Anti-CTLA-4 antibodies and methods of use thereof

Abstract

The present disclosure provides antibodies that specifically bind to CTLA-4 (e.g., human CTLA-4) and antagonize the function of CTLA-4. Also provided are pharmaceutical compositions comprising the antibodies, nucleic acids encoding the antibodies, expression vectors and host cells for producing the antibodies, and methods of treating subjects using the antibodies.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201780085649.X, with an application date of December 7, 2017 and an invention name of “Anti-CTLA-4 Antibodies and Methods of Use Thereof”.

[0002] Related Applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 431,272, filed December 7, 2016, which is incorporated herein by reference in its entirety. Technical Field

[0004] The present disclosure relates to antibodies that specifically bind to CTLA-4 (eg, human CTLA-4) and methods of use thereof. Background Art

[0005] T lymphocytes are the key to the adaptive immune response to antigens. The complete activation of primary T cells requires at least two signals (Bretscher 1999, "Proc Natl Acad Sci USA" 96: 185-90). The first antigen-specific signal is provided by the interaction of the MHC / peptide complex on the T cell receptor (TCR) and the antigen presenting cell (APC). The second co-stimulatory signal is provided by the interaction between the receptor on the T cell and its ligand on the antigen presenting cell (APC). The engagement of both TCR / MHC and co-stimulatory interactions causes T cell activation via a variety of intracellular pathways, including calcium-calcineurin and RAS mitogen-activated protein kinase, and the subsequent activation of the transcription factor of a variety of effector compounds (including cytokines, such as IL-2). These events cause T cell proliferation, generate CD4+ helper T cells (TH) pools, and expand activated CD8+ cytotoxic T cells. Not only is co-stimulation critical for full T cell activation, but its absence during TCR / MHC engagement can lead to anergy and / or apoptosis.

[0006] Multiple positive and negative co-stimulatory pathways are involved in T cell regulation; however, the most critical is between CD28 on T cells and B7-1 (CD80) and B7-2 (CD86) on APC. CD28 promotes T cell differentiation into TH1 phenotype cells and enhances antibody production of B cells and activation of T cells. B7-1 and B7-2 expressed on APC (such as dendritic cells (DC) and B cells) have overlapping but unique functions. B7-2 is constitutively expressed and rapidly upregulated on APC in concert with TCR / MHC participation (signal 1). B7-1 expression is extremely low on resting cells, but is typically induced after prolonged T cell stimulation. These differences indicate that, although B7-2 may be important at the start of T cell activation, B7-1 may play a greater role in the continuation of immune responses.

[0007] Following 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 structurally homologous to CD28 but binds more tightly to both the B7-1 and B7-2 ligands. CTLA-4 inhibits immune responses in several ways: it competes with CD28 for B7 ligands and thereby blocks co-stimulation; it signals negatively to inhibit T cell activation; and it can capture CD80 and CD86 from opposing cells by trans-endocytosis, resulting in impaired co-stimulation 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).

[0008] Given the critical role of the B7 co-stimulatory pathway in promoting and maintaining immune responses, therapeutic agents designed to antagonize this pathway hold promise for the treatment of autoimmune diseases and disorders. Summary of the invention

[0009] The present disclosure provides antibodies that specifically bind to human CTLA-4 and antagonize CTLA-4 function (e.g., CTLA-4-mediated immunosuppression). Pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for producing these antibodies, and methods for treating subjects using these antibodies are also provided. The antibodies described herein are particularly useful for increasing T cell activation in response to antigens (e.g., tumor antigens or infectious disease antigens) and / or reducing Treg-mediated immunosuppression, and are therefore useful for treating cancer in subjects or for treating or preventing infectious diseases in subjects.

[0010] Thus, in one aspect, the present disclosure provides an isolated 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:

[0011] (a) CDRH1 comprises the amino acid sequence SYSMN (SEQ ID NO: 10);

[0012] (b) CDRH2 comprises the amino acid sequence SISSSSSYIYYAXSVKG (SEQ ID NO: 18), wherein X is E or D;

[0013] (c) CDRH3 comprises the amino acid sequence VGLXGPFDI (SEQ ID NO: 19), wherein X is F or M;

[0014] (d) CDRL1 comprises the amino acid sequence RASQSVSRYLG (SEQ ID NO: 15);

[0015] (e) CDRL2 comprises the amino acid sequence GASTRAT (SEQ ID NO: 16); and

[0016] (f) CDRL3 comprises the amino acid sequence QQYGSSPWT (SEQ ID NO: 17),

[0017] And wherein the CDRH1, CDRH2 and CDRH3 sequences of the antibody are not SEQ ID NOs: 10, 11 and 13, respectively.

