in an activated state binds human CTLA-4 protein. In certain embodiments, the activatable
dy comprises a masking moiety that inhibits the binding of the antibody in an uncleaved
state to human CTLA-4 protein, and at least one ble moiety coupled to the antibody, e.g.,
wherein the cleavable moiety is a polypeptide that ons as a substrate for a protease that
is enriched in the tumor microenvironment. Exemplary activatable antibodies are described,
e.g., in U.S. Patent Nos. 8,513,390 and 8,518,404, and U.S. Patent Application Publication
Nos. US 255313, US 2014/0010810, US 2014/0023664, which are incorporated herein
by reference. In certain embodiments, the activatable antibody comprises a human IgG heavy
chain constant region that is a variant of a wild type human IgG heavy chain constant region,
wherein the variant human IgG heavy chain constant region binds to human FcγRIIIA with
higher affinity than the wild type human IgG heavy chain constant region binds to human
FcγRIIIA.
] In certain embodiments, one, two, or more mutations (e.g., amino acid tutions)
are introduced into the Fc region of an antibody described herein (e.g., CH2 domain (residues
231-340 of human IgG1) and/or CH3 domain (residues 341-447 of human IgG1) and/or the
hinge region, numbered according to the EU numbering system, to alter one or more functional
properties of the dy, such as serum half-life, complement fixation, Fc receptor binding
and/or antigen-dependent cellular xicity.
In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions)
are introduced into the hinge region of the Fc region (CH1 ) 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. Patent 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
ly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of
the antibody.
In a specific embodiment, one, two, or more amino acid mutations (e.g.,
substitutions, insertions or deletions) are introduced into an IgG nt domain, or FcRnbinding
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. Patent Nos. 5,869,046, 6,121,022,
375 and 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 some ments, one, two or more amino acid mutations ( e.g., substitutions,
insertions, or deletions) are introduced into an IgG constant , or FcRn-binding nt
thereof (preferably an Fc or hinge-Fc domain fragment) to decrease the half-life of the dy
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 ons
(e.g., substitutions) in the second constant (CH2) domain (residues 0 of human IgG1)
and/or the third constant (CH3) domain (residues 341-447 of human IgG1), numbered
according to the EU numbering system. In a specific embodiment, the constant region of the
IgG1 of an antibody described herein comprises a methionine (M) to tyrosine (Y) substitution
in on 252, a serine (S) to threonine (T) tution 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. Patent No. 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
cqua WF 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 tutions of amino acid residues at
positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU
numbering system.
In some 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 ues
231-340 of human IgG1) and/or CH3 domain (residues 341-447 of human IgG1) 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 ted 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 dy that can be made to alter the affinity of the antibody for an Fc
receptor are bed in, e.g., Smith P et al., (2012) PNAS 109: 6181-6186, U.S. Patent No.
6,737,056, and International Publication Nos. WO 919; WO 98/23289; and WO
97/34631, all of which are herein orated by reference in their entireties.
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, 297,
318, 320 and 322, 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 y of the parent antibody. The effector ligand to which
affinity is altered can be, for example, an Fc receptor or the C1 ent of complement.
This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, each
of which is herein incorporated by reference in its entirety. In some ments, 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 zation.
See, e.g., U.S. Patent 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 dy described herein to
remove potential ylation sites on Fc region, which may reduce Fc receptor binding (see,
e.g., Shields RL 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 bed herein may be made: an N297A substitution; an
N297Q substitution; a L235A substitution and a L237A substitution; a L234A substitution and
a L235A substitution; a E233P substitution; a L234V substitution; a L235A substitution; a
C236 deletion; a P238A substitution; a D265A tution; a A327Q substitution; or a P329A
substitution, numbered according to the EU ing . In certain embodiments, a
mutation ed from the group consisting of D265A, P329A, and a combination thereof,
numbered ing to the EU numbering system, may be made in the constant region of an
antibody described herein.
[00164] In a specific embodiment, an antibody described herein ses the constant
domain of an IgG1 with an N297Q or N297A amino acid tution, numbered ing to
the EU numbering system. In one embodiment, an antibody described herein comprises the
constant domain of an IgG1 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 IgG1
with a mutation selected from the group consisting of L234A, L235A, and a combination
thereof, ed 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 IgG1 heavy chain, numbered
according to the EU ing system, are not L, L, and D, respectively. This approach is
bed 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 IgG1 heavy chain are F, E, and
A; or A, A, and A, respectively, numbered according to the EU numbering system.
[00165] In certain embodiments, one or more amino acids ed 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. Patent No. 6,194,551
(Idusogie et al.), which is herein incorporated by reference in its entirety. In some
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 d to thereby
alter the y 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 dy described herein is ed to increase the ability of the antibody to mediate
antibody ent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody
for an Fcγ 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 ing to the EU numbering . This approach is bed further in
International Publication No. WO 00/42072, which is herein incorporated by reference in its
entirety.
In certain embodiments, an antibody described herein comprises the constant region
of an IgG4 antibody and the serine at amino acid e 228 of the heavy chain, numbered
according to the EU numbering system, is tuted for proline.
] 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.
In certain embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4) and functions as an antagonist.
In certain embodiments, the instant disclosure provides an isolated antibody that
ically binds to CTLA-4 (e.g., human CTLA-4) and decreases CTLA-4 (e.g., human
CTLA-4) 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 bed
herein and/or known to one of skill in the art, ve to CTLA-4 (e.g., human CTLA-4) activity
t any antibody or with an unrelated antibody (e.g., an antibody that does not specifically
bind to CTLA-4 (e.g., human CTLA-4)). In certain embodiments, the instant disclosure
provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) and
decreases CTLA-4 (e.g., human CTLA-4) 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,
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 and/or known to one of skill in the art, relative to
CTLA-4 (e.g., human CTLA-4) activity without any antibody or with an unrelated antibody
(e.g., an antibody that does not specifically bind to CTLA-4 (e.g., human )). Non-
limiting examples of CTLA-4 (e.g., human CTLA-4) activity can include CTLA-4 (e.g., human
CTLA-4) ing, CTLA-4 (e.g., human CTLA-4) binding to CTLA-4 (e.g., human CTLA-
4) ligand (e.g., CD80 or CD86), and inhibition of cytokine production (e.g., IL-2, IFN-γ, or
TNF-α). In certain embodiments, the instant disclosure es an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4) and deactivates, reduces, or inhibits a
CTLA-4 (e.g., human CTLA-4) activity. In specific embodiments, a decrease in a CTLA-4
(e.g., human CTLA-4) activity is assessed as described in the Examples, infra.
In ic embodiments, the instant disclosure provides an isolated dy that
specifically binds to CTLA-4 (e.g., human CTLA-4) and reduces CTLA-4 (e.g., human CTLA-
4) binding to its ligand (e.g., CD80 or CD86) by at least about 5%, 10%, 15%, 20%, 25%, 30%,
%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as
assessed by methods known to one of skill in the art, relative to CTLA-4 (e.g., human CTLA-
4) binding to its ligand (e.g., CD80 or CD86) without any antibody or with an unrelated
antibody (e.g., an antibody that does not specifically bind to CTLA-4 (e.g., human CTLA-4)).
In specific embodiments, the instant disclosure provides an isolated antibody that specifically
binds to CTLA-4 (e.g., human ) and reduces CTLA-4 (e.g., human CTLA-4) binding
to its ligand (e.g., CD80 or CD86) 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,
fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, as assessed by
methods known to one of skill in the art, relative to CTLA-4 (e.g., human CTLA-4) binding to
its ligand (e.g., CD80 or CD86) without any antibody or with an unrelated dy (e.g., an
dy that does not specifically bind to CTLA-4 (e.g., human CTLA-4)).
In specific embodiments, the instant disclosure provides an isolated antibody that
specifically binds to CTLA-4 (e.g., human CTLA-4) and increases cytokine tion (e.g.,
IL-2, IFN-γ, or TNF-α) 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
ne production without any dy or with an ted antibody (e.g., an dy that
does not specifically bind to CTLA-4 (e.g., human CTLA-4)). In specific embodiments, the
instant disclosure provides an isolated dy that specifically binds to CTLA-4 (e.g., human
CTLA-4) and increases cytokine production (e.g., IL-2, IFN-γ, or TNF-α) 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 cytokine production without any antibody or with
an unrelated antibody (e.g., an dy that does not specifically bind to CTLA-4 (e.g., human
CTLA-4)).
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 CTLA-4 (e.g., human CTLA-4), have increased 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 PBMCs only stimulated
with SEA without any antibody or with an unrelated antibody (e.g., an antibody that does not
specifically bind to CTLA-4 (e.g., human CTLA-4)), as assessed by s bed herein
(see the Examples, infra) or known to one of skill in the art.
6.3 Pharmaceutical Compositions
Provided herein are compositions comprising an anti-CTLA-4 antibody described
herein having the desired degree of purity in a physiologically acceptable carrier, excipient or
stabilizer (Remington’s ceutical Sciences (1990) Mack Publishing Co., Easton, PA).
Acceptable carriers, excipients, or stabilizers are nontoxic to ents at the s and
concentrations employed, and include buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid and nine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium
chloride, benzethonium chloride; phenol, butyl or benzyl l; 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; ns, such as serum albumin,
gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids
such as glycine, glutamine, asparagine, ine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates ing 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 xes (e.g., tein complexes); and/or non-ionic
surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
[00174] In a specific embodiment, pharmaceutical compositions comprise an anti-CTLA-4
antibody described herein, and optionally one or more additional prophylactic or therapeutic
agents, in a pharmaceutically acceptable carrier. In a specific embodiment, pharmaceutical
compositions se an effective amount of an antibody or antigen-binding fragment thereof
described herein, and optionally one or more additional prophylactic or therapeutic , in
a pharmaceutically acceptable r. In some embodiments, the antibody is the only active
ingredient included in the pharmaceutical composition. Pharmaceutical compositions
described herein can be useful in inhibiting, CTLA-4 activity and treating a condition, such as
cancer or an infectious disease.
In one aspect, provided herein is a pharmaceutical composition comprising an anti-
CTLA-4 antibody of the invention and a pharmaceutically able carrier or excipient, for
use as a medicament.
In one aspect, provided herein is a pharmaceutical composition comprising an anti-
CTLA-4 antibody of the invention and a pharmaceutically able carrier or excipient, for
use in a method for the treatment of cancer.
Pharmaceutically acceptable carriers used in eral preparations e
aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers,
antioxidants, local etics, suspending and sing agents, emulsifying agents,
sequestering or chelating agents and other pharmaceutically acceptable substances. Examples
of s 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 eral ations packaged in le-dose containers which include s
or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic
acid , thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents
include sodium de 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
nylpyrrolidone. Emulsifying agents e Polysorbate 80 (TWEEN® 80). A
sequestering or chelating agent of metal ions includes EDTA. Pharmaceutical carriers also
include ethyl alcohol, hylene glycol and propylene glycol for water le es;
and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.
[00178] A pharmaceutical composition may be formulated for any route of administration
to a subject. Specific examples of routes of administration include asal, 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 l. 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.
Preparations for parenteral stration of an antibody include sterile solutions
ready for injection, sterile dry soluble ts, such as lyophilized powders, ready to be
ed with a solvent just prior to use, ing rmic tablets, sterile suspensions
ready for injection, sterile dry ble products ready to be combined with a vehicle just prior
to use and sterile emulsions. The solutions may be either aqueous or nonaqueous.
If administered intravenously, suitable carriers include physiological saline or
phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents,
such as glucose, hylene glycol, and polypropylene glycol and mixtures f.
Topical mixtures comprising an antibody are prepared as described for the local and
systemic administration. The resulting mixture can be a solution, sion, emulsions or the
like and can be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions,
suspensions, tinctures, , foams, aerosols, irrigations, sprays, suppositories, bandages,
dermal patches or any other formulations suitable for topical administration.
An anti-CTLA-4 antibody described herein can be formulated as an aerosol for
topical application, such as by inhalation (see, e.g., U.S. Patent Nos. 126, 4,414,209 and
4,364,923, which describe ls 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 microfine 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 ment less than
microns.
[00183] An anti-CTLA-4 antibody described 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, , and lotions and for application to the eye or for intracisternal
or intraspinal ation. 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 able excipients can
also be administered.
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. Patent Nos. 6,267,983, 595, 6,256,533, 6,167,301,
6,024,975, 6,010715, 317, 5,983,134, 433, and 5,860,957, all of which are herein
incorporated by reference in their entireties.
In certain embodiments, a pharmaceutical composition comprising an antibody or
antigen-binding fragment thereof bed 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 or antigen-binding fragment thereof described herein, or a
pharmaceutically acceptable derivative thereof, in a suitable solvent. In some 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 t
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 on
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°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 e water or other suitable carrier. The
precise amount depends upon the selected compound. Such amount can be empirically
determined.
The TLA-4 antibodies bed herein and other compositions ed
herein can also be formulated to be targeted to a particular tissue, or, 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 ing methods are contemplated herein for use in the instant
compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Patent Nos.
6,316,652, 6,274,552, 6,271,359, 6,253,872, 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, 366, 252, 5,840,674,
542 and 5,709,874, all of which are herein incorporated by reference in their entireties.
In a specific embodiment, an antibody or antigen-binding fragment thereof described herein is
ed to a tumor.
The compositions to be used for in vivo administration can be sterile. This is readily
accomplished by filtration through, e.g., sterile tion membranes.
6.4 Methods of Use and Uses
In another aspect, the t disclosure provides a method of treating a subject
using the anti-CTLA-4 antibodies described herein. Any disease or disorder in a subject that
would benefit from inhibition of CTLA-4 function can be treated using the anti-CTLA-4
antibodies described herein. The anti-CTLA-4 antibodies described 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 activation in response to an antigen in a
t, the method sing administering to the subject an ive amount of an anti-
CTLA-4 antibody or pharmaceutical composition thereof, as described herein. In certain
embodiments, the instant sure provides a method of treating cancer in a subject, the
method comprising administering to the subject an effective amount of the antibody or
ceutical composition, as described herein.
[0001] Cancers that can be treated with the antibodies, therapeutic combinations, or
pharmaceutical compositions bed herein include, without limitation, solid cancer (e.g.,
relapsed or refractory solid cancer, and ed or metastatic solid cancer), carcinoma,
sarcoma, melanoma (e.g., stage III or stage IV melanoma), small cell lung cancer, non-small
cell lung cancer, urothelial cancer, ovarian cancer, prostate cancer (e.g., metastatic hormone-
tory prostate cancer and progressive metastatic prostate cancer), pancreatic cancer, breast
cancer (e.g., HER2+ breast cancer (e.g., relapsed/refractory HER2+ breast cancer)), head and
neck cancer (e.g., relapsed/refractory head and neck squamous cell carcinoma (HNSCC)),
, malignant glioma, glioblastoma multiforme, brain metastasis, merkel cancer, gastric
cancer, gastroesophageal cancer, renal cell carcinoma, uveal melanoma, colon cancer, cervical
cancer, ma (e.g., relapsed or tory lymphoma), non-Hodgkin's lymphoma,
Hodgkin's lymphoma, leukemia, and multiple myeloma. In certain embodiments, the cancer
is treated with intratumoral administration of an antibody, therapeutic ation, or
pharmaceutical composition described herein. Cancers that can be treated with umoral
administration of the antibodies, therapeutic combinations, or pharmaceutical compositions
described herein include, without limitation, solid tumors (e.g., advanced or metastatic solid
tumors), head and neck cancer (e.g., relapsed/refractory head and neck us cell
carcinoma (HNSCC)), and breast cancer (e.g., HER2+ breast cancer (e.g., relapsed/refractory
HER2+ breast cancer)).
In certain ments, the cancer treated in accordance with the methods
described herein is a solid tumor. In certain embodiments, the cancer treated in ance
with the methods described herein is a metastatic or locally advanced cancer (e.g., a metastatic
or locally advanced solid tumor). In certain embodiments, the cancer is treated in ance
with a method described herein as a first cancer therapy after diagnosis of the metastatic or
locally ed tumor (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1,
2, 3, 4, 6, 8, or 12 months after diagnosis). In certain embodiments, the cancer is treated in
accordance with a method described herein as the first cancer therapy after diagnosis of tumor
progression (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8,
or 12 months after diagnosis of tumor progression) that has occurred despite previous ent
of the tumor with a different cancer therapy, ally wherein the method described herein
is provided as the second cancer therapy stered. In certain embodiments, the cancer is
treated in accordance with a method described herein as the first cancer therapy after diagnosis
of toxicity of a different cancer therapy (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or
12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis of toxicity of the different cancer
therapy), optionally wherein the method described herein is provided as the second cancer
therapy administered. In certain ments, the cancer treated in accordance with the
methods described herein is a metastatic or locally advanced cancer (e.g., solid tumor) for
which no standard therapy is available. In other embodiments, the cancer treated in accordance
with the methods described herein is a metastatic or locally advanced cancer (e.g., solid tumor)
for which a standard therapy has failed (i.e., the cancer has progressed after the rd
therapy). In certain embodiments, a therapy fails if the cancer is refractory to the therapy. In
certain embodiments, a therapy fails if the cancer relapses after responding, fully or partially,
to the therapy. In certain embodiments, metastatic or locally advanced cancer (e.g., solid
tumor) has been confirmed histologically or cytologically.
In certain embodiments, the cancer is a solid tumor. In certain embodiments, the
cancer (e.g., solid tumor) expresses PD-L1. In certain embodiments, the tage of tumor
cells in a sample of the cancer (e.g., solid tumor) that exhibit able expression (e.g., partial
or complete expression) of PD-L1 is at least 1% (e.g., at least 2%, 3%, 4%, 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%). In certain embodiments,
the tage of tumor cells in a sample of the cancer (e.g., solid tumor) that exhibit detectable
membrane expression (e.g., partial or complete membrane expression) of PD-L1 is at least 1%
(e.g., at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,
80%, or 90%). In certain embodiments, the percentage of tumor cells in a sample of the cancer
(e.g., solid tumor) that exhibit detectable membrane expression (e.g., partial or complete
membrane expression) of PD-L1 is at least 1%. In certain embodiments, the percentage of
tumor cells in a sample of the cancer (e.g., solid tumor) that exhibit detectable membrane
sion (e.g., l or complete membrane expression) of PD-L1 is at least 5%. In certain
embodiments, the percentage of tumor cells in a sample of the cancer (e.g., solid tumor) that
exhibit detectable membrane expression (e.g., partial or complete membrane expression) of
PD-L1 is at least 25%. In certain embodiments, the percentage of tumor cells in a sample of
the cancer (e.g., solid tumor) that exhibit detectable ne expression (e.g., partial or
complete membrane expression) of PD-L1 is at least 50%.
[0004] In certain embodiments, the metastatic or locally advanced cancer (e.g., solid
tumor) expresses PD-L1. In certain embodiments, the percentage of tumor cells in a sample of
the metastatic or locally advanced cancer (e.g., solid tumor) that exhibit detectable expression
(e.g., partial or complete expression) of PD-L1 is at least 1% (e.g., at least 2%, 3%, 4%, 5%,
%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%). In certain
ments, the percentage of tumor cells in a sample of the metastatic or locally advanced
cancer (e.g., solid tumor) that exhibit detectable membrane expression (e.g., partial or complete
membrane expression) of PD-L1 is at least 1% (e.g., at least 2%, 3%, 4%, 5%, 10%, 15%, 20%,
%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%). In certain ments, the
percentage of tumor cells in a sample of the metastatic or locally advanced cancer (e.g., solid
tumor) that t detectable membrane expression (e.g., partial or complete membrane
expression) of PD-L1 is at least 1%. In certain embodiments, the percentage of tumor cells in
a sample of the metastatic or locally advanced cancer (e.g., solid tumor) that exhibit detectable
membrane expression (e.g., partial or complete membrane expression) of PD-L1 is at least 5%.
In certain embodiments, the percentage of tumor cells in a sample of the metastatic or locally
advanced cancer (e.g., solid tumor) that exhibit detectable membrane expression (e.g., partial
or complete membrane expression) of PD-L1 is at least 25%. In certain embodiments, the
percentage of tumor cells in a sample of the atic or y advanced cancer (e.g., solid
tumor) that exhibit detectable membrane expression (e.g., partial or te membrane
expression) of PD-L1 is at least 50%.
[0005] For each and every one of the methods bed herein that requires a certain
percentage of cells in a sample exhibit detectable expression (e.g., membrane expression,
partial or complete membrane expression) of PD-L1, the expression of PD-L1 can be detected
by any method well known in the art, ing but not limited to immunohistochemistry.
ary immunohistochemistry assays for measuring PD-L1 expression in tumor cells are
provided in Hirsch et al. (2017, J. Thoracic Oncol. 12(2): 208-222), Rimm et al. (2017, JAMA
Oncol. 3(8): 1051-1058), and Diggs and Hsueh (2017, Biomarker Res. 5:12), which are
incorporated by nce herein in their entirety.
In certain embodiments, the cancer treated in accordance with a method bed
herein is a metastatic or locally advanced non-small cell lung cancer (NSCLC). In n
embodiments, the cancer treated in ance with a method described herein is a metastatic
non-small cell lung cancer (NSCLC). In certain embodiments, the cancer d in accordance
with a method described herein is a Stage IV, metastatic or locally advanced NSCLC. In certain
embodiments, the cancer treated in accordance with a method described herein is a Stage IV,
metastatic NSCLC. In certain embodiments, the percentage of tumor cells in a sample of the
metastatic or locally advanced NSCLC that exhibit detectable expression (e.g., partial or
complete expression) of PD-L1 is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%,
%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%. In certain embodiments, the percentage of
tumor cells in a sample of the metastatic or locally advanced NSCLC that t detectable
membrane sion (e.g., l or complete membrane expression) of PD-L1 is at least 1%,
2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or
90%. In certain embodiments, the percentage of tumor cells in a sample of the metastatic or
locally advanced NSCLC that exhibit detectable ne expression (e.g., partial or
complete membrane expression) of PD-L1 is at least 1%. In certain embodiments, the
percentage of tumor cells in a sample of the atic or locally advanced NSCLC that exhibit
detectable membrane expression (e.g., l or complete membrane expression) of PD-L1 is
at least 5%. In certain embodiments, the percentage of tumor cells in a sample of the metastatic
or y advanced NSCLC that exhibit able membrane expression (e.g., partial or
complete membrane sion) of PD-L1 is at least 25%. In certain embodiments, the
percentage of tumor cells in a sample of the metastatic or locally advanced NSCLC that exhibit
detectable membrane expression (e.g., partial or complete membrane expression) of PD-L1 is
at least 50%. In certain embodiments, the metastatic or locally advanced NSCLC has no EGFR
or ALK genomic tumor aberrations. In certain embodiments, the atic or locally
advanced NSCLC has no EGFR sensitizing mutation (e.g., mutation that is amenable to
treatment with a tyrosine kinase inhibitor including erlotinib, nib, or afatanib) or ALK
translocation. In certain embodiments, the subject having the metastatic or locally advanced
NSCLC has received no prior systemic chemotherapy treatment for metastatic or locally
advanced NSCLC. In certain embodiments, the metastatic or locally advanced NSCLC is
treated in accordance with a method described herein as a first cancer therapy after diagnosis
(e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months
after diagnosis) of the metastatic or y ed NSCLC. In certain embodiments, the
method comprises treating a subject having NSCLC (e.g., Stage IV, metastatic, or locally
advanced NSCLC) using an TLA-4 antibody described herein, e.g., AGEN1884.H3
(IgG1 A330L/I332E), or pharmaceutical composition comprising such TLA-4
antibody, wherein the percentage of tumor cells in a sample of the NSCLC that exhibit
detectable membrane expression (e.g., partial or complete membrane expression) of PD-L1 is
at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,
80%, or 90%, and wherein the method is provided as a first cancer therapy after diagnosis of
the cervical cancer (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3,
4, 6, 8, or 12 months after diagnosis).
In certain embodiments, the cancer treated in accordance with the methods
described herein is a cervical . In certain embodiments, the cancer treated in accordance
with the methods bed herein is a metastatic or locally advanced, unresectable squamous
cell carcinoma, adenosquamous carcinoma, or adenocarcinoma of the cervix. In certain
embodiments, the cancer treated in accordance with the methods described herein is an
unresectable or metastatic cervical cancer. In certain embodiments, the cervical cancer has
progressed after a standard therapy (e.g., has relapsed after the standard therapy, or is refractory
to the standard therapy). In certain embodiments, the standard y comprises a platinum-
containing herapy. In certain embodiments, the platinum-containing chemotherapy is
selected from the group ting of cisplatin, latin, oxaliplatin, nedaplatin, satraplatin,
picoplatin, tin, phenanthriplatin, iproplatin, lobapatin, heptaplatin, lipoplatin, and a
combination thereof. In certain ments, the standard y further comprises a second
chemotherapy. In certain embodiments, the second chemotherapy is selected from the group
consisting of a nucleotide analog (e.g., gemcitabine), a folate antimetabolite (e.g., pemetrexed),
and a taxane (e.g., paclitaxel). In certain embodiments, the standard therapy is any platinumbased
doublet chemotherapy (PT-DC) (also known as platinum-containing doublet) known in
the art. In certain embodiments, the PT-DC comprises cisplatin and abine, cisplatin and
pemetrexed, cisplatin and paclitaxel, carboplatin and paclitaxel, or tin and topotecan. The
standard therapy (e.g., one comprising a PT-DC) can optionally further comprise one or more
additional therapies, such as bevacizumab. In certain embodiments, the standard therapy
comprises paclitaxel and topotecan. In n embodiments, the cervical cancer is HPV
positive. In certain embodiments, the cervical cancer is associated with microsatellite
instability. In certain embodiments, the cancer treated in accordance with the methods
described herein is a metastatic or locally advanced, ctable squamous cell carcinoma,
adenosquamous carcinoma, or adenocarcinoma of the cervix that has relapsed after a platinumcontaining
doublet administered for treatment of ed (recurrent, unresectable, or
metastatic) disease. In certain embodiments, the cancer of the cervix is treated in ance
with a method described herein as a first cancer y after diagnosis of the cervical cancer
(e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months
after diagnosis). In certain ments, the cancer of the cervix is treated in accordance with
a method bed herein as the first cancer therapy after diagnosis of tumor progression (e.g.,
within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after
diagnosis of tumor progression) that has occurred despite previous treatment of the cancer of
the cervix with a different cancer therapy, optionally wherein the method described herein is
provided as the second cancer therapy administered. In certain embodiments, the cancer of the
cervix is treated in accordance with a method described herein as the first cancer therapy after
diagnosis of toxicity of a different cancer y (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3,
4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after sis of toxicity of the different
cancer therapy), ally wherein the method described herein is provided as the second
cancer therapy administered. In certain embodiments, the method comprises treating a subject
having cervical cancer (e.g., a metastatic or locally advanced, unresectable squamous cell
carcinoma, adenosquamous carcinoma, or adenocarcinoma of the cervix) using an anti-CTLA-
4 antibody described , e.g., AGEN1884.H3 (IgG1 S239D/A330L/I332E), or
pharmaceutical composition comprising such anti-CTLA-4 antibody, wherein the method is
provided as a first cancer therapy after diagnosis of the cervical cancer (e.g., within 1, 2, 3, 4,
, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis). In
certain embodiments, the method ses treating a subject having cervical cancer (e.g., a
metastatic or y advanced, unresectable squamous cell oma, adenosquamous
carcinoma, or adenocarcinoma of the cervix) using an anti-CTLA-4 antibody described herein,
e.g., AGEN1884.H3 (IgG1 S239D/A330L/I332E), or pharmaceutical composition comprising
such anti-CTLA-4 antibody, wherein the method is provided after sis of tumor
progression (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8,
or 12 months after diagnosis of tumor progression) that has occurred despite previous treatment
of the cervical cancer with a different cancer therapy, or provided after diagnosis of toxicity of
a different cancer therapy (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or,
1, 2, 3, 4, 6, 8, or 12 months after diagnosis of toxicity of the different cancer therapy), and
wherein the method described herein is provided as the second cancer therapy administered.
In certain embodiments, the cancer treated in accordance with the methods
described herein is a cutaneous squamous-cell oma (cSCC). In certain embodiments, the
cancer treated in ance with the methods described herein is a Stage IV cutaneous
squamous-cell carcinoma (cSCC). In certain embodiments, the cSCC (e.g., Stage IV cSCC) is
not e with radiation therapy. In certain embodiments, the Stage IV cSCC is sed
histologically or cytologically according to the eighth edition of the American Joint Committee
on Cancer staging manual (AJCC-8). In certain embodiments, the cSCC (e.g., Stage IV cSCC)
is treated in accordance with a method described herein as a first cancer therapy after diagnosis
of the cSCC (e.g., Stage IV cSCC) (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12
weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis).. In certain embodiments, the cSCC
(e.g., Stage IV cSCC) is treated in accordance with a method bed herein as the first cancer
therapy after diagnosis of tumor progression (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6,
8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis of tumor progression) that has
occurred e previous treatment of the cSCC (e.g., Stage IV cSCC) with a ent cancer
y, optionally wherein the method described herein is provided as the second cancer
therapy administered. In certain embodiments, the cSCC (e.g., Stage IV cSCC) is treated in
according with a method described herein as the first cancer y after diagnosis of toxicity
of a different cancer therapy (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks;
or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis of toxicity of the different cancer therapy),
optionally wherein the method described herein is provided as the second cancer therapy
administered.
In certain embodiments, the cancer treated in accordance with the methods
described herein is B cell lymphoma (e.g., B cell chronic lymphocytic leukemia, B cell non-
Hodgkin ma, cutaneous B cell lymphoma, diffuse large B cell lymphoma), basal cell
carcinoma, bladder cancer, blastoma, brain metastasis, breast cancer, Burkitt ma,
carcinoma (e.g., adenocarcinoma (e.g., of the gastroesophageal junction)), cervical ,
colon cancer, colorectal cancer (colon cancer and rectal cancer), trial carcinoma,
esophageal cancer, Ewing sarcoma, follicular lymphoma, gastric cancer, esophageal
junction carcinoma, gastrointestinal cancer, glioblastoma (e.g., glioblastoma multiforme, e.g.,
newly diagnosed or recurrent), glioma, head and neck cancer (e.g., head and neck squamous
cell carcinoma), hepatic metastasis, Hodgkin’s and non-Hodgkin’s lymphoma, kidney cancer
(e.g., renal cell carcinoma and Wilms’ tumors), laryngeal cancer, leukemia (e.g., chronic
myelocytic ia, hairy cell leukemia), liver cancer (e.g., hepatic carcinoma and hepatoma),
lung cancer (e.g., non-small cell lung cancer and cell lung cancer), lymphblastic
lymphoma, lymphoma, mantle cell ma, metastatic brain tumor, metastatic cancer,
myeloma (e.g., multiple myeloma), neuroblastoma, ocular melanoma, oropharyngeal cancer,
osteosarcoma, ovarian cancer, pancreatic cancer (e.g., pancreatis ductal adenocarcinoma),
prostate cancer (e.g., hormone refractory (e.g., castration resistant), metastatic, metastatic
hormone refractory (e.g., castration resistant, androgen independent)), renal cell oma
(e.g., metastatic), salivary gland carcinoma, a (e.g., rhabdomyosarcoma), skin cancer
(e.g., melanoma (e.g., atic melanoma)), soft tissue sarcoma, solid tumor, squamous cell
carcinoma, synovia sarcoma, testicular cancer, thyroid cancer, transitional cell cancer
(urothelial cell cancer), uveal melanoma (e.g., metastatic), verrucous carcinoma, vulval cancer,
and Waldenstrom macroglobulinemia.
In certain ments, the cancer treated in accordance with the s
described herein is human a or carcinoma, e.g., fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, osarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast , ovarian cancer, prostate cancer, us cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, enocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma (e.g., metastatic), hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, nal carcinoma, Wilms' tumor, cervical cancer,
testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
oma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma,
pharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma, or retinoblastoma.
[0011] In certain embodiments, the cancer treated in accordance with the methods
described herein is angiosarcoma.
In certain embodiments, the cancer treated in accordance with the methods
described herein is an acute lymphocytic leukemia or acute myelocytic leukemia (e.g.,
myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic
leukemia (chronic myelocytic (granulocytic) leukemia or chronic lymphocytic leukemia);
n's e; non-Hodgkin's disease; acute d leukemia; B-cell lymphoma; T-cell
lymphoma; anaplastic large cell lymphoma; intraocular lymphoma; follicular ma; small
intestine lymphoma; or nic marginal zone lymphoma.
In certain embodiments, the cancer treated in accordance with the methods
described herein is multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain
disease, gastrointestinal stromal tumors, head and/or neck cancer (e.g., squamous cell
oma of the hypopharynx, squamous cell carcinoma of the larynx, cell carcinoma of the
oropharynx, or verrucous carcinoma of the ), endometrial stromal sarcoma, mast cell
sarcoma, adult soft tissue sarcoma, uterine sarcoma, merkel cell carcinoma, urothelial
carcinoma, melanoma with brain metastases, uveal melanoma, uveal melanoma with liver
metastases, non-small cell lung cancer, rectal cancer, or myelodysplastic syndrome. In some
embodiments, the cancer treated in accordance with the methods is metastatic.
In certain ments, the cancer treated in accordance with the methods
described herein is prostate cancer, breast cancer, lung cancer, colorectal cancer, melanoma,
bronchial cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous
system cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, non-
n’s lymphoma, thyroid cancer, kidney , biliary tract cancer, small bowel or
appendix cancer, salivary gland , thyroid gland cancer, adrenal gland cancer, squamous
cell cancer, mesothelioma, osteocarcinoma, thyoma/thymic oma, glioblastoma,
myelodysplastic syndrome, soft tissue sarcoma, DIPG, adenocarcinoma, osteosarcoma,
chondrosarcoma, leukemia, or pancreatic cancer. In some embodiments, the cancer treated in
accordance with the methods described herein includes a oma (e.g., an adenocarcinoma),
lymphoma, blastoma, melanoma, sarcoma, or leukemia.
[0015] In certain ments, the cancer treated in accordance with the methods
described herein is squamous cell cancer, small-cell lung cancer, non-small cell lung ,
gastrointestinal , Hodgkin's lymphoma, non-Hodgkin's lymphoma, pancreatic ,
glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer (e.g., hepatic carcinoma and
hepatoma), bladder cancer, breast , inflammatory breast cancer, Merkel cell carcinoma,
colon cancer, colorectal cancer, h cancer, urinary bladder cancer, endometrial
carcinoma, myeloma (e.g., multiple myeloma), salivary gland, carcinoma, kidney cancer (e.g.,
renal cell carcinoma and Wilms’ tumors), basal cell carcinoma, melanoma, prostate cancer,
vulval cancer, d cancer, testicular cancer, esophageal cancer, serous adenocarcinoma or
various types of head and neck cancer. In certain embodiments, the cancer treated in
ance with the methods described herein includes desmoplastic ma, inflammatory
breast cancer, thymoma, rectal cancer, anal cancer, or surgically treatable or non-surgically
treatable brain stem . In a specific embodiment, the cancer is a solid tumor. In another
ic ment, the cancer is glioblastoma multiforme. In some embodiments, the
glioblastoma multiforme is recurrent. In some ments, the glioblastoma multiforme is
newly diagnosed. In some embodiments, the glioblastoma multiforme is in a subject having
non-methylated MGMT promoters. In some embodiments, the glioblastoma multiforme is
refractory to Bevacizumab therapy. In some embodiments, the glioblastoma multiforme is in
a subject that has not received Bevacizumab therapy.
[0016] In certain embodiments, the cancer d in accordance with the methods
described herein is metastatic melanoma (e.g., resistant metastatic melanoma), atic
ovarian , or metastatic renal cell carcinoma. In certain embodiments, the cancer treated
in accordance with the methods described herein is ma that is resistant to Ipilimumab.
In some ments, the cancer treated in accordance with the methods described herein is
melanoma that is resistant to Nivolumab or Pembrolizumab. In some embodiments, the cancer
treated in accordance with the methods described herein is melanoma that is resistant to
Ipilimumab and Nivolumab or Pembrolizumab.
] In certain embodiments, the cancer treated in accordance with the s
described herein is breast cancer (e.g., herceptin resistant breast cancer and trastuzumab-DM1
) resistant breast cancer), prostate cancer, glioblastoma multiforme, colorectal cancer,
sarcoma, r , cervical cancer, HPV-associated cancers, cancers of the vagina,
cancers of the vulva, cancers of the penis, cancer of the anus, cancer of the rectum, cancer of
the oropharynx, multiple myeloma, renal cell carcinoma, ovarian cancer, hepatocellular cancer,
endometrial cancer, pancreatic cancer, lymphoma, and leukemia (e.g., elderly leukemia, acute
myeloid leukemia (AML), and y AML).
In certain embodiments, the cancer treated in accordance with the s
described herein is metastatic malignant ma (e.g., cutaneous or intraocular malignant
melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone tory
prostate adenocarcinoma), breast cancer, colon cancer, lung cancer (e.g., non-small cell lung
cancer), bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, e cancer,
ovarian , rectal cancer, cancer of the anal region, stomach , testicular cancer,
uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of
the cervix, carcinoma of the , carcinoma of the vulva, cancer of the esophagus, cancer
of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of
the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra,
cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic
myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors
of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter,
carcinoma of the renal , neoplasm of the central nervous system (CNS), primary CNS
lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, glioma, pituitary
adenoma, Kaposi's a, epidermoid cancer, squamous cell cancer, T-cell lymphoma,
environmentally induced cancers including those induced by asbestos, esophageal cancer, liver
cancer, refractory or recurrent ancies, metastatic cancers, s that express PD-L1,
and combinations of said cancers.
In certain embodiments, the subject has previously received an therapy. In
certain embodiments, the subject has not previously received any immunotherapy. In certain
embodiments, the cancer is an advanced or atic .