[0018] In certain embodiments, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 11. In certain embodiments, 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 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: 10, 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, 15, 16, and 17; 10, 12, 13, 15, 16, and 17; or 10, 12, 14, 15, 16, and 17, respectively. In certain 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.

[0019] In another aspect, the present disclosure provides an isolated antibody that specifically 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:

[0020] (a) CDRH1 comprises the amino acid sequence SYSMN (SEQ ID NO: 10);

[0021] (b) CDRH2 comprises the amino acid sequence SISSSSSYIYYAXSVKG (SEQ ID NO: 18), wherein X is E or D;

[0022] (c) CDRH3 comprises the amino acid sequence VGLXGPFDI (SEQ ID NO: 19), wherein X is F or M;

[0023] (d) CDRL1 comprises the amino acid sequence RASQSVSRYLG (SEQ ID NO: 15);

[0024] (e) CDRL2 comprises the amino acid sequence GASTRAT (SEQ ID NO: 16); and

[0025] (f) CDRL3 comprises the amino acid sequence QQYGSSPWT (SEQ ID NO: 17),

[0026] And wherein the CDRH1, CDRH2 and CDRH3 sequences of the antibody are not SEQ ID NOs: 10, 11 and 13, respectively.

[0027] In certain embodiments, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 11. In certain embodiments, 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 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: 10, 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, 15, 16, and 17; 10, 12, 13, 15, 16, and 17; or 10, 12, 14, 15, 16, and 17, respectively. In certain 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.

[0028] In another aspect, the present disclosure provides an isolated antibody that specifically binds to human CTLA-4, the antibody comprising a heavy chain variable region comprising complementary determining regions CDRH1, CDRH2 and CDRH3 and a light chain variable region comprising complementary 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.

[0029] 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 that 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 NO: 2 and 4-8. In certain embodiments, the antibody comprises a heavy chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 3. In certain embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 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 antibody comprises a heavy chain comprising 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 certain embodiments, the antibody comprises a heavy chain comprising 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 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 the amino acid sequence of SEQ ID NO: 21).

[0030] In certain embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence 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 comprising the amino acid sequence of SEQ ID NO: 27. In certain 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 the amino acid sequence of SEQ ID NO: 22).

[0031] In certain embodiments, the present 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 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), 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 variable region having an amino acid sequence derived from a human IGKV3-20 germline sequence (e.g., IGKV3-20*01, e.g., having the amino acid sequence of SEQ ID NO: 22).

[0032] In another aspect, the present disclosure provides an isolated antibody that specifically binds to human CTLA-4, the antibody comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2-8. In certain embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence 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 NO: 23-26. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence SEQ ID NO: 23. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence SEQ ID NO: 24. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence SEQ ID NO: 25. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence SEQ ID NO: 26.

[0033] In another aspect, the present disclosure provides 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 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, respectively. In certain embodiments, the antibody comprises 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 sequence of SEQ ID NO: 9. In certain embodiments, 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 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 antibody comprises 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.

[0034] In another aspect, the present 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 derived from a human IGHV3-21*01 germline sequence (e.g., IGHV3-21*01, e.g., having the 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 the amino acid sequence of SEQ ID NO:22).

[0035] 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 embodiments, 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.

[0036] In certain embodiments, 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 embodiments, 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 sequence 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 certain embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:30. In certain embodiments, the amino acid sequence 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 antibody 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 an afucosylated IgG1.

[0037] 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 constant region binds to FcγRIIIA. In certain embodiments, the variant human IgG heavy chain constant region is a variant human IgG1 heavy chain constant region.

[0038] 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 SEQ ID NO:32.

[0039] In another aspect, the disclosure provides an isolated antibody that cross-competes with an antibody described herein for binding to human CTLA-4. In another aspect, the disclosure provides an isolated antibody that cross-competes with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 8 and 9, respectively, for binding to human CTLA-4.

[0040] In another aspect, the disclosure provides an isolated antibody that binds to the same epitope on human CTLA-4 as the antibodies described herein. In another aspect, the 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.

[0041] In another aspect, the present 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 sequence selected from the group consisting 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 an 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 an 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 an amino acid sequence of SEQ ID NO: 35. In certain embodiments, the antibody binds to an epitope located within a region of human CTLA-4 consisting of an 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 an 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 an amino acid sequence of SEQ ID NO: 39.