In certain embodiments, the instant disclosure provides a method of preventing or
treating an infectious disease in a subject, the method sing stering to the subject
an effective amount of an anti-CTLA-4 antibody or pharmaceutical composition thereof, as
described herein. In one ment, provided herein are s 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 . In a specific embodiment,
an anti-CTLA-4 dy described herein or a composition f is the only active agent
administered to a subject. In some embodiments, an anti-CTLA-4 antibody described herein
or a composition thereof is used in combination with nfective interventions (e.g.,
antivirals, antibacterials, antifungals, or anti-helminthics) for the treatment of infectious
diseases.
ious diseases that can be treated and/or prevented by anti-CTLA-4 antibodies
or pharmaceutical compositions described 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-CTLA-4 antibodies or pharmaceutical
compositions described herein is caused by a virus. Viral diseases or viral infections that can
be prevented and/or treated in accordance with the s described herein include, but are
not d 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, irus, 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
Bacterial infections that can be prevented and/or treated e infections caused
by Escherichia coli, Klebsiella pneumoniae, lococcus , Enterococcus faecalis,
Proteus is, Staphylococcus viridans, and Pseudomonas aeruginosa. Bacterial diseases
caused by bacteria (e.g., Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus,
Enterococcus faecalis, Proteus vulgaris, lococcus 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.
Protozoal diseases or protozoal infections caused by protozoa that can be ted
and/or treated in accordance with the methods described herein include, but are not limited to,
ania, 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 bed herein include, but are not limited to, chlamydia and rickettsia.
Fungal diseases or fungal ions that can be prevented and/or treated in
accordance with the methods described herein include, but are not limited to, those caused by
a infections, cosis, Candida mastitis, progressive disseminated
trichosporonosis with latent trichosporonemia, disseminated candidiasis, pulmonary
paracoccidioidomycosis, pulmonary aspergillosis, Pneumocystis carinii nia,
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
ion, staphylococcal endophthalmitis, richosis, and dermatophytosis.
In certain embodiments, the infectious e is acute. In certain embodiments,
the infectious e is chronic. In certain ments, the infectious disease is caused by
flavivirus, e.g., West Nile virus, Saint Louis alitis virus, Powassan virus, tick-borne
encephalitis virus, dengue virus, zika virus, Kyasanur Forest disease virus, yellow fever virus,
and chikungunya virus. In certain embodiments, the infectious disease is caused by Ebola
virus. In certain embodiments, the infectious disease is caused by influenza virus. In n
embodiments, the infectious disease is caused by Human Immunodeficiency Virus (HIV),
Hepatitis B virus (HBV) or Hepatitis C virus (HCV). In certain embodiments, the anti-CTLA-
4 antibody or pharmaceutical composition thereof, as described herein, promotes viral control.
In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition thereof, as
described herein, eliminates viral reservoirs.
[00198] The present invention relates in one aspect to an anti-CTLA-4 antibody of the
invention and/or a ceutical composition of the invention sing an anti-CTLA-4
antibody of the invention and a pharmaceutically acceptable carrier or excipient, for use as a
medicament.
The present invention relates, in one aspect, to an anti-CTLA-4 antibody of the
invention, and/or its use in combination with pharmaceutically acceptable carriers or
excipients, for preparing pharmaceutical compositions or medicaments for immunotherapy
(e.g., an immunotherapy for increasing T-cell activation in response to an n in a subject,
treating cancer, or treating or preventing infectious diseases).
] The present invention relates in one aspect to an TLA-4 antibody of the
invention and/or a pharmaceutical composition of the ion comprising an anti-CTLA-4
antibody of the invention and a pharmaceutically acceptable carrier or excipient, for use in a
method for the treatment of cancer.
The present invention relates in one aspect to an TLA-4 antibody of the
invention and/or a ceutical composition of the invention comprising an anti-CTLA-4
dy of the invention and a pharmaceutically acceptable carrier or excipient, for use in a
method for inhibiting immune system tolerance to tumors and/or for immunotherapy for
subjects with cancer.
The present invention relates in one aspect to an anti-CTLA-4 antibody of the
invention and/or a pharmaceutical composition of the invention comprising an anti-CTLA-4
dy of the ion and a pharmaceutically acceptable carrier or excipient, for use in a
method for the treatment of an infectious disease.
] In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition
described herein is administered as a monotherapy.
] In certain embodiments, these methods further se administering an
additional therapeutic agent to the subject. In certain embodiments, the additional therapeutic
agent is a herapeutic 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 nist anti-TIM-3 antibody, an antagonist anti-LAG-3 antibody,
an nist anti-CEACAM1 antibody, an agonist anti-GITR antibody, an agonist anti-OX40
antibody, an agonist anti-CD137 antibody, an t anti-DR3 antibody, an agonist anti-
TNFSF14 antibody, and an agonist D27 antibody. In certain embodiments, the
checkpoint targeting agent is an antagonist anti-PD-1 antibody. In certain embodiments, the
checkpoint targeting agent is an antagonist D-L1 antibody. In certain embodiments, the
oint targeting agent is an antagonist anti-LAG-3 dy. In certain embodiments, the
additional therapeutic agent is an agonist to a tumor necrosis factor receptor superfamily
member or a tumor necrosis factor superfamily member.
In certain ments, the present invention relates to (a) an anti-CTLA-4
antibody of the ion and/or a pharmaceutical ition of the invention comprising an
anti-CTLA-4 antibody of the ion and a pharmaceutically acceptable carrier or excipient
and (b) an additional therapeutic agent, for use as a medicament. In a preferred ment,
the additional therapeutic agent is a chemotherapeutic or a checkpoint targeting agent.
In certain embodiments, the present invention relates to (a) an anti-CTLA-4
antibody of the invention and/or a pharmaceutical composition of the invention comprising an
anti-CTLA-4 antibody of the invention and a pharmaceutically acceptable carrier or excipient
and (b) an additional therapeutic agent, for use in a method for the treatment of .
In certain embodiments, the present invention relates to (a) an TLA-4
antibody of the ion and/or a ceutical composition of the invention comprising an
anti-CTLA-4 dy of the invention and a pharmaceutically acceptable carrier or excipient
and (b) an additional therapeutic agent, for use in a method for the treatment of an infectious
disease.
In certain embodiments, an anti-CTLA-4 dy described 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). In
certain embodiments, such compound is selected from the group consisting of epacadostat
(Incyte Corp; see, e.g.,
entirety), F001287 (Flexus Biosciences), indoximod (NewLink Genetics), and NLG919
(NewLink Genetics). In one embodiment, the compound is epacadostat. In another
ment, the compound is F001287. In another embodiment, the compound is indoximod.
In another embodiment, the compound is NLG919.
] In certain embodiments, the present invention relates to (a) an anti-CTLA-4
antibody of the invention and/or a pharmaceutical composition of the invention comprising an
anti-CTLA-4 antibody of the invention and a pharmaceutically acceptable carrier or excipient
and (b) a compound that targets an immunomodulatory , for use as a medicament. In
a preferred embodiment, the compound targets IDO and/or TDO.
In certain embodiments, the present invention relates to (a) an anti-CTLA-4
antibody of the invention and/or a pharmaceutical composition of the invention comprising an
anti-CTLA-4 dy of the invention and a pharmaceutically acceptable carrier or excipient
and (b) a compound that targets an modulatory enzyme, for use in a method for the
treatment of cancer. In a preferred embodiment, the nd targets IDO and/or TDO.
In certain embodiments, an anti-CTLA-4 antibody described herein is administered
to a subject in combination with a vaccine. In certain embodiments, the vaccine is a heat shock
protein based tumor e or a heat shock protein based pathogen vaccine. In a specific
embodiment, an anti-CTLA-4 antibody described herein is stered 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 s. 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 ation. Five families have
been classified according to lar 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 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 antitumor
CD8+ and CD4+ T cells. The immunity elicited by HSP complexes d from tumor
preparations is specifically directed against the unique antigenic peptide repertoire expressed
by the cancer of each subject.
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 ve immune responses. In a specific embodiment, the nic
peptide(s) ys antigenicity for the cancer being treated. HSPPCs are efficiently seized by
APCs via membrane receptors (mainly CD91) or by g to ike receptors. HSPPC
alization 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
mediated immune responses. In certain embodiments, HSPPCs used in s described
herein comprise one or more heat shock proteins from the hsp60, hsp70, or hsp90 family of
stress ns complexed with antigenic peptides. In certain embodiments, HSPPCs comprise
hsc70, hsp70, hsp90, hsp110, grp170, gp96, calreticulin, or combinations of two or more
thereof.
In a specific embodiment, an anti-CTLA-4 antibody described herein is
administered to a subject in combination with a heat shock protein peptide x (HSPPC),
e.g., heat shock protein e 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 t’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.
In certain embodiments, the HSPPC, e.g., HSPPC-96, is produced from the tumor
tissue of a subject. In a specific ment, 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., 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 al resection, non-necrotic tumor tissue is frozen
prior to use in vaccine ation. In some embodiments, the HSPPC, e.g., HSPPC-96, is
ed from the tumor tissue by cation 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 stered
weekly for the first 4 doses and then biweekly for the 2-8 additional doses.
Further es of HSPPCs that may be used in accordance with the methods
described herein are disclosed in the following patents and patent applications, which are
incorporated herein by reference herein in their entireties, U.S. Patent Nos. 6,391,306,
6,383,492, 095, 6,410,026, 6,436,404, 6,447,780, 6,447,781 and 6,610,659, all of which
are herein incorporated by reference in their entireties.
In certain embodiments, the present ion relates to (a) an anti-CTLA-4
antibody of the invention and/or a pharmaceutical composition of the invention comprising an
anti-CTLA-4 antibody of the invention and a ceutically acceptable r or excipient
and (b) a vaccine, for use as a medicament. In a preferred embodiment, the vaccine is a heat
shock protein based tumor vaccine or a heat shock protein based pathogen vaccine. In a
preferred ment, the vaccine is a heat shock protein based viral vaccine.
In certain embodiments, the present invention relates to (a) an anti-CTLA-4
antibody of the invention and/or a pharmaceutical composition of the ion comprising an
anti-CTLA-4 dy of the invention and a pharmaceutically acceptable carrier or excipient
and (b) a vaccine, for use in a method for the treatment of cancer. In a preferred embodiment,
the e is a heat shock protein based tumor vaccine.
The anti-CTLA-4 antibody and the additional therapeutic agent (e.g.,
chemotherapeutic, checkpoint targeting agent, IDO inhibitor, and/or vaccine) can be
administered separately, sequentially or concurrently as separate dosage forms. In one
embodiment, an anti-CTLA-4 antibody is administered parenterally, and an IDO inhibitor is
administered orally.
In n embodiments, an anti-CTLA-4 antibody described herein is stered
to a subject intratumorally. In certain embodiments, an TLA-4 antibody described herein
is administered to a subject umorally in combination with an additional therapeutic agent.
In certain embodiments, the additional therapeutic agent is administered systemically. In
certain embodiments, the subject has solid tumors. In certain ments, the subject has
head and neck squamous cell carcinoma (HNSCC). In certain embodiments, the subject has
HER2+ breast . In certain embodiments, the additional therapeutic agent that is
administered systemically is an D-1 antibody (e.g., pembrolizumab or nivolumab). In
certain embodiments, the additional therapeutic agent that is administered systemically is an
anti-EGFR antibody (e.g., cetuximab). In certain embodiments, the additional therapeutic
agent that is administered systemically is an anti-HER2 antibody (e.g., trastuzumab). In certain
embodiments, the onal therapeutic agent that is stered systemically is a
chemotherapeutic agent (e.g., gemcitabine). In certain embodiments, the t has solid
tumors and the additional therapeutic agent that is administered systemically is an anti-PD-1
dy (e.g., pembrolizumab or nivolumab). In n embodiments, the anti-PD-1 antibody
is pembrolizumab administered at 200 mg every three weeks. In certain embodiments, the
subject has head and neck squamous cell carcinoma (HNSCC) and the additional therapeutic
agent that is administered systemically is an anti-EGFR antibody (e.g., cetuximab). In certain
embodiments, the subject has HER2+ breast cancer and the additional therapeutic agent that is
administered systemically is an anti-HER2 antibody (e.g., zumab). In certain
embodiments, the subject further received a chemotherapeutic agent (e.g., gemcitabine). In one
, the present invention relates to an anti-CTLA-4 antibody and/or pharmaceutical
composition of the present invention, and optionally an additional therapeutic agent, for use in
a method for the treatment of cancer, wherein the anti-CTLA-4 antibody and/or pharmaceutical
composition of the present invention is administered intratumorally to the subject. In one
preferred embodiment, an additional eutic agent is administered to the subject, more
preferably, an additional therapeutic agent is administered systemically to the subject.
] In certain ments, an anti-PD-1 antibody is used in methods described 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, ped 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, ped 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 ped by Regeneron Pharmaceuticals. In certain
ments, the anti-PD-1 antibody is 01591 developed by Pfizer. In certain
embodiments, the anti-PD-1 antibody is 17 developed by BeiGene. In n
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.
Further non-limiting examples of D-1 antibodies that may be used in
treatment methods described herein are disclosed in the following patents and patent
applications, which are incorporated herein by reference in their entireties for all purposes: U.S.
Patent No. 6,808,710; U.S. Patent No. 7,332,582; U.S. Patent No. 7,488,802; U.S. Patent No.
8,008,449; U.S. Patent No. 8,114,845; U.S. Patent No. 8,168,757; U.S. Patent No. 8,354,509;
U.S. Patent No. 8,686,119; U.S. Patent No. 553; U.S. Patent No. 8,747,847; U.S. Patent
No. 8,779,105; U.S. Patent No. 8,927,697; U.S. Patent No. 8,993,731; U.S. Patent No.
9,102,727; U.S. Patent No. 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 33091 A1; PCT Publication No.
Publication No. WO 79664 A2; PCT Publication No.
Publication No.
Publication No.
Publication No.
In certain embodiments, an anti-PD-L1 antibody is used in s described
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 ments, the anti-PD-L1 antibody is
avelumab, also known as MSB0010718C, developed by Merck Serono and Pfizer. In certain
embodiments, the D-L1 dy is MDX-1105 developed by Bristol-Myers Squibb. In
n ments, the anti-PD-L1 antibody is AMP-224 developed by Amplimmune and
miting examples of anti-PD-L1 antibodies that may be used in ent
methods described herein are sed in the following patents and patent applications, which
are incorporated herein by reference in their entireties for all purposes: US Patent No.
7,943,743; US Patent No. 8,168,179; US Patent No. 8,217,149; U.S. Patent No. 8,552,154;
U.S. Patent No. 8,779,108; U.S. Patent No. 8,981,063; U.S. Patent No. 9,175,082; U.S.
Publication No. US 2010/0203056 A1; U.S. Publication No. US 2003/0232323 A1; U.S.
Publication No. US 323249 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.
Publication No.
Publication No. WO 61668 A1; PCT Publication No.
Publication No.
Publication No.
In certain embodiments, an anti-LAG-3 antibody is used in methods described
herein. In certain embodiments, the AG-3 antibody is BMS-986016 developed by
Bristol-Myers Squibb. In certain embodiments, the anti-LAG-3 antibody is LAG525
developed by Novartis. In certain embodiments, the anti-LAG-3 antibody is GSK2831781
developed by GSK.
] Non-limiting examples of anti-LAG-3 antibodies that may be used in treatment
methods bed herein are disclosed in the following s and patent ations, which
are incorporated herein by reference in their entireties for all purposes: US Patent No.
9,244,059; U.S. Publication No. US 2011/0150892 A1; U.S. Publication No. US 2014/0093511
A1; U.S. Publication No. US 2014/0286935 A1; U.S. Publication No. US 2015/0259420 A1;
and PCT Publication No.
PCT Publication No.
In certain embodiments, an anti-EGFR antibody is used in s described
herein. In certain embodiments, the anti-EGFR antibody is cetuximab developed by Bristol-
Myers Squibb and ImClone, panitumumab developed by x and Amgen, nimotuzumab
developed by CMI Cuba and YM BioSciences, necitumumab ped by e,
zalutumumab ped by Genmab, matuzumab developed by Takeda, Sym004 developed
by Merck Serono and Symphogen, imgatuzumab developed by t and Roche,
duligotumab developed by Genentech and Roche, depatuxizumab developed by Abbott,
depatuxizumab mafodotin developed by Abbvie, MM-151 developed by Adimab and
Merrimack, GC1118 developed by Green Cross, AMG 595 developed by Amgen and
ImmunoGen, CetuGEX developed by Glycotope, uximab emtansine developed by
ImmunoGen, JNJ-61186372 developed by Genmab and Janssen Biotech, SCT200 developed
by Sinocelltech, LY3164530 developed by Lilly, HLX07 developed by Shanghai Henlius, or
SYN004 developed by Synermore.
In certain embodiments, an anti-HER2 antibody is used in s described
herein. In n embodiments, the anti-HER2 antibody is trastuzumab developed by
Genentech and Roche, trastuzumab ine ped by Genentech and Roche,
pertuzumab developed by Genentech, ertumaxomab developed by Fresenius, margetuximab
developed by MacroGenics, MM-111 developed by ack, CT-P06 developed by
Celltrion, PF-05280014 developed by Pfizer, MM-302 developed by Merrimack, SB3
developed by Merck & Co, CMAB302 developed by Shanghai CP Guojian, TrasGEX
developed by Glycotope, ARX788 developed by Ambrx and Zhejiang Medicine, SYD985
developed by Synthon, FS102 developed by Bristol-Myers Squibb and f-star, BCD-022
developed by Biocad, ABP 980 developed by Amgen, DS-8201a developed by Daiichi Sankyo,
HLX02 developed by Shanghai Henlius, or CANMAb developed by Biocon and Mylan.
An antibody or ceutical 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 and aneous routes. Pulmonary administration
can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an
lizing 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 ceutical ition bed herein is delivered
intratumorally. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical
composition described herein is delivered to a tumor draining lymph node. In certain
embodiments, the antibody or pharmaceutical composition described herein is delivered via a
localized administration (e.g., subcutaneous administration). In certain embodiments, the anti-
CTLA-4 antibody or pharmaceutical ition described herein is delivered systemically.
In certain embodiments, the TLA-4 antibody or pharmaceutical composition described
herein is delivered locally.
[00229] In one aspect, the present invention relates to an anti-CTLA-4 antibody and/or
pharmaceutical composition of the present invention, and optionally an additional therapeutic
agent, for use in a method for the treatment of cancer, wherein the anti-CTLA-4 antibody and/or
pharmaceutical composition of the present invention is delivered intratumorally to the subject,
is delivered to a tumor ng lymph node of a subject, or is delivered via a localized
administration (e.g., aneous administration) to a t.
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 e, and can be ined
by standard clinical techniques.
The precise dose to be employed in a composition will also depend on the route of
administration, and the seriousness of the infection or e 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 ), whether the patient
is human or an animal, other medications administered, or r treatment is prophylactic
or therapeutic. Usually, the patient is a human but non-human mammals ing transgenic
mammals can also be treated. Treatment dosages are optimally titrated to optimize safety and
efficacy.
In certain embodiments, an anti-CTLA-4 antibody or pharmaceutical composition
described herein is administered to a subject (e.g., via intravenous injection) at 0.01 mg/kg,
0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, 10 mg/kg, about 0.1 mg/kg,
about 0.3 mg/kg, about 1 mg/kg, about 3 mg/kg, about 6 mg/kg, or about 10 mg/kg. In certain
embodiments, an anti-CTLA-4 antibody or pharmaceutical composition described herein is
administered to a subject (e.g., via intravenous injection) every week, every two weeks, every
three weeks, every four weeks, every six weeks, every eight weeks, every twelve weeks, every
month, every two months, every three months, every four months, every five months, every six
months, every eight months, and every year, e.g., at the doses described above. In certain
embodiments, an anti-CTLA-4 dy or pharmaceutical composition bed herein is
administered to a subject (e.g., via intravenous injection) every three weeks at the doses
described above.
In one aspect, the present invention s to an anti-CTLA-4 antibody and/or
pharmaceutical composition of the present invention, and ally an additional therapeutic
agent, for use in a method for the treatment of cancer, wherein the TLA-4 antibody and/or
pharmaceutical composition of the t invention is administered to a t at 0.01 mg/kg,
0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, 10 mg/kg, about 0.1 mg/kg,
about 0.3 mg/kg, about 1 mg/kg, about 3 mg/kg, about 6 mg/kg, or about 10 mg/kg, more
preferably every two weeks, every three weeks, every four weeks, every six weeks, or every
twelve weeks.
In certain embodiments, an anti-CTLA-4 antibody or pharmaceutical composition
described herein is administered to a t (e.g., via intravenous injection) at 0.1 mg/kg every
two weeks, every three weeks, every four weeks, every six weeks, or every twelve weeks. In
certain ments, an anti-CTLA-4 antibody or pharmaceutical composition described
herein is stered to a subject (e.g., via intravenous injection) at 0.3 mg/kg every two
weeks, every three weeks, every four weeks, every six weeks, or every twelve weeks. In certain
embodiments, an anti-CTLA-4 antibody or pharmaceutical composition bed herein is
administered to a subject (e.g., via intravenous injection) at 1 mg/kg every two weeks, every
three weeks, every four weeks, every six weeks, or every twelve weeks. In n
embodiments, an anti-CTLA-4 dy or pharmaceutical composition described herein is
administered to a subject (e.g., via intravenous injection) at 3 mg/kg every two weeks, every
three weeks, every four weeks, every six weeks, or every twelve weeks. In certain
embodiments, an anti-CTLA-4 antibody or pharmaceutical composition described herein is
administered to a subject (e.g., via intravenous injection) at 6 mg/kg every two weeks, every
three weeks, every four weeks, every six weeks, or every twelve weeks. In certain
embodiments, an anti-CTLA-4 antibody or pharmaceutical composition described herein is
administered to a subject (e.g., via intravenous injection) at 10 mg/kg every two weeks, every
three weeks, every four weeks, every six weeks, or every twelve weeks.
In certain embodiments, an anti-CTLA-4 antibody or pharmaceutical composition
described herein is administered to a subject (e.g., via intravenous injection) at 0.1 mg/kg or
about 0.1 mg/kg every three weeks. In certain ments, an anti-CTLA-4 antibody or
pharmaceutical composition described herein is administered to a subject (e.g., via intravenous
injection) at 0.3 mg/kg or about 0.3 mg/kg every three weeks. In certain embodiments, an anti-
CTLA-4 antibody or pharmaceutical composition bed herein is administered to a subject
(e.g., via intravenous injection) at 1 mg/kg or about 1 mg/kg every three weeks. In certain
embodiments, an anti-CTLA-4 antibody or pharmaceutical composition described herein is
administered to a subject (e.g., via intravenous injection) at 3 mg/kg or about 3 mg/kg every
three weeks. In certain embodiments, an TLA-4 antibody or pharmaceutical composition
described herein is administered to a subject (e.g., via intravenous injection) at 6 mg/kg or
about 6 mg/kg every three weeks. In certain embodiments, an anti-CTLA-4 antibody or
pharmaceutical composition described herein is administered to a subject (e.g., via intravenous
injection) at 10 mg/kg or about 10 mg/kg every three weeks.
In n embodiments, an anti-CTLA-4 antibody or pharmaceutical ition
described herein is administered to a subject via umoral injection at 0.01 mg/kg, 0.03
mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, about 0.01 mg/kg, about 0.03 mg/kg, about
0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, or about 3 mg/kg. In certain embodiments, an
anti-CTLA-4 antibody or pharmaceutical ition bed herein is administered to a
subject via intratumoral injection every week, every two weeks, every three weeks, every four
weeks, every six weeks, every eight weeks, every twelve weeks, every month, every two
months, every three months, every four months, every five , every six months, every
eight months, and every year, e.g., at the doses described above. In certain embodiments, an
anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a
subject via umoral injection every three weeks at the doses described above.
In certain embodiments, an anti-CTLA-4 antibody or pharmaceutical composition
described herein is administered to a subject via intratumoral injection at 0.01 mg/kg or about
0.01 mg/kg every three weeks. In certain ments, an TLA-4 antibody or
pharmaceutical composition described herein is administered to a subject via intratumoral
injection at 0.03 mg/kg or about 0.03 mg/kg every three weeks. In certain embodiments, an
anti-CTLA-4 antibody or pharmaceutical ition described herein is administered to a
subject via intratumoral injection at 0.1 mg/kg or about 0.1 mg/kg every three weeks. In certain
embodiments, an anti-CTLA-4 antibody or pharmaceutical composition described herein is
administered to a subject via intratumoral ion at 0.3 mg/kg or about 0.3 mg/kg every three
weeks. In certain embodiments, an anti-CTLA-4 antibody or pharmaceutical composition
described herein is administered to a subject via intratumoral ion at 1 mg/kg or about 1
mg/kg every three weeks. In certain embodiments, an anti-CTLA-4 antibody or
pharmaceutical composition described herein is administered to a subject via intratumoral
injection at 3 mg/kg or about 3 mg/kg every three weeks.
In certain embodiments, an anti-CTLA-4 antibody or pharmaceutical composition
described herein is administered to a subject via a localized administration (e.g., aneous
administration) at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, about
0.01 mg/kg, about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, or about 3
mg/kg. In certain embodiments, an TLA-4 antibody or pharmaceutical composition
described herein is administered to a subject via a localized administration (e.g., subcutaneous
administration) every week, every two weeks, every three weeks, every four weeks, every six
weeks, every eight weeks, every twelve weeks, every month, every two months, every three
months, every four months, every five months, every six months, every eight months, and every
year, e.g., at the doses bed above.
In certain embodiments, the therapeutic combination is administered to a subject
for at least 3, 6, 9, 12, 18, or 24 months. In certain embodiments, the therapeutic combination
is administered to a subject for up to 3, 6, 9, 12, 18, or 24 months.
In certain ments, the instant disclosure provides a method of treating a
subject having angiosarcoma, the method comprising stering to the subject (e.g.,
intravenously, intratumorally, or subcutaneously) an effective amount of an anti-CTLA-4
antibody or pharmaceutical composition described herein. In certain embodiments, the t
disclosure provides a method of treating a subject having angiosarcoma, the method comprising
administering to the subject intravenously an antibody that specifically binds to human CTLA-
4 at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, about 0.01 mg/kg, about
0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, or about 3 mg/kg, optionally
every one, two or three weeks. In certain embodiments, the instant disclosure provides a
method of treating a subject having angiosarcoma, the method comprising administering to the
subject intravenously an dy that ically binds to human CTLA-4 at 0.1 mg/kg once
every three weeks. In certain embodiments, the antibody comprises a heavy chain le
region comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 9. In n embodiments, the antibody
comprises a heavy chain sing the amino acid sequence of SEQ ID NO: 23; and a light
chain comprising the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the
antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24; and
a light chain comprising the amino acid sequence of SEQ ID NO: 27. In certain embodiments,
the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25;
and a light chain comprising the amino acid sequence of SEQ ID NO: 27. In certain
embodiments, the dy ses a heavy chain comprising the amino acid sequence of
SEQ ID NO: 26; and a light chain comprising the amino acid sequence of SEQ ID NO: 27.
An anti-CTLA-4 antibody described herein can also be used to assay CTLA-4
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 (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium ),
and technetium (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 or an
antigen-binding fragment thereof described herein. Alternatively, a second antibody that
recognizes an anti-CTLA-4 antibody or antigen-binding fragment thereof described herein can
be labeled and used in combination with an anti-CTLA-4 antibody or antigen-binding fragment
f to detect CTLA-4 n levels. In one embodiment, the present invention relates to
the use of an anti-CTLA-4 antibody of the invention, for assaying and/or detecting CTLA-4
protein levels in a biological sample in vitro.
Assaying for the expression level of CTLA-4 protein is intended to include
qualitatively or quantitatively measuring or estimating the level of a CTLA-4 n in a
first biological sample either directly (e.g., by determining or estimating absolute n
level) or relatively (e.g., by comparing to the disease associated protein level in a second
ical sample). CTLA-4 polypeptide expression level in the first biological sample can
be measured or ted and compared to a standard CTLA-4 protein level, the standard
being taken from a second ical 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 ard” CTLA-4 polypeptide level
is known, it can be used repeatedly as a standard for comparison.
] As used herein, the term “biological sample” refers to any biological sample
obtained from a t, cell line, tissue, or other source of cells potentially expressing
CTLA-4. Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans)
are well known in the art. Biological samples include peripheral clear blood cells.
An anti-CTLA-4 antibody or antigen-binding fragment thereof 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 d artisan and based on the present
description. Prognostic, diagnostic, monitoring and screening assays and kits for in vitro
assessment and tion 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 -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 or an antibody or antigen-binding fragment thereof, including
combinations thereof, versus a different agent or antibody or antigen-binding fragment thereof.
This type of prognostic and diagnostic monitoring and assessment is already in practice
ing dies against the HER2 protein in breast cancer pTestTM, Dako) where the
assay is also used to te patients for antibody therapy using Herceptin®. In vivo
applications e directed cell therapy and immune system tion and radio imaging
of immune responses.
[00245] In one aspect, the present invention relates to an anti-CTLA-4 antibody and/or
pharmaceutical composition of the present invention for use as a diagnostic.
In one , the present invention relates to an anti-CTLA-4 antibody and/or
pharmaceutical composition of the present ion for use in a method for the prediction,
diagnosis and/or monitoring of an immune system-dysfunction and/or .
[00247] In one embodiment, the present invention relates to the use of an anti-CTLA-4
antibody of the invention, for predicting, diagnosing and/or monitoring an immune systemdysfunction
and/or cancer in a subject by ng and/or detecting CTLA-4 protein levels in
a ical sample of the subject of in vitro.
In one embodiment, an anti-CTLA-4 antibody or antigen-binding fragment thereof
can be used in immunohistochemistry of biopsy samples. In another embodiment, an anti-
CTLA-4 antibody or antigen-binding fragment thereof can be used to detect levels of CTLA-
4, or levels of cells which contain CTLA-4 on their membrane surface, which levels can then
be linked to certain disease symptoms. Anti-CTLA-4 antibodies or antigen-binding fragments
thereof described herein may carry a detectable or functional label. When fluorescence labels
are used, currently available microscopy and fluorescence-activated cell sorter analysis (FACS)
or combination of both methods ures known in the art may be utilized to identify and to
quantitate the specific binding s. Anti-CTLA-4 antibodies or antigen-binding
fragments thereof described herein may carry 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-CTLA-4 antibody or
antigen-binding nt thereof may carry a radioactive label, such as the es 3H, 14C,
32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 67Cu, 90Y, 99Tc, 111In, 117Lu, 121I, 124I, 125I, 131I, 198Au, 211At,
213Bi, 225Ac and 186Re. When radioactive labels are used, currently available counting
ures known in the art may be utilized to identify and quantitate the specific binding of
anti-CTLA-4 antibody or antigen-binding fragment f to CTLA-4 (e.g., human CTLA-4).
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 ed by contacting a sample or
a control sample with an anti-CTLA-4 antibody or n-binding fragment thereof under
conditions that allow for the formation of a complex between the dy or antigen-binding
fragment f and CTLA-4. Any complexes formed between the antibody or antigenbinding
fragment thereof and CTLA-4 are detected and compared in the sample and the control.
In light of the specific binding of the antibodies described herein for CTLA-4, the dies
or antigen-binding fragments thereof can be used to specifically detect CTLA-4 expression on
the surface of cells. The antibodies or antigen-binding fragments thereof described herein can
also be used to purify CTLA-4 via immunoaffinity purification. Also included herein is an
assay system which may be prepared in the form of a test kit for the quantitative analysis of the
extent of the ce of, for instance, CTLA-4 or CTLA-4/CTLA-4 ligand complexes. The
system or test kit may se a labeled ent, e.g., a labeled antibody, and one or more
additional immunochemical reagents.
In one embodiment, the present invention relates to an in vitro method for assaying
and/or detecting CTLA-4 protein levels in a biological sample comprising (1) ting a
sample and optionally a control sample with an anti-CTLA-4 antibody or antigen-binding
fragment thereof of the invention under conditions that allow for the ion of a complex
between the antibody or antigen-binding fragment thereof and CTLA-4, and (2) detecting and
comparing the complexes formed in the sample and optionally the control.
6.5 Polynucleotides, Vectors and Methods of Producing Anti-CTLA-4
Antibodies
[00250] In another aspect, provided herein are cleotides 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 CTLA-4 (e.g.,
human CTLA-4) antigen, and vectors, e.g., vectors comprising such polynucleotides for
recombinant expression in host cells (e.g., E. coli and ian cells). Provided herein are
cleotides comprising nucleotide sequences encoding any of the antibodies provided
herein, as well as vectors comprising such polynucleotide sequences, e.g., sion vectors
for their efficient sion in host cells, e.g., mammalian cells.
] 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 al, or
e 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, e medium, other nucleic acid molecules, chemical
precursors and/or other als. In a specific embodiment, a nucleic acid molecule(s)
encoding an antibody described herein is ed or purified.
[00252] In particular aspects, ed herein are polynucleotides comprising tide
sequences encoding dies or antigen-binding fragments thereof, which specifically bind
to a CTLA-4 polypeptide (e.g., human CTLA-4) and comprises an amino acid sequence as
described , as well as antibodies which compete with such antibodies for binding to a
CTLA-4 polypeptide (e.g., in a dose-dependent manner), or which binds to the same epitope
as that of such antibodies.
In certain aspects, ed herein are polynucleotides sing 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).
[00254] Also provided herein are polynucleotides ng an anti-CTLA-4 antibody that
are optimized, e.g., by codon/RNA optimization, ement with heterologous signal
sequences, and elimination of mRNA instability elements. Methods to generate optimized
nucleic acids encoding an anti-CTLA-4 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 bed in, e.g., U.S. Patent 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 some embodiments, it can be desirable to alter one or more codons to
encode a vative 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-CTLA-4 antibody or fragment thereof by at least 1 fold, 2 fold, 3 fold, 4 fold, 5 fold,
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-CTLA-4 antibody encoded by cleotides that have
not been optimized.
[00255] In certain embodiments, an optimized polynucleotide sequence encoding an anti-
CTLA-4 antibody described herein or a fragment f (e.g., VL domain and/or VH domain)
can ize to an antisense (e.g., complementary) polynucleotide of an unoptimized
polynucleotide sequence encoding an anti-CTLA-4 antibody described herein or a fragment
f (e.g., VL domain and/or VH domain). In specific embodiments, an zed
nucleotide sequence encoding an anti-CTLA-4 antibody described herein or a fragment
izes under high stringency conditions to antisense polynucleotide of an unoptimized
polynucleotide sequence encoding an anti-CTLA-4 antibody described herein or a fragment
thereof. In a specific embodiment, an optimized nucleotide sequence ng an anti-CTLA-
4 antibody described herein or a fragment thereof hybridizes under high ency,
ediate or lower stringency hybridization conditions to an antisense cleotide of an
unoptimized nucleotide sequence encoding an anti-CTLA-4 antibody described herein or a
fragment thereof. Information regarding ization conditions has been described, see, e.g.,
U.S. Patent Application ation No. US 2005/0048549 (e.g., paragraphs 72-73), which is
incorporated herein by reference.
[00256] The polynucleotides can be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. Nucleotide sequences encoding
antibodies bed 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, incorporated by reference in its entirety), which, briefly, involves
the synthesis of overlapping oligonucleotides ning portions of the sequence encoding the
antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated
oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an dy bed 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 e, 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 c acids comprising the sequence encoding
the variable light chain region and/or the variable heavy chain region of an antibody. The
ied nucleic acids can be cloned into vectors for expression in host cells and for further
cloning, for example, to generate chimeric and humanized antibodies.
If a clone containing a c 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 sized 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, ed from, any tissue or cells expressing the antibody, such as hybridoma cells
ed to express an antibody described ) by PCR ication 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 ted by PCR can then be cloned into
replicable cloning vectors using any method well known in the art.
DNA encoding anti-CTLA-4 antibodies described herein can be readily isolated and
sequenced using tional procedures (e.g., by using oligonucleotide probes that are
capable of binding specifically to genes encoding the heavy and light chains of the anti-CTLA-
4 antibodies). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA
can be placed into expression s, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese r ovary (CHO) cells (e.g., CHO cells from the CHO
GS System™ (Lonza)), or myeloma cells that do not otherwise produce immunoglobulin
protein, to obtain the synthesis of anti-CTLA-4 antibodies in the recombinant host cells.
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 nt region, e.g., the human gamma 4 constant region, and the PCR amplified
VL domains can be cloned into vectors sing 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-1α promoter, a secretion signal, a cloning site for the variable
region, constant domains, and a selection marker such as in. 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 s 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.
[00261] The DNA also can be modified, for example, by substituting the coding sequence
for human heavy and light chain constant s in place of the murine sequences, or by
covalently joining to the globulin coding sequence all or part of the coding sequence
for a non-immunoglobulin polypeptide.
Also provided are polynucleotides that hybridize under high stringency,
ediate or lower stringency hybridization conditions to polynucleotides that encode an
antibody described . In specific embodiments, polynucleotides described herein
hybridize under high stringency, ediate or lower stringency hybridization conditions to
polynucleotides encoding a VH domain and/or VL domain ed herein.
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° C followed by
one or more washes in 0.2xSSC/0.1% SDS at about 50-65° C; hybridization under highly
stringent conditions can involve hybridization to filter-bound nucleic acid in 6xSSC at about
45° C followed by one or more washes in 0.1xSSC/0.2% SDS at about 68° C. Hybridization
under other stringent hybridization conditions are known to those of skill in the art and have
been described, see, for example, Ausubel FM et al., eds., (1989) t 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, incorporated by reference in its ty.
] In certain aspects, provided herein are cells (e.g., host cells) expressing (e.g.,
recombinantly) antibodies described herein (or an antigen-binding nt thereof) which
specifically bind to CTLA-4 (e.g., human ) and related polynucleotides and expression
vectors. ed herein are vectors (e.g., expression vectors) comprising polynucleotides
comprising nucleotide sequences encoding TLA-4 antibodies or a fragment for
recombinant expression in host cells, preferably in mammalian cells. Also provided herein are
host cells sing such vectors for recombinantly expressing anti-CTLA-4 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.
Recombinant expression of an dy 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 CTLA-4 (e.g., human CTLA-4) involves construction of an
sion vector containing a polynucleotide that encodes the antibody. Once a
polynucleotide encoding an antibody le, 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
ed, 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) ng 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 riptional and translational control signals. These methods
include, for example, in vitro recombinant DNA techniques, tic techniques, and in vivo
genetic recombination. Also provided are replicable vectors comprising a tide 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
ce encoding the constant region of the antibody molecule (see, e.g., International
Publication Nos. WO 86/05807 and WO 89/01036; and U.S. Patent 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.