[0042] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to the same epitope of human CTLA-4 as any of the antibodies 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 consisting 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 an 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 an 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 an amino acid sequence of SEQ ID NO: 35. In certain embodiments, the antibody binds to an epitope located within a region of human CTLA-4 consisting of an 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 an amino acid sequence of SEQ ID NO: 38. In certain embodiments, the antibody binds to an epitope located within the region of human CTLA-4 consisting of the amino acid sequence of SEQ ID NO:39.

[0043] In another aspect, the disclosure provides an antibody that, when bound to a human CTLA-4 protein or a fragment thereof (e.g., an amino acid sequence comprising 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:34 relative to hydrogen / deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO:34 in the absence of the antibody, as determined by hydrogen / deuterium analysis. In another aspect, the disclosure provides an antibody that, when bound to a human CTLA-4 protein or a fragment thereof (e.g., an amino acid sequence comprising 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:35 relative to hydrogen / deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO:35 in the absence of the antibody, as determined by hydrogen / deuterium analysis. In another aspect, the disclosure provides an antibody that, when bound to a human CTLA-4 protein or a fragment thereof (e.g., an amino acid sequence comprising 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 a region consisting of the amino acid sequence set forth in SEQ ID NO:36 in the absence of the antibody, as determined by hydrogen / deuterium analysis. In another aspect, the disclosure provides an antibody that, when bound to a human CTLA-4 protein or a fragment thereof (e.g., an amino acid sequence comprising 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 a region consisting of the amino acid sequence set forth in SEQ ID NO:37 in the absence of the antibody, as determined by hydrogen / deuterium analysis. In another aspect, the disclosure provides an antibody that, when bound to a human CTLA-4 protein or a fragment thereof (e.g., an amino acid sequence comprising 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 hydrogen / deuterium analysis.In another aspect, the disclosure provides an antibody that, when bound to a human CTLA-4 protein or a fragment thereof (e.g., an amino acid sequence comprising 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: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 hydrogen / deuterium analysis.

[0044] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to the same epitope of human CTLA-4 as any of the antibodies of the present invention. In certain embodiments, the antibody, when bound to a human CTLA-4 protein or a fragment thereof (e.g., an amino acid sequence comprising 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:34 relative to hydrogen / deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO:34 in the absence of the antibody, as determined by hydrogen / deuterium analysis. In certain embodiments, the antibody, when bound to a human CTLA-4 protein or a fragment thereof (e.g., an amino acid sequence comprising 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:35 relative to hydrogen / deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO:35 in the absence of the antibody, as determined by hydrogen / deuterium analysis. In certain embodiments, the antibody, when bound to a human CTLA-4 protein or a fragment thereof (e.g., an amino acid sequence comprising residues 37-162 of SEQ ID NO:33), reduces hydrogen / deuterium exchange in the 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 hydrogen / deuterium analysis. In certain embodiments, the antibody, when bound to a human CTLA-4 protein or a fragment thereof (e.g., an amino acid sequence comprising residues 37-162 of SEQ ID NO:33), reduces hydrogen / deuterium exchange in the 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 hydrogen / deuterium analysis. In certain embodiments, the antibody, when bound to a human CTLA-4 protein or a fragment thereof (e.g., an amino acid sequence comprising residues 37-162 of SEQ ID NO:33), reduces hydrogen / deuterium exchange in the 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 hydrogen / deuterium analysis.In certain embodiments, the antibody, when bound to a human CTLA-4 protein or a fragment thereof (e.g., an amino acid sequence comprising residues 37-162 of SEQ ID NO:33), reduces hydrogen / deuterium exchange in the region consisting 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 hydrogen / deuterium analysis.

[0045] In certain embodiments, the antibody is a human antibody. In certain embodiments, the antibody is a bispecific antibody.

[0046] In certain embodiments, the antibody is antagonistic to human CTLA-4. In certain embodiments, the antibody inactivates, reduces or inhibits the activity of human CTLA-4. In certain embodiments, the antibody inhibits the binding of human CTLA-4 to human CD80 or human CD86. In certain embodiments, the antibody induces IL-2 production in peripheral blood mononuclear cells (PBMCs) stimulated with Staphylococcal enterotoxin A (SEA).

[0047] In certain embodiments, the antibody is conjugated to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label.

[0048] 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 converted to pyroglutamic acid.

[0049] In one embodiment, the invention relates to an antibody of the invention for use as a medicament.

[0050] In one embodiment, the present invention relates to a use of an antibody of the present invention for preparing a pharmaceutical composition or medicament for immunotherapy. In certain embodiments, the immunotherapy is used to increase T cell activation in response to an antigen in a subject, optionally for treating cancer, or treating or preventing an infectious disease.