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 bed herein or a fragment f. Thus, provided herein are host cells
containing a polynucleotide encoding an dy described herein or fragments thereof, or a
heavy or light chain f, or fragment thereof, or a single chain antibody described herein,
operably linked to a promoter for sion of such sequences in the host cell. In certain
embodiments, for the expression of double-chained dies, vectors encoding both the
heavy and light chains, dually, 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 dy 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-CTLA-4 antibody described
herein or an antigen-binding nt f. In certain embodiments, provided herein is a
population of host cells comprising such first host cell and such second host cell.
In a particular embodiment, provided herein is a population of vectors sing
a first vector comprising a polynucleotide encoding a light light chain variable region of
an TLA-4 antibody described herein, and a second vector sing a polynucleotide
encoding a heavy chain/heavy chain variable region of an anti-CTLA-4 antibody described
herein.
A y of host-expression vector systems can be ed to express antibody
molecules described herein (see, e.g., U.S. Patent No. 5,807,715). Such host-expression
systems represent vehicles by which the coding sequences of interest can be produced and
uently 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 recombinant bacteriophage DNA, plasmid DNA or cosmid DNA
sion vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia)
transformed with recombinant yeast expression vectors containing antibody coding ces;
insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus)
containing antibody coding sequences; plant cell systems (e.g., green algae such as
Chlamydomonas reinhardtii) infected with recombinant virus expression s ( e.g.,
lower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or ian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK,
HEK 293, NS0, 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
inant sion constructs containing promoters derived from the genome of
mammalian cells (e.g., metallothionein promoter) or from mammalian s (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter). In a specific ment, cells
for expressing antibodies described herein or an antigen-binding fragment thereof are CHO
cells, for e CHO cells from the CHO GS System™ (Lonza). In a particular
embodiment, cells for expressing antibodies described herein are human cells, e.g., human cell
lines. In a specific embodiment, a mammalian sion vector is pOptiVEC™ or pcDNA3.3.
In a particular embodiment, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g.,
mammalian cells), ally for the sion of whole recombinant antibody molecule, are
used for the expression of a recombinant antibody molecule. For example, mammalian cells
such as Chinese hamster ovary (CHO) cells, in conjunction with a vector such as the major
intermediate early gene promoter t from human cytomegalovirus is an effective
expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-5; and
Cockett MI et al., (1990) Biotechnology 8(7): 662-7, which are herein incorporated by
reference in their entireties). In certain embodiments, antibodies described herein are produced
by CHO cells or NS0 cells. In a specific embodiment, the expression of nucleotide sequences
encoding antibodies described herein which specifically bind CTLA-4 (e.g., human )
is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.
In bacterial systems, a number of expression vectors can be advantageously selected
ing upon the use ed for the antibody molecule being expressed. For example,
when a large quantity of such an antibody is to be produced, for the generation of
ceutical 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 & r-
Hill B (1983) EMBO J 2: 1791-1794, herein incorporated by reference in its entirety), 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 SM (1989) J Biol Chem 24: 5503-
5509, which are herein incorporated by reference in their entireties); and the like. For example,
pGEX vectors can also be used to express foreign ptides as fusion proteins with
glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be
purified from lysed cells by tion 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.
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
erda cells. The antibody coding sequence can be cloned individually into non-essential
regions (for e the polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems can be
utilized. In cases where an adenovirus is used as an expression , the antibody coding
sequence of interest can be ligated to an adenovirus transcription/translation l complex,
e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be ed
in the irus genome by in vitro or in vivo recombination. Insertion in a non-essential
region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable
and capable of expressing the antibody le in infected hosts (e.g., see Logan J & Shenk
T (1984) PNAS 81(12): 3655-9, herein incorporated by reference in its entirety). Specific
initiation signals can also be required for efficient translation of inserted antibody coding
sequences. These signals e the ATG initiation codon and nt sequences.
Furthermore, the initiation codon must be in phase with the g frame of the desired coding
sequence to ensure translation of the entire insert. These ous 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, herein incorporated by reference in its entirety).
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 d.
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
isms 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, NS0 (a murine myeloma cell line that does
not endogenously produce any immunoglobulin chains), O, COS (e.g., COS1 or
COS), PER.C6, VERO, st, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1,
BSC40, YB/20, BMT10 and HsS78Bst cells. In certain embodiments, anti-CTLA-4 antibodies
described herein are produced in mammalian cells, such as CHO cells.
In a specific embodiment, the antibodies described herein or antigen-binding
fragments thereof 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 α1,6-fucosyltransferase can be used to produce
antibodies or antigen-binding fragments thereof with reduced fucose content. The Potelligent®
system (Lonza) is an example of such a system that can be used to produce antibodies or
antigen-binding fragments thereof with reduced fucose content.
For erm, high-yield production of recombinant proteins, stable expression
cells can be generated. For example, cell lines which stably express an anti-CTLA-4 antibody
described herein or an antigen-binding fragment thereof can be engineered. In specific
ments, a cell provided herein stably expresses a light chain/light chain variable region
and a heavy heavy chain variable region which associate to form an antibody described
herein or an antigen-binding fragment thereof.
In certain aspects, rather than using expression vectors which contain viral s
of replication, host cells can be transformed with DNA controlled by riate 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 ed
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 ate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned and ed into cell
lines. This method can advantageously be used to engineer cell lines which s an anti-
CTLA-4 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.
A number of selection systems can be used, including but not limited to the herpes
simplex virus thymidine kinase r M et al., (1977) Cell 11(1): 223-32),
hypoxanthineguanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS
48(12): 2026-2034, herein incorporated by nce in its entirety) and adenine
phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23, herein incorporated by
reference in its entirety) genes in tk-, hgprt- or aprt-cells, respectively. 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 RC
& Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance to the aminoglycoside
G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev
Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA
& Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ & Felgner PL (1993) Trends
hnol 11(5): 211-5); and hygro, which confers resistance to hygromycin (Santerre RF et
al., (1984) Gene 30(1-3): ), 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 d recombinant clone and such methods are described, for
e, in Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, NY (1993); Kriegler M, Gene er and Expression, A Laboratory Manual, Stockton
Press, NY (1990); and in Chapters 12 and 13, Dracopoli NC et al., (eds.), Current Protocols in
Human cs, John Wiley & Sons, NY (1994); Colbère-Garapin F et al., (1981) J Mol Biol
150: 1-14, which are incorporated by nce herein in their entireties.
The expression levels of an antibody molecule can be sed by vector
amplification (for a review, see Bebbington CR & Hentschel CCG, 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), herein orated by reference in its entirety).
When a marker in the vector system expressing antibody is amplifiable, se 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 GF et al., (1983) Mol Cell Biol 3: 257-66, herein incorporated by
reference in its entirety).
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
ng 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 sion vector: 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.
Alternatively, a single vector can be used which s, 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 NJ
(1986) Nature 322: 562-565; and Köhler G (1980) PNAS 77: 2197-2199, which are herein
incorporated by reference in their ties). The coding sequences for the heavy and light
chains can comprise cDNA or genomic DNA. The expression vector can be stronic or
multicistronic. A multicistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more, 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.
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 e, 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 ns. r, the antibodies described herein can be fused to
heterologous polypeptide sequences described herein or otherwise known in the art to tate
purification.
In specific embodiments, an antibody or an antigen-binding fragment thereof
described herein is isolated or purified. Generally, an isolated antibody is one that is
substantially free of other dies with different antigenic specificities than the isolated
dy. For example, in a particular embodiment, a preparation of an antibody bed
herein is substantially free of cellular material and/or chemical precursors. The language
“substantially free of ar 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 ar 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 minating
protein”) and/or variants of an antibody, for example, different post-translational modified
forms of an antibody or other different versions of an dy (e.g., antibody nts).
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 dy is produced by chemical synthesis,
it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated
from chemical sors or other chemicals which are ed in the sis 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.
Antibodies or fragments thereof that specifically bind to CTLA-4 (e.g., human
CTLA-4) can be produced by any method known in the art for the synthesis of antibodies, for
e, by chemical synthesis or by recombinant expression techniques. The methods
described herein employs, unless otherwise indicated, conventional techniques in molecular
biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry,
PCR, oligonucleotide sis and cation, 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 , NY; Ausubel FM et al., Current
Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current
ols 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 ch, IRL Press; Birren B et al., (eds.)
(1999) Genome is: A Laboratory Manual, Cold Spring Harbor Laboratory Press, all of
which are herein incorporated by reference in their entireties.
In a specific embodiment, an antibody described herein is an antibody (e.g.,
recombinant antibody) prepared, expressed, created or isolated by any means that es
creation, e.g., via synthesis, genetic engineering of DNA ces. In certain embodiments,
such antibody comprises sequences (e.g., DNA ces or amino acid sequences) that do not
naturally exist within the antibody germline repertoire of an animal or mammal (e.g., human)
in vivo.
In one aspect, ed herein is a method of making an antibody or an antigenbinding
fragment thereof which specifically binds to CTLA-4 (e.g., human CTLA-4)
comprising culturing a cell or host cell described herein. In a certain aspect, provided herein
is a method of making an antibody or an antigen-binding fragment f which specifically
binds to CTLA-4 (e.g., human CTLA-4) comprising expressing (e.g., recombinantly
expressing) the antibody or antigen-binding fragment thereof 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 r comprises the step of purifying the antibody or antigen-binding nt
thereof obtained from the cell or host cell. Preferably, the method is performed in vitro.
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 FM et al., eds.,
John Wiley and Sons, New York, herein incorporated by reference in its ty).
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 GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier,
N.Y., 1981), which are herein incorporated by reference in their entireties. The term
lonal antibody” as used herein is not d to antibodies produced h hybridoma
technology. For example, monoclonal antibodies can be produced recombinantly from host
cells exogenously sing an antibody described herein or a fragment thereof, for example,
light chain and/or heavy chain of such dy.
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 CTLA-4 (e.g., human CTLA-4) as determined, e.g.,
by ELISA or other antigen-binding or competitive g assay known in the art or in the
examples provided herein. In particular ments, a monoclonal antibody can be a
chimeric antibody or a humanized antibody. In n 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 dy).
Monoclonal dies described herein can, for example, be made by the hybridoma method
as described in Kohler G & Milstein C (1975) Nature 256: 495, herein incorporated by
reference in its ty, or can, e.g., be isolated from phage libraries using the ques as
bed 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 FM et al., supra).
Methods for producing and screening for specific dies using oma
technology are e 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., CTLA-4 (e.g., human CTLA-4)) used
for immunization. Alternatively, lymphocytes may be immunized in vitro. cytes then
are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to
form a hybridoma cell (Goding JW (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 KE et al., (1997) Hybridoma 16:381-9, herein orated by reference in
its entirety).
[00289] In some embodiments, mice (or other s, such as rats, monkeys, donkeys, pigs,
sheep, r, or dogs) can be immunized with an antigen (e.g., CTLA-4 (e.g., human CTLA-
4)) and once an immune response is detected, e.g., antibodies specific for the n are
detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The
splenocytes are then fused by nown ques to any suitable myeloma cells, for
example cells from cell line SP20 available from the American Type Culture Collection
(ATCC®) (Manassas, VA), to form hybridomas. Hybridomas are selected and cloned by
limited on. In certain embodiments, lymph nodes of the immunized mice are harvested
and fused with NS0 myeloma cells.
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, al 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 e hypoxanthine, aminopterin, and thymidine
(HAT medium), which substances prevent the growth of deficient cells.
[00291] Specific ments employ myeloma cells that fuse efficiently, support stable
evel 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 NS0 cell line or those derived from MOPC-21 and MPC-11 mouse tumors
available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or
X63-Ag8.653 cells available from the American Type Culture Collection, lle, MD,
USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described
for the production of human onal antibodies (Kozbor D (1984) J Immunol 133: 3001-
; Brodeur et al., Monoclonal Antibody Production ques and Applications, pp. 51-63
(Marcel Dekker, Inc., New York, 1987), which are herein incorporated by reference in their
entireties).
e medium in which hybridoma cells are growing is assayed for production of
monoclonal antibodies ed against CTLA-4 (e.g., human CTLA-4). The binding
specificity of monoclonal antibodies ed 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 absorbent assay (ELISA).
After oma 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 JW (Ed), Monoclonal Antibodies:
Principles and Practice, supra). le culture media for this purpose include, for example,
D-MEM or RPMI 1640 . In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an .
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.
Antibodies described herein e antibody fragments which recognize specific
CTLA-4 (e.g., human CTLA-4) and can be generated by any technique known to those of skill
in the art. For example, Fab and F(ab’)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’)2 fragments). A Fab nt 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’)2 nt contains the two
antigen-binding arms of an antibody molecule linked by disulfide bonds in the hinge region.
[00296] Further, the antibodies described herein or antigen-binding fragments thereof 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 ng 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 id . The vector is
electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these
s 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 ed or identified with
antigen, e.g., using labeled antigen or antigen bound or ed 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
RS et al., (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al., (1994) Eur J
Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; Burton DR & Barbas CF (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 93/1 1236,
WO 95/15982, WO 01, and WO 97/13844; and U.S. Patent 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,
,780,225, 727, 5,733,743 and 5,969,108, all of which are herein incorporated by
reference in their entireties.
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 nt, 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 nts such as Fab, Fab’ and F(ab’)2
fragments can also be employed using methods known in the art such as those disclosed in PCT
publication No. WO 92/22324; Mullinax RL et al., (1992) BioTechniques 12(6): 864-9; Sawai
H et al., (1995) Am J Reprod l 34: 26-34; and Better M et al., (1988) e 240:
1041-1043, all of which are herein incorporated by reference in their entireties.
In certain embodiments, to generate whole antibodies, PCR primers including VH
or VL nucleotide ces, a restriction site, and a flanking sequence to protect the restriction
site can be used to amplify the VH or VL sequences from a te, e.g., scFv clones.
Utilizing cloning ques 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.
A chimeric antibody is a molecule in which ent portions of the antibody are
derived from different immunoglobulin molecules. For example, a chimeric antibody can
n a variable region of a mouse or rat monoclonal antibody fused to a constant region of
a human antibody. Methods for producing chimeric dies are known in the art. See, e.g.,
Morrison SL (1985) Science 229: 1202-7; Oi VT & Morrison SL (1986) BioTechniques 4:
214-221; Gillies SD et al., (1989) J Immunol Methods 125: 191-202; and U.S. Patent 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.
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 ular embodiments, a humanized
dy also ses at least a portion of an immunoglobulin constant region (Fc), typically
that of a human immunoglobulin. The dy 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 IgG1,
IgG2, IgG3 and IgG4. zed antibodies can be produced using a variety of ques
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. Patent Nos. 5,225,539, 101, and
,585,089), veneering or resurfacing (European Patent Nos. EP 592106 and EP 519596; Padlan
EA (1991) Mol Immunol 28(4/5): 489-498; Studnicka GM et al., (1994) Prot Engineering 7(6):
805-814; and Roguska MA et al., (1994) PNAS 91: 3), chain shuffling (U.S. Patent No.
,565,332), and techniques disclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886,
ational Publication No. WO 93/17105; Tan P et al., (2002) J Immunol 169: 5;
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 MA et al., (1996) Protein
Eng 9(10): 895 904; Couto JR et al., (1995) Cancer Res. 55 (23 Supp): 5973s-5977s; Couto JR
et al., (1995) Cancer Res 55(8): 1717-22; Sandhu JS (1994) Gene 150(2): 409-10 and Pedersen
JT et al., (1994) J Mol Biol 235(3): 959-73, all of which are herein incorporated by nce
in their entireties. See also U.S. Application Publication No. US 2005/0042664 A1 (Feb. 24,
2005), which is incorporated by reference herein in its entirety.
Methods for making multispecific (e.g., bispecific antibodies) have been described,
see, for example, U.S. Patent Nos. 7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830;
,869,620; 6,132,992 and 8,586,713, all of which are herein incorporated by nce in their
ties.
[00302] Single domain antibodies, for e, 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 SD et al., (2000) Curr Pharm Biotechnol 1(3): 253-263;
Muyldermans S, (2001) J Biotechnol 74(4): 277-302; U.S. Patent No. 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.
Further, dies that ically bind to a CTLA-4 antigen can, in turn, be
utilized to te anti-idiotype antibodies that “mimic” an antigen using techniques well
known to those skilled in the art. (See, e.g., Greenspan NS & Bona CA (1989) FASEB J 7(5):
437-444; and Nissinoff A (1991) J l 147(8): 2429-2438, which are herein incorporated
by reference in their entireties).
In ular embodiments, an dy described herein, which binds to the same
epitope of CTLA-4 (e.g., human CTLA-4) as an anti-CTLA-4 antibody described herein, is a
human antibody or an antigen-binding fragment thereof. In particular embodiments, an
antibody bed herein, which competitively blocks (e.g., in a dose-dependent manner) any
one of the antibodies described herein, from binding to CTLA-4 (e.g., human CTLA-4), is a
human antibody or an antigen-binding fragment thereof. Human antibodies can be produced
using any method known in the art. For example, transgenic mice which are incapable of
expressing onal 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,
nt 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 gous recombination. In particular,
homozygous deletion of the JH region prevents endogenous antibody production. The modified
embryonic stem cells are ed 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 n with a selected antigen,
e.g., all or a portion of an antigen (e.g., CTLA-4). Monoclonal antibodies directed against the
antigen can be ed from the immunized, transgenic mice using tional hybridoma
technology. The human immunoglobulin transgenes harbored by the enic 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, which is 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.
Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 016, 5,545,806, 5,814,318 and
,939,598. Examples of mice capable of producing human antibodies include the
XenomouseTM (Abgenix, Inc.; U.S. Patent Nos. 6,075,181 and 6,150,184), the HuAb-MouseTM
(Mederex, Inc./Gen Pharm; U.S. Patent Nos. 5,545,806 and 5,569, 825), the Trans Chromo
MouseTM (Kirin) and the KM MouseTM (Medarex/Kirin), all of which are herein incorporated
by reference in their entireties.
Human antibodies which specifically bind to CTLA-4 (e.g., human CTLA-4) can
be made by a variety of methods known in the art including phage display methods described
above using antibody libraries derived from human immunoglobulin sequences. See also U.S.
Patent Nos. 4,444,887, 4,716,111, and 5,885,793; and International ation Nos. WO
98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 35, and
WO 91/10741, all of which are herein incorporated by reference in their entireties.
In some embodiments, human antibodies can be produced using human
hybridomas. For example, human eral blood lymphocytes ormed 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 dies that ically
bind to a target antigen (e.g., CTLA-4 (e.g., human CTLA-4)). Such methods are known and
are described in the art, see, e.g., to H et al., (2004) chnology 46: 19-23;
Naganawa Y et al., (2005) Human Antibodies 14: 27-31, which are herein incorporated by
reference in their entireties.
6.6 Kits
Also provided, are kits sing one or more antibodies bed herein, or
pharmaceutical composition 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
dies provided herein or an antigen-binding fragment thereof. In some 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 x stimulating antibody, such as an anti-CD3 antibody and anti-CD28 antibody.
Optionally ated with such container(s) can be a notice in the form prescribed by a
governmental agency regulating the cture, use or sale of pharmaceuticals or biological
products, which notice reflects al by the agency of manufacture, use or sale for human
administration.
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
ners. In a ic embodiment, kits bed herein contain a substantially isolated
CTLA-4 antigen (e.g., human CTLA-4) as a control. In another specific embodiment, the kits
described herein further comprise a control antibody which does not react with a CTLA-4
antigen. In another specific embodiment, kits described herein contain one or more ts
for detecting the binding of an antibody to a CTLA-4 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 ments, a kit
provided herein can include a inantly ed or chemically synthesized CTLA-4
antigen. The CTLA-4 antigen ed in the kit can also be attached to a solid support. In a
more specific embodiment, the detecting means of the above bed kit includes a solid
support to which a CTLA-4 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 dy to the CTLA-4 antigen can be detected by g of the said reporter-labeled
antibody.
[00309] In one embodiment, the present ion relates to the use of a kit of the present
invention for in vitro assaying and/or detection of human CTLA-4 in a biological sample.
7. EXAMPLES
The examples in this Section (i.e., Section 6) are offered by way of illustration, and
not by way of limitation.
7.1 Example 1: Characterization of novel anti-CTLA-4 antibodies
This example bes the characterization of antibodies that bind to human
. In particular, this example describes the characterization of antibodies that
specifically bind to human CTLA-4 and inhibit the function of CTLA-4. The sequence
ation of the variable regions of these dies is provided in Table 4. All the antibodies
were expressed as IgG1 antibodies and analyzed in the assays described below.
7.1.1 Binding of anti-CTLA-4 antibodies to CTLAexpressing cells
] Jurkat cells engineered to constitutively s human CTLA-4 (Promega) were
used to analyze the binding of anti-CTLA-4 antibodies. Briefly, the cells were stained at 5x105
cells/well using 2 µg/ml of antibody in a 96-well plate for 30 minutes at 4°C. The cells were
washed twice and incubated for 20 s at 4°C with an anti-human IgG secondary antibody
(Thermo Scientific, Cat #31529). The cells were washed and suspended in 50 μl of 2%
paraformaldehyde (Alfa Aesar, Cat #43368) prepared in PBS. Data were collected with BD
FACS Canto II.
As shown in Figures 1A-1G, all the anti-CTLA-4 antibodies tested bound to CTLA-
essing cells.
7.1.2 Effect of anti-CTLA-4 antibody on human PBMCs ing Staphylococcal
Enterotoxin A (SEA) stimulation
[00314] The functional activities of the anti-CTLA-4 antibody AGEN1884.H3 (IgG1) on
primary human PBMCs were assessed following Staphylococcal Enterotoxin A (SEA)
stimulation. Briefly, cryopreserved PBMCs were stimulated with 100 ng/ml of the SEA
superantigen (Toxin Technologies, Cat# at101red) in the absence or ce of 10 µg/ml of
an anti-CTLA-4 dy or an isotype control antibody (IgG1) for 5 days at 37°C and 5% CO2.
IL-2 concentrations in the culture supernatant were analyzed by AlphaLISA (Perkin Elmer,
Cat# AL221F).
] The anti-CTLA-4 antibody AGEN1884.H3 (IgG1) increased IL-2 production in
human PBMCs stimulated with the SEA superantigen (Figure 2).
7.1.3 Effect of anti-CTLA-4 antibody on ILluciferase reporter cell line
[0017] Next, the functional activities of the anti-CTLA-4 antibody AGEN1884.H3 (IgG1)
were further analyzed using an ILluciferase reporter assay. Briefly, a human T cell line
t) that endogenously expressed CD3 and CD28 was engineered to constitutively express
cell surface CTLA-4 and a luciferase reporter gene driven by an IL-2 promoter. The Jurkat
reporter cell line was co-cultured with an antigen ting cell line (Raji) that endogenously
expressed CD80 and CD86 and was engineered to express a proprietary T cell activator
(Promega). T cell receptor (TCR) triggering (signal 1) was achieved by the T cell activator;
and costimulatory signaling (signal 2) was provided in trans by CD80 and CD86 expressed on
Raji cells. TCR signaling in the Jurkat T cell line red IL-2 expression, leading to
rase production, a surrogate marker for T cell activation. ture of these two cell
lines resulted in engagement of the inhibitory co-receptor CTLA-4 (expressed on Jurkat cells)
with its natural s CD80 and CD86 (expressed on Raji cells) inhibiting T cell activation,
demonstrated by a lack of luciferase expression. This inhibition was ed upon addition of
increasing concentrations of anti-CTLA-4 blocking antibodies. Luciferase expression was
quantified using Bio-GloTM reagent and the resulting data were used to determine fold response
values (fold increase with AGEN1884.H3 (IgG1) compared with an isotype control antibody
(IgG1)).
As shown in Figure 3, the anti-CTLA-4 antibody 84.H3 (IgG1) dose-
dependently released CTLA-4 mediated inhibition of T cells in this ILluciferase er
assay.
7.1.4 Effect of anti-CTLA-4 antibody on Fc gamma receptor IIIA reporter cell line
The y of anti-CTLA-4 antibody to co-engage CTLA-4 and signal via activating
Fc gamma receptors was evaluated using a reporter cell line expressing Fc gamma receptor
IIIA (FcγRIIIA) (Promega). Briefly, Jurkat cells were ered to constitutively express
human CTLA-4 on the cell surface. These target cells were co-cultured with an effector cell
line (Jurkat) engineered to express FcγRIIIA (the V158 variant) upstream of an NFAT response
element (RE) g expression of firefly luciferase. A titrated dose of AGEN1884.H3 (IgG1)
or an isotype l antibody (IgG1) was added to the co-culture and incubated at 37°C
ght. Simultaneous engagement of AGEN1884.H3 by the target cell line (binding to
CTLA-4 by the Fab region) and effector cell line (binding to FcγRIIIA by the Fc region)
triggers NFAT RE reporter gene tion and luciferase expression. The next day, Bio-Glo
reagent (Promega) was added to the co-culture, luminescence was measured by n
Multimode Plate Reader (Perkin Elmer), and relative light units (RLU) were recorded to
calculate fold response values (fold increase with 84.H3 (IgG1) compared with an
isotype control antibody (IgG1)).
When bound to target cells expressing human CTLA-4 on the cell surface, the IgG1
antibody AGEN1884.H3 activated FcγRIIIA signaling in the effector cells (Figure 4).
7.2 Example 2: Characterization of anti-CTLA-4 antibodies with different Fc regions
This example analyzes the impact of Fc/Fc receptor interaction on the functional
activity of anti-CTLA-4 antibodies. 84.H3 was expressed as antibodies in which the
IgG1 Fc region comprises the S239D/I332E, S239D/A330L/I332E, or
L235V/F243L/R292P/Y300L/P396L mutations, numbered according to the EU numbering
, and tested in functional assays described below. The antibody AGEN1884.H3 (IgG1
S239D/I332E) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:
24 and a light chain comprising the amino acid ce of SEQ ID NO: 27. The antibody
AGEN1884.H3 (IgG1 S239D/A330L/I332E) comprises a heavy chain comprising the amino
acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ
ID NO: 27. The antibody AGEN1884.H3 (IgG1 L235V/F243L/R292P/Y300L/P396L)
comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 26 and a light
chain sing the amino acid sequence of SEQ ID NO: 27. For ison, AGEN1884
was also expressed as a wild type IgG1 antibody, an IgG1 antibody sing S239D/I332E
or S239D/A330L/I332E mutations, numbered according to the EU numbering system, or an
ylated IgG1 antibody, and tested in some functional assays.
7.2.1 Binding of anti-CTLA-4 dies to CTLAexpressing cells
The binding of anit-CTLA-4 antibodies AGEN1884.H3 (IgG1 S239D/I332E),
AGEN1884.H3 (IgG1 S239D/A330L/I332E), and AGEN1884.H3 (IgG1
L235V/F243L/R292P/Y300L/P396L) to CTLAexpressing cells was characterized similarly
as described above. Briefly, Jurkat cells engineered to s human CTLA-4 (Promega)
were stained first with 5 µg/ml of an anti-CTLA-4 antibody or an isotype control antibody and
then with an uman IgG secondary antibody (Thermo Scientific, Cat #31529). The cells
were analyzed using BD FACS Canto II.
[00320] As shown in Figures 5A-5D, AGEN1884.H3 antibodies with different Fc regions
all bound to cells expressing human CTLA-4.
7.2.2 Effect of anti-CTLA-4 antibody on ligand binding to human CTLA-4
In this example, the y of an Fc variant anti-CTLA-4 dy AGEN1884.H3
(IgG1-S239D/A330E/I332E) to block binding between human CTLA-4 and its ligands, CD80
and CD86, was .
Briefly, recombinant CD80-Fc and CD86-Fc proteins were conjugated to the
fluorochrome Alexa Fluor 647 (Invitrogen, A20186). Jurkat cells were uced with
trCTLA4 (truncated intracellular domain) under the control of the EF1α promoter, as described
in Nakaseko et al. (J Exp Med. 1999 Sep 20; 190(6): 765–774), thus producing a cell line that
constitutively expressed human CTLA-4 on the cell surface. CTLAexpressing cells were
incubated with a dose titration of anti-CTLA-4 antibody AGEN1884.H3 (IgG1-
S239D/A330E/I332E), a reference anti-CTLA-4 antibody, or an isotype control antibody
(IgG1). The cells were then stained with fluorescently ed c or CD86-Fc n.
Following staining, fluorescence was ed using the LSRFortessa flow ter (BD
Biosciences). FACS plots were analyzed using a combination of FACS DIVA and WEHI
Weasel software. Values were plotted using Graphpad Prism software.
As shown in Figure 6A, AGEN1884.H3 (IgG1-S239D/A330E/I332E) and the
reference anti-CTLA-4 antibody each blocked binding between human CTLA-4 and CD80 in
a dose-dependent manner, whereas isotype control antibody (IgG1) had no effect. As shown
in Figure 6B, AGEN1884.H3 (IgG1-S239D/A330E/I332E) and the reference anti-CTLA-4
antibody each also blocked g between human CTLA-4 and CD86 in a dose-dependent
manner, whereas isotype control antibody (IgG1) had no effect. These data show that
AGEN1884.H3 (IgG1-S239D/A330E/I332E) functions as a ligand-blocking antibody for
CTLA-4.
7.2.3 Effect of anti-CTLA-4 antibodies on human PBMCs following Staphylococcal
Enterotoxin A (SEA) stimulation
In this e, the impact of Fc s on the functional activity of anti-CTLA-4
antibodies was analyzed using the SEA ation assay described above. In brief, human
PBMCs were ed in vitro with 100 ng/ml of the SEA peptide (Toxin Technologies, Cat#
at101red) in the absence or presence of TLA-4 antibodies with different Fc regions or an
isotype control antibody. After five days, concentrations of IL-2 in the culture supernatant, a
marker of T cell activation, were measured using AlphaLISA (Perkin Elmer, Cat# AL221F).
[00325] As shown in Figure 7A, the three 84.H3 dies containing mutations
in the IgG1 Fc regions, all of which enhanced binding to FcγRIIIA, stimulated more IL-2
secretion than AGEN1884.H3 with a wild type IgG1 Fc region.
In similar studies, AGEN1884.H3 or 84 antibodies with different Fc
regions were tested in the SEA stimulation assay. Introducing S239D/I332E,
S239D/A330L/I332E, or L235V/F243L/R292P/Y300L/P396L substitutions in the IgG1 Fc
region significantly enhanced the functional activity of AGEN1884.H3 (Figure 7B). Similarly,
84 (IgG1 S239D/I332E), AGEN1884 (IgG1 A330L/I332E), and afucosylated
AGEN1884 (IgG1) enhanced IL-2 production at substantially lower concentrations compared
to AGEN1884 with a wild type IgG1 Fc region (Figure 7C).
7.2.4 Effect of anti-CTLA-4 antibodies on ZAP70 phosphorylation
In this example, the impact of Fc regions on the onal activity of anti-CTLA-4
antibodies in the T cell-antigen presenting cell (APC) e was analyzed using an assay that
measures extent of phosphorylation of the protein tyrosine kinase ZAP70, which is ted
to the TCR following TCR engagement, where it becomes phosphorylated and facilitates
downstream signaling events.
y, human PBMCs were incubated with a suboptimal concentration of SEA
peptide and 10 µg/mL of isotype control antibody (IgG1) or the anti-CTLA-4 antibodies
AGEN1884.H3 (IgG1), AGEN1884.H3 (IgG1 S239D/A330L/I332E), or AGEN1884.H3 (IgG1
N297A). Cells were then incubated at 37°C for 0 (pre) 1, 5, 10, 30, or 60 minutes. At the end
of the tion, cells were lysed with cold 1×RIPA buffer supplemented with a
phosphatase/protease inhibitor cocktail (Cell Signaling Technologies). Following supernatant
clarification, n concentration was quantified using bicinchoninic acid (BCA) analysis
(Pierce Biotechnology). Cell lysates (20 µg/lane) were prepared in Bolt LDS sample buffer
diluted and heated for 10 minutes at 70°C before being loaded onto a 4-12% Bolt Bis Tris gels
(Novex). Proteins were separated in 1x Bolt MOPS-buffer (ThermoFisher) and then blotted
onto a PVDF membrane. Following blockade with 5% bovine serum albumin (BSA, 1 hour),
samples were incubated with primary anti-human rabbit phospho-ZAP70 (Tyr493)/Syk
6) antibody (Cell Signaling Technologies) in blocking buffer overnight at 4°C.
Membranes were probed with goat anti-rabbit secondary HRP-conjugate and visualized with
SignalFire ECL reagent (Cell Signaling Technology). Images were ed using the
Chemidoc imaging system (BioRad). As a control, total ZAP70 protein was evaluated
following membrane stripping with Restore™ PLUS n Blot Stripping Buffer.
Densitometric analysis of phospho-ZAP70 normalized to that of total ZAP70 was med
using Image J (Wayne Rasband; National Institute of Mental Health, Bethesda, MD, USA) and
expressed as the fold change relative to the isotype control d samples that was incubated
for 1 .
As shown in Figures 8A-8B, in the isotype control antibody sample, ZAP70
phosphorylation was transiently increased within ten minutes after stimulation and rapidly
diminished with no detectable levels after 15 minutes. In contrast, the on of anti-CTLA-
4 antibodies AGEN1884.H3 (IgG1) or 84.H3 (IgG1 S239D/A330L/I332E) extended
detectable ZAP70 activation to 30 minutes, with the most pronounced activity and relative
nce observed with AGEN1884.H3 (IgG1 S239D/A330L/I332E).
7.2.5 Effect of murine anti-CTLA-4 dies on tumor growth and intratumoral
tory T cell depletion in a mouse model
In this example, the impact of Fc regions on the antitumor and intratumoral
regulatory T cell (Treg) depletion activities of anti-CTLA-4 antibodies was analyzed using a
mouse model for colon cancer (CT26 tumor-bearing mice).
Briefly, 5x104 CT26 tumor cells were suspended in 100 ml PBS and injected
subcutaneously into 6-8 week old female BALB/cJ mice (Jackson Laboratories). Following
tment to a tumor volume of 50-80mm3, mice were treated with a single 100 µg dose of
murine anti-CTLA-4 antibody 9D9 (mIgG2a), an ent variant of anti-CTLA-4 antibody
9D9 (mIgG2a-N297A), an Fc variant of anti-CTLA-4 antibody 9D9 a-
S239D/A330L/I332E), or an isotype control antibody (mIgG2a). Amino acid sequences for
the murine antibodies tested are shown in Table 7.
Table 7. Amino acid sequences of murine anti-CTLA-4 antibodies
Description Sequence SEQ ID NO
SGPVLVKPGASVKMSCKASGYTFTDY
YMNWVKQSHGKSLEWIGVINPYNGDTSYNQKF
KGKATLTVDKSSSTAYMELNSLTSEDSAVYYC
WFAYWGQGTLVTVSSAKTTAPSVYPL
APVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSG
Murine anti-CTLA-4 SLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQ
antibody SITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCP
(mIgG2a) APNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVD
heavy chain VSEDDPDVQISWFVNNVEVHTAQTQTHREDYN
STLRVVSALPIQHQDWMSGKEFKCKVNNKDLP
APIERTISKPKGSVRAPQVYVLPPPEEEMTKKQV
TLTCMVTDFMPEDIYVEWTNNGKTELNYKNTE
PVLDSDGSYFMYSKLRVEKKNWVERNSYSCSV
NHHTTKSFSRTPG
EAKLQESGPVLVKPGASVKMSCKASGYTFTDY
YMNWVKQSHGKSLEWIGVINPYNGDTSYNQKF
KGKATLTVDKSSSTAYMELNSLTSEDSAVYYC
ARYYGSWFAYWGQGTLVTVSSAKTTAPSVYPL
APVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSG
Murine anti-CTLA-4
SLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQ
antibody
SITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCP
(mIgG2a- 50
APNLLGGPDVFIFPPKIKDVLMISLSPIVTCVVVD
S239D/A330L/I332E)
DVQISWFVNNVEVHTAQTQTHREDYN
heavy chain
STLRVVSALPIQHQDWMSGKEFKCKVNNKDLP
LPEERTISKPKGSVRAPQVYVLPPPEEEMTKKQ
VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNT
EPVLDSDGSYFMYSKLRVEKKNWVERNSYSCS
VVHEGLHNHHTTKSFSRTPG
Murine anti-CTLA-4 EAKLQESGPVLVKPGASVKMSCKASGYTFTDY
antibody YMNWVKQSHGKSLEWIGVINPYNGDTSYNQKF
(mIgG2a-N297A) KGKATLTVDKSSSTAYMELNSLTSEDSAVYYC
heavy chain ARYYGSWFAYWGQGTLVTVSSAKTTAPSVYPL
APVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSG
SLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQ
SITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCP
GPSVFIFPPKIKDVLMISLSPIVTCVVVD
VSEDDPDVQISWFVNNVEVHTAQTQTHREDYA
STLRVVSALPIQHQDWMSGKEFKCKVNNKDLP
APIERTISKPKGSVRAPQVYVLPPPEEEMTKKQV
TDFMPEDIYVEWTNNGKTELNYKNTE
PVLDSDGSYFMYSKLRVEKKNWVERNSYSCSV
VHEGLHNHHTTKSFSRTPG
DIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNG
NTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRF
Murine anti-CTLA-4
SGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP
antibody
YTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGG 52
(mIgG2a)
ASVVCFLNNFYPKDINVKWKIDGSERQNGVLNS
light chain
WTDQDSKDSTYSMSSTLTLTKDEYERHNSYTC
STSPIVKSFNRNEC
In a first experiment, mice d with antibodies were then measured biweekly for
tumor growth. As shown in Figure 9A, an Fc variant of anti-CTLA-4 antibody 9D9 (shown as
mIgG2a-S239D/A330L/I332E) d complete regression in all CT26 tumor-bearing mice
(eight out of eight mice tested). In contrast, other variants of dy 9D9 failed to elicit the
same efficacy: antibody 9D9 (mIgG2a) itself induced complete regressions in three out of nine
mice , and the Fc-silent variant of antibody 9D9 (mIgG2a-N297A) failed to induce
sion in any of the nine mice tested.
In a second experiment, CT26 tumor-bearing mice were treated as described above
and then sacrificed at 0, 24, 72, or 240 hours post-treatment for collection of tumor tissue,
tumor draining lymph nodes, and spleens. Collected tissues were evaluated for FoxP3+ Treg
expansion by flow cytometry. Single cell suspensions were obtained by mechanical
dissociation followed by filtration (70 µM cell strainer). To reduce non-specific binding, the
cells were incubated with an FcγR blocking antibody (Biolegend) in FACS buffer (PBS, 2mM
EDTA, 0.5% BSA, pH 7.2) for 15 minutes at ambient temperature. Samples were then washed
twice in FACS buffer and stained for a lineage panel of CD3, CD4, CD8, and CD25, as well
as a fixable live/dead marker, for 30 minutes at 4ºC. For Treg delineation, samples were then
washed twice, fixed, permeabilized, and then incubated with an anti-FoxP3 dy 6s)
for 30 minutes at 4ºC. Samples were analyzed using the tessa flow cytometer (BD
Biosciences). FACS plots were analyzed using a combination of FACS DIVA and WEHI
Weasel software. As shown in Figure 9B, the anti-CTLA-4 antibody 9D9 (mIgG2a) and the
Fc variant anti-CTLA-4 antibody 9D9 a-S239D/A330L/I332E) each reduced the
quantities of intratumoral FoxP3+ Tregs compared to the isotype control antibody, with the Fc
variant anti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E) decreasing the quantity of
intratumoral FoxP3+ Tregs most icantly. The Fc-silent variant of anti-CTLA-4 antibody
9D9 (mIgG2a-N297A) did not substantially reduce the quantity of intratumoral FoxP3+ Tregs
relative to the isotype control antibody. None of the treatment groups showed substantial
changes in the quantities of intratumoral CD45+ leukocytes or CD4+ non-Tregs. The Fc
t anti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E) induced the largest
increase in intratumoral CD8/Treg ratio over time, followed by antibody 9D9 (mIgG2a), and
then by the Fc-silent variant antibody 9D9 (mIgG2a-N297A) and the isotype l antibody
(mIgG2a).
As shown in Figure 9C, the anti-CTLA-4 antibody 9D9 (mIgG2a), the Fc variant
anti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E) and the Fc-silent t of anti-
CTLA-4 antibody 9D9 (mIgG2a-N297A) had no substantial effect on the quantities of tumor
ng lymph node (TDLN) FoxP3+ Tregs compared to the isotype control antibody.
Similarly, as shown in Figure 9D, the TLA-4 dy 9D9 (mIgG2a), the Fc variant
anti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E) and Fc-silent variant of anti-
CTLA-4 antibody 9D9 (mIgG2a-N297A) had no ntial effect on the quantities of splenic
FoxP3+ Tregs compared to the isotype control antibody.
7.2.6 Effect of murine anti-CTLA-4 antibodies in ation with a tumor vaccine on
tumor growth
In this example, the effect on tumor growth of a combination of murine anti-CTLA-
4 antibodies and an HPV tumor vaccine was tested in the HPV+ TC-1 syngeneic tumor mouse
model.
[00336] The TC-1 cell line was developed by co-transformation of primary lung epithelial
cells /6) with c-Ha-ras and HPV-16 (E6/E7) oncogenes, as described in Lin et al. (1996,
Cancer Res. 56(1): 21-26). For tumor implantation, 2x105 TC-1 cells were injected
subcutaneously into 6-8 week old female C57BL/6 mice (Jackson Laboratories). At each of
days day 5, 10 and 15 post-tumor implantation, mice were administered 100 µg of anti-CTLA-
4 antibody 9D9 (mIgG2a), an Fc variant anti-CTLA-4 antibody 9D9 (mIgG2a-
S239D/A330L/I332E), or an isotype control antibody (mIgG2a), in combination with a dose
of HPV vaccine (HPV+ tumor, viral ns E6/E7) or no additional treatment. Each dose of
HPV e contained 30 µg of HSP protein (0.4 nM) complexed with HPV pool peptide (1.2
nM) and was mented with 10 µg QS-21 Stimulon® adjuvant. After treatment, mice were
assessed biweekly for tumor growth and were sacrificed when tumors reached 2000 mm3 or
upon ulceration.
As shown in Figure 10, the antitumor efficacy of anti-CTLA-4 antibody 9D9
(mIgG2a) and the Fc t anti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E) each
showed improvement when stered in combination with HPV tumor vaccine. This effect
was greater for the Fc variant anti-CTLA-4 antibody (mIgG2a-S239D/A330L/I332E). In
particular, the Fc t anti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E) induced
a noticeable additional decrease in TC-1 tumor growth when combined with the HPV tumor
vaccine, relative to when the antibody was administered as a single agent. This additional
decrease in tumor growth was greater than that observed for the combinations of antibody 9D9
(mIgG2a) or the isotype control antibody (mIgG2a) with the HPV tumor vaccine.
7.2.7 Characterization of ed and activated T cell populations
In this example, ed and activated T cell tions were characterized for
gene expression and CpG methylation. In brief, natural CD4+ CD25+ FOXP3+ regulatory T
cells or CD4+ CD25+/- FOXP3- non-regulatory T cells were isolated from peripheral blood of
a healthy human donor, expanded, and activated. The T cells were then characterized for
expression of FOXP3 and CTLA-4 by flow cytometry, and assessed for lineage stability by
examining DNA CpG ation at CpG regions within the FOXP3 and CTLA4 loci. As
known in the art, hypomethylation at these CpG sites can be used to accurately delineate
effector versus regulatory T cell lineages t et al., 2015, J. l. 194(3): 878-882).
PBMCs were isolated via Ficoll gradient from y donor buffy coats rch
Blood Components, LLC) and were then enriched for effector T cells (Teffs) or natural
regulatory T cells (Tregs) using magnetic bead ion (MACS, Miltenyi). The enriched
Teffs or Tregs were activated with CD3-CD28 microbeads (1:1 bead:cell ratio; Invitrogen) and
with recombinant human IL-2 for seven days in RPMI media mented with 10% heatinactivated
FBS at 37ºC and 5% CO2. Following stimulation, the cells were evaluated for
FOXP3 and CTLA-4 expression via flow cytometry. To reduce ecific binding, the cells
were pre-incubated with an FcγR blocking antibody (Biolegend) in FACS buffer (PBS, 2mM
EDTA, 0.5% BSA, pH 7.2) for 15 minutes at ambient temperature. Samples were then washed
twice in FACS buffer and stained with a lineage panel of CD3, CD4, CD8, CD25, as well as a
fixable cell death marker, for 30 minutes at 4ºC. To assess membrane CTLA-4 expression,
CTLA-4 staining was conducted at 37ºC. For intracellular FOXP3 and CTLA-4 staining,
samples were washed twice, fixed, permeabilized, and incubated with an anti-FOXP3 antibody
(PCH101) and TLA-4 antibody (BNI3), respectively, for 30 minutes at 4ºC. Samples
were then washed twice and analyzed using the LSRFortessa flow cytometer (BD Biosciences).
FACS plots were analyzed using a combination of FACS DIVA and WEHI Weasel software.
For CpG methylation analysis, total DNA was isolated from approximately 1x105 naïve CD4+
T cells, activated Teffs, or activated Tregs and subjected to pyrosequencing.
] As shown in Figure 11A, a high level of FOXP3 expression was detected on
activated Tregs, as well as high levels of both intracellular and membrane CTLA-4 expression.
In st, activated Teffs showed reduced levels of FOXP3, intracellular CTLA-4, and
membrane CTLA-4 relative to activated Tregs. In particular, substantially less membrane
CTLA-4 sion was observed for activated Teffs compared to activated Tregs. Figure 11B
further shows that activated Tregs also exhibited hypomethylated FOXP3 and CTLA4 CpG
regions compared to naïve and activated Teffs from the same donor.
7.2.8 Effect of anti-CTLA-4 dies on antibody dependent cellular cytotoxicity of
CTLAexpressing human T cells
[00341] In this e, the effect of anti-CTLA-4 antibody AGEN1884.H3 (IgG1) or Fc
variants thereof on antibody dependent cellular xicity (ADCC) of human CTLA
expressing T cells was ed using high content microscopy of caspase 3/7 activation to
quantify ADCC activity.
y, CTLAexpressing target cells were co-cultured with NK-92 cells
expressing FcγRIIIA, following opsonization with 10 μg/ml of TLA-4 antibody or Fc
variants thereof, as bed below. In a first experiment, Jurkat cells engineered to
constitutively express urface human CTLA-4 were used as target cells. CTLA
expressing Jurkat cells were generated by transducing the Jurkat cell line with trCTLA4
(intracellular domain removed) under the control of the EF1α promoter, as bed in
Nakaseko et al. (1999, J. Exp. Med. 190(6): 765-774). In a second experiment, y human
activated effector and regulatory T cells were used as target cells. CTLAexpressing target
cells and FcγRIIIA-158V-expressing NK-92 cells were differentially stained using red and blue
live-cell tracers (Thermo Fisher) and co-cultured at a 1:1 cell ratio (1.5x103 cells/well in 384-
well plates). s were treated with 10 µg/ml of AGEN1884.H3 (IgG1), AGEN1884.H3
(IgG1 N297A), AGEN1884.H3 (IgG1 S239D/A330L/I332E), AGEN1884.H3 (IgG1
S267E/L328F), afucosylated AGEN1884.H3 (IgG1), or an isotype control antibody (IgG1).
Samples were then evaluated for the induction of apoptosis over time by live confocal imaging
of caspase 3/7 substrate, which fluoresces following cleavage by activated caspase. Sample
images were acquired every 20 minutes for six hours. Percentage ADCC activity is measured
as the number of apoptotic cells relative to the total cell count under each condition.
As shown in Figure 12A, the Fc variant AGEN1884.H3 (IgG1
S239D/A330L/I332E) antibody, the afucosylated AGEN1884.H3 antibody, and the
AGEN1884.H3 (IgG1) dy each induced substantially greater ADCC activity in Jurkat
cells engineered to express cell-surface CTLA-4 relative to the AGEN1884.H3 (IgG1 N297A)
variant, the AGEN1884.H3 (IgG1 S267E/L328F) variant, and isotype l antibody (IgG1).
The AGEN1884.H3 (IgG1 S239D/A330L/I332E) Fc variant antibody and the afucosylated
AGEN1884.H3 dy induced greater increases in ADCC ty compared to the
AGEN1884.H3 (IgG1) antibody. As shown in Figure 12B, the AGEN1884.H3 (IgG 1
S239D/A330L/I332E) Fc variant antibody induced the highest levels of ADCC in both y
human activated effector T cells (left panel) and y human activated regulatory T cells
(right panel), followed by afucosylated AGEN1884.H3 antibody. The AGEN1884.H3 (IgG1)
antibody also induced slightly higher levels of ADCC compared to ls. The remaining
antibodies tested induced little to no ADCC activity in either effector or regulatory T cells.
Notably, the AGEN1884.H3 (IgG1 S239D/A330L/I332E) Fc variant antibody and the
afucosylated AGEN1884.H3 antibody each induced substantially greater ADCC in regulatory
T cells compared to effector T cells.
7.2.9 Effect of anti-CTLA-4 antibodies in combination with an anti-PD-1 dy on T
cell onality
[00344] In this example, the effect of anti-CTLA-4 antibodies in combination with an anti-
PD-1 antibody on primary human T cell function was examined.
Briefly, PBMCs were isolated via Ficoll gradient from healthy donor buffy coats
(Research Blood Components, LLC) of two human . This ment was performed
twice on PBMCs collected from each donor, for a total of two replicates per donor. For each
replicate, isolated PBMCs were incubated for four days under stimulatory culture conditions
with a dosage titration of anti-CTLA-4 antibody 84.H3 (IgG1), an Fc variant anti-
CTLA-4 antibody AGEN1884.H3 (IgG1 S239D/A330L/I332E), or an isotype control antibody
(IgG1), in combination with a fixed dosage (5 µg/ml) of a reference anti-PD-1 antagonist
antibody or an isotype control antibody (IgG4). Stimulatory culture conditions were defined as
cells suspended in RPMI media, supplemented with 100 ng/ml SEA superantigen -
Aldrich), 10% nactivated FBS at 37ºC, and 5% CO2. Following incubation, ree
supernatants were assayed for IL-2 production using an AlphaLISA immunoassay (Perkin-
Elmer). Data was collected using the on® Multilabel Plate Reader (Perkin-Elmer), and
the concentration of IL-2 was determined using an IL-2 standard curve. Values were
interpolated and plotted using Graphpad Prism software.
As shown in Figures 13A-13D, the anti-CTLA-4 dy AGEN1884.H3 (IgG1)
and the Fc t anti-CTLA-4 antibody AGEN1884.H3 (IgG1 S239D/A330L/I332E) each
induced increased IL-2 production ve to isotype controls or reference anti-PD-1 antibody
alone. IL-2 production was further enhanced when AGEN1884.H3 or AGEN1884.H3 (IgG1
S239D/A330L/I332E) was combined with reference anti-PD-1 dy. Whether
administered with isotype control antibody or in combination with anti-PD-1 reference
antibody, the Fc variant anti-CTLA-4 antibody AGEN1884.H3 (IgG1 A330L/I332E)
induced a greater se in IL-2 production compared to AGEN1884.H3 (IgG1). This effect
was consistent in replicates for the first donor (Figures 13A and 13B) and the second donor
(Figures 13C and 13D).
7.3 Example 3: Epitope mapping of anti-CTLA-4 antibody
The interaction of the Fab fragment of AGEN1884 (AGEN1884-Fab) with the
extracellular domain of human CTLA-4 was studied by hydrogen-deuterium exchange (HDX)
mass spectrometry. CTLA-4 extracellular domain alone or in combination with AGEN1884-
Fab, in phosphate buffered saline solution at pH 7.4, was d with a ten-fold volume of
deuterium oxide labeling buffer and incubated for varying periods of time (0, 60, 300, 1800,
and 7200 seconds) at room temperature. ge of deuterium for hydrogen was ed
by adding one volume of 4 M guanidine hydrochloride, 0.85 M TCEP (tris(2-
carboxyethyl)phosphine) buffer and the final pH was 2.5. Samples were then subjected to oncolumn
pepsin/protease type XIII digestion and LC-MS analysis. Mass spectra were ed
in MS only mode. For the calculation of deuterium incorporation, the mass spectra for a given
peptide were combined across the extracted ion chromatogram peak and the ed average
m/z was calculated. The mass increase from the mass of the native peptide (0 minute) to the
weighted ed mass corresponds to the level of deuterium incorporation. The ium
buildup curves over exchange time for all the peptides were plotted for further analysis and
were compared with HDExaminer re.
Most of the CTLA-4 peptides displayed identical or similar deuterium levels with
and without the anti-human CTLA-4 Fab t. Several peptide segments, r, were
found to have significantly decreased deuterium incorporation upon Fab binding. All the
residues in this paragraph are numbered according to SEQ ID NO: 33. Two regions, residues
80-82 (QVT, SEQ ID NO: 39) and residues 135-149 (YPPPYYLGIGNGTQI, SEQ ID NO:
37), experienced strong deuterium protection when human CTLA-4 was bound to Fab. The
strongest decrease in deuterium uptake was observed at residues 140-141 (YL) which thus
appeared to be a main feature of the e of AGEN1884 on CTLA-4. Inspection of the
ces of human and cynomolgus monkey CTLA-4, both of which AGEN1884 binds
strongly (data not shown), reveals almost complete sequence identity in the two regions
described above, except for a methionine tution for e at position 141 (Figure 14A).
In contrast, AGEN1884 does not bind to any significant extent to either mouse or rat CTLA-4
(data not shown) which differ from human CTLA-4 at residues 140-143 (YLGI, SEQ ID NO:
34) at three out of four positions e 14A). Further selectivity data show that AGEN1884
binds with high specificity to human and cynomolgus monkey CTLA-4 and not to other related
CD28 family members ing CD28, ICOS, BTLA, and PD-1 (data not shown). Sequence
comparison among these related proteins shows that the non-CTLA-4 proteins all differ at
residues 3 (YLGI, SEQ ID NO: 34) (Figure 14B), further supporting the importance of
this epitope to the binding of AGEN1884.
* * *
] 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 cations are intended to fall within the scope of the appended claims.
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.
The following numbered paragraphs define particular aspects of the present invention:
1. An isolated antibody that ically binds to human CTLA-4, the antibody comprising a
heavy chain variable region comprising complementarity determining regions 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 SYSMN (SEQ ID NO: 10);
(b) CDRH2 comprises the amino acid ce of SISSSSSYIYYAXSVKG (SEQ ID NO:
18), n X is E or D;
(c) CDRH3 comprises the amino acid sequence of VGLXGPFDI (SEQ ID NO: 19),
wherein X is F or M;
(d) CDRL1 comprises the amino acid sequence of RASQSVSRYLG (SEQ ID NO: 15);
(e) CDRL2 comprises the amino acid sequence of GASTRAT (SEQ ID NO: 16); and
(f) CDRL3 comprises the amino acid sequence of QQYGSSPWT (SEQ ID NO: 17),
and wherein the CDRH1, CDRH2, and CDRH3 sequences of the antibody are not SEQ
ID NOs: 10, 11, and 13, respectively.
2. The isolated antibody of paragraph 1, wherein CDRH2 comprises the amino acid sequence
of SEQ ID NO: 12.
3. The isolated antibody of paragraph 1 or 2, wherein CDRH3 ses the amino acid
sequence of SEQ ID NO: 14.
4. An isolated antibody that specifically binds to human CTLA-4, the dy comprising a
heavy chain variable region comprising complementarity determining regions CDRH1,
CDRH2, and CDRH3 and a light chain variable region comprising complementarity
ining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3,
CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences set forth in SEQ ID
NOs: 10, 12, 14, 15, 16, and 17, respectively.
. The isolated antibody of aph 1, wherein the antibody comprises a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO: 20.
6. The isolated dy of aph 1, wherein the antibody comprises a heavy chain
variable region comprising an amino acid sequence which is at least 75%, 80%, 85%, 90%,
95%, 99%, or 100% identical to an amino acid ce selected from the group consisting
of SEQ ID NOs: 2 and 4-8.
7. The ed antibody of paragraph 6, wherein the heavy chain variable region comprises
an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4-8.
8. The ed antibody of paragraph 7, wherein the heavy chain variable region comprises
the amino acid sequence of SEQ ID NO: 8.
9. The isolated antibody of any one of the preceding paragraphs, wherein the antibody
comprises a light chain variable region comprising an amino acid sequence which is at least
75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ
ID NO: 9.
. The ed antibody of paragraph 9, wherein the antibody comprises a light chain variable
region comprising the amino acid sequence of SEQ ID NO: 9.
11. An isolated antibody that specifically binds to human CTLA-4, the antibody sing a
heavy chain variable region comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 2-8.
12. The isolated antibody of paragraph 11, wherein the heavy chain variable region comprises
the amino acid sequence of SEQ ID NO: 8.
13. The isolated antibody of paragraph 12, wherein the antibody comprises a heavy chain
comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:
23-26.
14. An isolated antibody that specifically binds to human CTLA-4, the antibody comprising a
heavy chain variable region and a light chain variable region, wherein the heavy chain
variable region and the light chain variable region comprise the amino acid ces set
forth in SEQ ID NOs: 2 and 9; 3 and 9; 4 and 9; 5 and 9; 6 and 9; 7 and 9; or 8 and 9,
respectively.
. The isolated antibody of aph 14, wherein the heavy chain variable region and the
light chain variable region comprise the amino acid ces set forth in SEQ ID NOs: 8
and 9, tively.
16. An isolated antibody that specifically binds to human CTLA-4, the dy comprising a
heavy chain le region having an amino acid sequence derived from a human IGHV3-
21 germline ce, wherein the heavy chain variable region comprises the amino acid
sequence set forth in SEQ ID NO: 14.
17. The isolated dy of paragraph 16, n the antibody comprises a light chain
variable region having an amino acid sequence derived from a human IGKV3-20 germline
sequence.
18. An isolated antibody that specifically binds to human CTLA-4, the antibody comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO: 23, and a light chain
comprising the amino acid sequence of SEQ ID NO: 27.
19. An isolated antibody that specifically binds to human CTLA-4, the antibody comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO: 24, and a light chain
comprising the amino acid sequence of SEQ ID NO: 27.
. An isolated antibody that specifically binds to human CTLA-4, the antibody comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and a light chain
comprising the amino acid sequence of SEQ ID NO: 27.
21. An isolated antibody that ically binds to human CTLA-4, the dy comprising a
heavy chain comprising the amino acid sequence of SEQ ID NO: 26, and a light chain
comprising the amino acid sequence of SEQ ID NO: 27.
22. The isolated antibody of any one of paragraphs 1-12 or 14-17, wherein the antibody
comprises a heavy chain constant region selected from the group consisting of human IgG1,
IgG2, IgG3, IgG4, IgA1, and IgA2.
23. The isolated antibody of any one of paragraphs 1-12 or 14-17, wherein the antibody
comprises an IgG1 heavy chain constant region.
24. The isolated antibody of paragraph 23, wherein the antibody comprises a heavy chain
constant region sing the amino acid sequence of SEQ ID NO: 28.
. The isolated dy of paragraph 23, wherein the amino acid sequence of the IgG1 heavy
chain constant region ses S239D/I332E mutations, numbered according to the EU
numbering system.
26. The isolated antibody of paragraph 25, wherein the antibody comprises a heavy chain
constant region comprising the amino acid ce of SEQ ID NO: 29.
27. The isolated antibody of paragraph 23, wherein the amino acid sequence of the IgG1 heavy
chain constant region comprises S239D/A330L/I332E mutations, numbered according to
the EU numbering system.
28. The isolated antibody of paragraph 27, wherein the antibody comprises a heavy chain
constant region comprising the amino acid sequence of SEQ ID NO: 30.
29. The isolated antibody of paragraph 23, wherein the amino acid sequence of the IgG1 heavy
chain constant region comprises L235V/F243L/R292P/Y300L/P396L mutations,
numbered according to the EU ing system.
. The isolated dy of paragraph 29, wherein the antibody comprises a heavy chain
constant region sing the amino acid sequence of SEQ ID NO: 31.
31. The ed antibody of paragraph 23, wherein the IgG1 heavy chain constant region is
afucosylated IgG1.
32. The isolated antibody of any one of paragraphs 1-12 or 14-17, wherein the antibody
comprises a human IgG heavy chain constant region that is a variant of a wild type human
IgG heavy chain constant , wherein the variant human IgG heavy chain constant
region binds to FcγRIIIA with a higher affinity than the wild type human IgG heavy chain
constant region binds to FcγRIIIA.
33. The isolated dy of paragraph 32, wherein the variant human IgG heavy chain constant
region is a variant human IgG1 heavy chain constant .
34. The isolated antibody of any one of paragraphs 1-17 or 22-33, wherein the dy
comprises a light chain constant region selected from the group consisting of human Igκ
and Igλ.
. The isolated antibody of paragraph 34, n the antibody comprises an Igκ light chain
constant region.
36. The ed antibody of paragraph 35, wherein the antibody ses a light chain
constant region comprising the amino acid sequence of SEQ ID NO: 32.
37. The isolated antibody of any one of the preceding paragraphs, wherein the antibody binds
to the same epitope of human CTLA-4 as an antibody comprising a heavy chain variable
region comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable
region comprising the amino acid ce of SEQ ID NO: 9.
38. The isolated antibody of any one of the preceding paragraphs, n the antibody binds
to an epitope located within a region of human CTLA-4 ting of an amino acid
sequence selected from the group consisting of SEQ ID NOs: 34-39.
39. The isolated antibody of any one of the ing paragraphs, wherein the antibody is a
human antibody.
40. The isolated antibody of any one of the preceding paragraphs, wherein the antibody is a
bispecific antibody.
41. The isolated antibody of any one of the preceding paragraphs, wherein the antibody is
antagonistic to human CTLA-4.
42. The isolated antibody of any one of the preceding paragraphs, wherein the antibody
vates, reduces, or inhibits an activity of human CTLA-4.
43. The isolated antibody of paragraph 42, n the antibody inhibits binding of human
CTLA-4 to human CD80 or human CD86.
44. The isolated dy of paragraph 42, wherein the antibody induces IL-2 production by
peripheral blood mononuclear cells (PBMCs) stimulated with staphylococcal enterotoxin
A (SEA).
45. The ed dy of any one of the preceding paragraphs conjugated to a cytotoxic
agent, cytostatic agent, toxin, radionuclide, or detectable label.
46. The isolated antibody of any one of the preceding paragraphs, wherein the N-terminal
amino acid residue of the heavy chain variable region and/or the light chain variable region
of the antibody has been converted to pyroglutamate.
47. A pharmaceutical composition comprising the antibody of any one of the preceding
paragraphs and a pharmaceutically acceptable carrier or ent.
48. An isolated polynucleotide encoding a heavy and/or light chain of the dy of any one
of paragraphs 1-45.
49. A vector comprising the polynucleotide of paragraph 48.
50. A recombinant host cell comprising the polynucleotide of paragraph 48 or the vector of
paragraph 49.
51. A method of producing an antibody that specifically binds to human CTLA-4, the method
comprising culturing the host cell of paragraph 50 so that the polynucleotide is expressed
and the antibody is produced.
52. A method of sing T cell activation in se to an antigen in a subject, the method
comprising administering to the subject an effective amount of the antibody or
pharmaceutical composition of any one of paragraphs 1-47.
53. A method of treating cancer in a t, the method comprising administering to the
subject an effective amount of the dy or pharmaceutical ition of any one of
paragraphs 1-47.
54. The method of aph 53, wherein the cancer is a metastatic or locally ed tumor.
55. The method of paragraph 53 or 54, n the cancer is a solid tumor.
56. The method of any one of paragraphs 53-55, wherein the cancer is a metastatic or locally
advanced, unresectable squamous cell carcinoma, adenosquamous oma, or
adenocarcinoma of the cervix.
57. The method of any one of paragraphs 53-56, wherein no standard therapy is available for
the cancer.
58. The method of any one of paragraphs 53-56, wherein the cancer is refractory to a standard
therapy.
59. The method of any one of paragraphs 53-56, wherein the cancer has relapsed after a
standard therapy.
60. The method of paragraph 58 or 59, wherein the standard therapy comprises a platinumcontaining
chemotherapy.
61. The method of paragraph 60, wherein the standard therapy comprises a platinumcontaining
doublet.
62. The method of any one of aphs 53-61, wherein the cancer is HPV positive.
63. The method of any one of paragraphs 53-55, wherein the cancer is a non-small cell lung
cancer (NSCLC).
64. The method of paragraph 63, wherein the cancer is a metastatic NSCLC.
65. The method of paragraph 63 or 64, wherein the NSCLC has no EGFR or ALK genomic
tumor aberrations.
66. The method of any one of paragraphs 63-65, wherein the NSCLC has no EGFR sensitizing
mutation or ALK translocation.
67. The method of any one of paragraphs 63-66, wherein the subject has received no prior
systemic chemotherapy treatment for the NSCLC.
68. The method of any one of aphs 53-55, n the cancer is a cutaneous squamouscell
carcinoma .
69. The method of paragraph 68, wherein the cancer is a Stage IV cSCC.
70. The method of paragraph 69, wherein the cSCC is diagnosed histologically or cytologically
according to the eighth edition of the American Joint Committee on Cancer staging manual.
71. The method of any one of paragraphs 68-70, wherein the cSCC is not curable with radiation
therapy.
72. The method of any one of paragraphs 53-71, wherein the percentage of tumor cells in a
sample of the cancer that exhibit detectable membrane expression of PD-L1 is at least 1%,
2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or
73. The method of any one of paragraphs 53-72, wherein the antibody is administered as a
first cancer therapy after diagnosis of the cancer.
74. The method of any one of paragraphs 53-73, wherein the antibody is administered as the
first cancer therapy after:
(a) diagnosis of tumor ssion that has occurred despite previous ent of the
cancer with a different cancer therapy; or
(b) diagnosis of toxicity of a different cancer y,
optionally wherein the antibody is the second cancer therapy administered to the subject.
75. The method of any one of paragraphs 52-75, wherein the antibody or pharmaceutical
composition is administered intravenously.
76. The method of paragraph 75, wherein the antibody or pharmaceutical composition is
administered intravenously at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3
mg/kg, 6 mg/kg, or 10 mg/kg, optionally at an interval of once every two weeks.
77. The method of paragraph 75, wherein the antibody or pharmaceutical composition is
administered intravenously at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3
mg/kg, 6 mg/kg, or 10 mg/kg, optionally at an interval of once every three weeks.
78. The method of paragraph 75, wherein the antibody or pharmaceutical composition is
administered intravenously at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3
mg/kg, 6 mg/kg, or 10 mg/kg, optionally at an al of once every four weeks.
79. The method of aph 75, wherein the dy or pharmaceutical composition is
administered enously at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3
mg/kg, 6 mg/kg, or 10 mg/kg, optionally at an interval of once every six weeks.
80. The method of paragraph 75, wherein the antibody or pharmaceutical composition is
administered intravenously at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3
mg/kg, 6 mg/kg, or 10 mg/kg, optionally at an interval of once every twelve weeks.
81. The method of any one of paragraphs 52-75, wherein the antibody or pharmaceutical
composition is administered aneously.
82. The method of any one of paragraphs 53-75, wherein the antibody or pharmaceutical
composition is administered intratumorally.
83. The method of paragraph 82, wherein the antibody or pharmaceutical composition is
administered intratumorally at 0.03 mg/kg, 0.1 mg/kg, or 0.3 mg/kg, optionally at an
interval of once every three weeks.
84. The method of any one of paragraphs 52-75, wherein the antibody or pharmaceutical
composition is delivered to a tumor draining lymph node.
85. The method of any one of paragraphs 81-84, wherein the antibody is the antibody of any
one of aphs 25-33.
86. The method of any one of paragraphs 52-85, wherein the antibody is administered as a
monotherapy.
87. The method of any one of aphs 52-85, further comprising administering an additional
therapeutic agent to the subject.
88. The method of paragraph 87, wherein the additional therapeutic agent is administered
ically.
89. The method of paragraph 87 or 88, wherein the additional eutic agent is an anti-PD-
1 antibody, optionally wherein the anti-PD-1 dy is pembrolizumab or mab.
90. The method of paragraph 89, wherein the additional therapeutic agent is pembrolizumab
administered at 200 mg every three weeks.
91. The method of aph 87 or 88, wherein the subject has head and neck squamous cell
carcinoma and wherein the additional therapeutic agent is an GFR antibody,
optionally wherein the anti-EGFR antibody is cetuximab,
optionally wherein the method further comprises stering a chemotherapeutic agent
to the subject, optionally wherein the chemotherapeutic agent is administered systemically,
optionally wherein the chemotherapeutic agent is gemcitabine.
92. The method of paragraph 87 or 88, wherein 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 n the herapeutic agent is administered systemically,
ally wherein the herapeutic agent is gemcitabine.
93. The method of paragraph 87 or 88, wherein the additional therapeutic agent is a
chemotherapeutic or a checkpoint targeting agent.
94. The method of paragraph 93, wherein 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 dy, an antagonist
anti-TIM-3 antibody, an antagonist anti-LAG-3 antibody, an nist anti-CEACAM1
dy, an t anti-GITR antibody, an agonist anti-OX40 antibody, and an agonist
anti-CD137 antibody.
95. The method of paragraph 87 or 88, wherein the additional therapeutic agent is an inhibitor
of indoleamine-2,3-dioxygenase (IDO).
96. The method of paragraph 95, wherein the inhibitor is selected from the group ting of
epacadostat, F001287, indoximod, and NLG919.
97. The method of paragraph 87 or 88, wherein the additional therapeutic agent is a vaccine.
98. The method of aph 97, wherein the vaccine comprises a heat shock protein peptide
x (HSPPC) comprising a heat shock protein complexed with an antigenic peptide.
99. The method of paragraph 98, wherein the heat shock protein is hsc70 and is complexed
with a tumor-associated antigenic peptide.
100. The method of paragraph 98, wherein the heat shock protein is gp96 protein and is
complexed with a tumor-associated antigenic peptide, n the HSPPC is derived from
a tumor obtained from a subject.
101. A method of treating an infectious disease in a subject, the method comprising
stering to the subject an effective amount of the antibody or pharmaceutical
composition of any one of paragraphs 1-47.
] Other embodiments are within the following claims.
Claims (29)
1. An isolated antibody that specifically binds to human CTLA-4, the antibody comprising a heavy chain variable region comprising mentarity determining regions CDRH1, CDRH2, and CDRH3 and a light chain variable region comprising complementarity 5 determining regions CDRL1, CDRL2, and CDRL3, wherein: (a) CDRH1 comprises the amino acid sequence of SYSMN (SEQ ID NO: 10); (b) CDRH2 comprises the amino acid sequence of SISSSSSYIYYAXSVKG (SEQ ID NO: 18), wherein X is E or D; (c) CDRH3 comprises the amino acid sequence of FDI (SEQ ID NO: 19), 10 wherein X is F or M; (d) CDRL1 comprises the amino acid sequence of SRYLG (SEQ ID NO: 15); (e) CDRL2 ses the amino acid sequence of GASTRAT (SEQ ID NO: 16); and (f) CDRL3 comprises the amino acid sequence of PWT (SEQ ID NO: 17), and wherein the CDRH1, CDRH2, and CDRH3 sequences of the antibody are not SEQ 15 ID NOs: 10, 11, and 13, respectively.
2. The isolated antibody of claim 1, wherein CDRH2 ses the amino acid sequence of SEQ ID NO: 12.
3. The isolated antibody of claim 1 or 2, wherein CDRH3 comprises the amino acid sequence of SEQ ID NO: 14.
4. An isolated dy that specifically binds to human CTLA-4, the antibody comprising a heavy chain variable region comprising complementarity determining s CDRH1, CDRH2, and CDRH3 and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, 5 CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 10, 12, 14, 15, 16, and 17, respectively.
5. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20.
6. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence which is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4-8.
7. The isolated antibody of claim 6, wherein the heavy chain variable region ses an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4-8.
8. The isolated antibody of claim 7, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8.
9. The isolated antibody of any one of the preceding claims, wherein the antibody comprises a light chain variable region comprising an amino acid sequence which is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:
10. The isolated antibody of claim 9, wherein the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9.
11. An isolated antibody that specifically binds to human CTLA-4, the antibody comprising a heavy chain variable region comprising an amino acid ce selected from the group consisting of SEQ ID NOs: 2-8.
12. The ed antibody of claim 11, wherein the heavy chain le region comprises the amino acid sequence of SEQ ID NO: 8.
13. The isolated antibody of claim 12, n the antibody comprises a heavy chain comprising an amino acid sequence ed from the group consisting of SEQ ID NOs: 23-26.
14. An isolated dy that ically binds to human CTLA-4, the antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth in SEQ ID NOs: 2 and 9; 3 and 9; 4 and 9; 5 and 9; 6 and 9; 7 and 9; or 8 and 9, 5 respectively.
15. The isolated antibody of claim 14, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth in SEQ ID NOs: 8 and 9, respectively.
16. An isolated antibody that specifically binds to human CTLA-4, the antibody comprising a heavy chain variable region having an amino acid sequence d from a human IGHV3- 21 germline sequence, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14.
17. An isolated antibody that ically binds to human CTLA-4, the dy comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 23, and a light chain comprising the amino acid sequence of SEQ ID NO: 27.
18. An isolated antibody that specifically binds to human CTLA-4, the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 24, and a light chain comprising the amino acid sequence of SEQ ID NO: 27.
19. An isolated antibody that ically binds to human CTLA-4, the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and a light chain comprising the amino acid sequence of SEQ ID NO: 27.
20. An isolated antibody that specifically binds to human CTLA-4, the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO: 27.
21. A pharmaceutical composition comprising the antibody of any one of the preceding claims and a ceutically acceptable carrier or ent.
22. An isolated polynucleotide encoding a heavy and/or light chain of the antibody of any one of claims 1-20.
23. A vector comprising the polynucleotide of claim 22.
24. A recombinant host cell comprising the polynucleotide of claim 22 or the vector of claim 23, with the proviso that said host cell is not present in a human being.
25. A method of producing an antibody that specifically binds to human CTLA-4, the method sing ing the host cell of claim 24 so that the polynucleotide is expressed and the antibody is produced.
26. A use of the antibody or pharmaceutical composition of any one of claims 1-21, in the manufacture of a medicament for increasing T cell activation in response to an antigen in a subject in need thereof.
27. A use of the antibody or ceutical composition of any one of claims 1-21, in the manufacture of a medicament for ng cancer in a subject in need thereof.
28. The use of claim 27, wherein the cancer is a metastatic or locally advanced tumor.
29. A use of the antibody or pharmaceutical composition of any one of claims 1-21, in the manufacture of a ment for treating an infectious disease in a subject in need thereof. (£961) H lsotype AGEN1884.H2.1 s... E] [3 (‘0 A (‘0 A I. I {5 E5 ‘3' .4: 2’ 0° C) V w G a) CD F a) v— o. 2 Q) Z? “J (D _ < a) 8 m s... S [I I: .30 E] E! .@ N w. A A N 55 I 235 I- 9 § .9 g v a) V (D ; EV- ‘éi gV- m LL w: 0 8 H MW“ g“ g} (<9 .52 3 G) E 3 . CID a) CH] no '— L‘: f 3 3 in 5 :H snrf i“ LL. T- N :3 E x— x (D V; 0 v 0') co 9 CO a) V" 3 E o. 2 a m L“ UJ < .. 3‘ o (D V'“ ‘ O 0 H 0') WW. ?‘ (I) < "" < 9 Wi3 5 CID as "¥ CHI! &0 {“{$!{S=‘${it:$t‘$2;§.‘ Ll- >¥l:i§>i€‘€)igat:§l:’; W0 06862 PCT/U
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US62/431,272 | 2016-12-07 |
Publications (1)
Publication Number | Publication Date |
---|---|
NZ794974A true NZ794974A (en) | 2022-12-23 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10912831B1 (en) | Anti-CTLA-4 antibodies and methods of use thereof | |
US11993651B2 (en) | Anti-lag-3 antibodies and methods of use thereof | |
NZ794974A (en) | Anti-ctla-4 antibodies and methods of use thereof | |
EA044966B1 (en) | ANTIBODIES AGAINST CTLA-4 AND METHODS OF THEIR APPLICATION | |
NZ751913B2 (en) | Anti-lag-3 antibodies and methods of use thereof | |
NZ792355A (en) | Anti-lag-3 antibodies and methods of use thereof | |
NZ792356A (en) | Anti-lag-3 antibodies and methods of use thereof | |
EA044026B1 (en) | ANTIBODIES AGAINST LAG-3 AND METHODS OF THEIR APPLICATION | |
NZ788279A (en) | Anti-tim-3 antibodies and methods of use thereof |