[0051] In one embodiment, the invention relates to an antibody of the invention for use as a diagnostic agent.

[0052] In one embodiment, the present invention relates to a use of the antibody of the present invention for detecting human CTLA-4 in a biological sample in vitro.

[0053] In another aspect, the present disclosure provides a pharmaceutical composition comprising an anti-CTLA-4 antibody described herein and a pharmaceutically acceptable carrier or excipient.

[0054] In another aspect, the present disclosure provides an isolated polynucleotide encoding the heavy chain and / or light chain of the antibody described herein. In another aspect, the present disclosure provides a vector comprising the polynucleotide. In another aspect, the present disclosure provides a recombinant host cell comprising the polynucleotide or the vector. In another aspect, the present disclosure provides a method for producing an antibody that binds to human CTLA-4, the method comprising culturing the host cell so as to express the polynucleotide and produce the antibody.

[0055] In another aspect, the present disclosure provides a method of increasing T cell activation in a subject in response to an antigen, 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 present 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.

[0056] In certain embodiments, the subject suffers from cancer. In certain embodiments, the subject suffers from metastatic or locally advanced tumors (e.g., solid tumors). In certain embodiments, after the diagnosis of metastatic or locally advanced tumors (e.g., 1, 2, 3, 4, 5 or 6 days after diagnosis; 1, 2, 3, 4, 6, 8 or 12 weeks; or within 1, 2, 3, 4, 6, 8 or 12 months), cancer is treated as the first cancer therapy according to the methods described herein. In certain embodiments, after the diagnosis of tumor progression that occurred despite the previous treatment of tumors with different cancer therapies (e.g., 1, 2, 3, 4, 5 or 6 days after the diagnosis of tumor progression; 1, 2, 3, 4, 6, 8 or 12 weeks; or within 1, 2, 3, 4, 6, 8 or 12 months), cancer is treated as the first cancer therapy according to the methods described herein, optionally wherein the methods described herein are provided as the second cancer therapy administered. In certain embodiments, after the diagnosis of toxicity of different cancer therapies (e.g., within 1, 2, 3, 4, 5, or 6 days after the diagnosis of toxicity of different cancer therapies; 1, 2, 3, 4, 6, 8, or 12 weeks; or within 1, 2, 3, 4, 6, 8, or 12 months), cancer is treated according to the methods described herein as a first cancer therapy, optionally wherein the methods described herein are provided as a second cancer therapy administered. In certain embodiments, the cancer treated according to the methods described herein is a metastatic or locally advanced cancer (e.g., a solid tumor) for which no standard therapy is available. In other embodiments, the cancer treated according to the methods described herein is a metastatic or locally advanced cancer (e.g., a solid tumor) for which standard therapy has failed (i.e., after standard therapy, the cancer has progressed). In certain embodiments, if the cancer is refractory to therapy, then the therapy fails. In certain embodiments, if the cancer recurs after a complete or partial response to therapy, then the therapy fails. In certain embodiments, metastatic or locally advanced cancer (e.g., a solid tumor) has been diagnosed histologically or cytologically.

[0057] In certain embodiments, cancer expresses PD-L1. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in a cancer sample 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 that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in a cancer sample is at least 1%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in a cancer sample is at least 5%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in a cancer sample is at least 25%. In certain embodiments, the percentage of tumor cells in a cancer sample that exhibit detectable membrane expression (eg, partial or complete membrane expression) of PD-L1 is at least 50%.

[0058] In certain embodiments, metastatic or locally advanced tumors express PD-L1. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced tumor samples 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 that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced tumor samples is at least 1%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced tumor samples is at least 5%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced tumor samples is at least 25%. In certain embodiments, the percentage of tumor cells in a metastatic or locally advanced tumor sample that exhibit detectable membrane expression (eg, partial or complete membrane expression) of PD-L1 is at least 50%.

[0059] In certain embodiments, cancer is cervical cancer. In certain embodiments, cancer is metastatic or locally advanced cancer (e.g., solid tumor). In certain embodiments, metastatic or locally advanced cancer (e.g., solid tumor) is metastatic or locally advanced unresectable squamous cell carcinoma, adenosquamous carcinoma or cervical adenocarcinoma. In certain embodiments, no standard therapy is available for cancer (e.g., cervical cancer) or metastatic or locally advanced tumors (e.g., solid tumors). . In certain embodiments, cancer (e.g., cervical cancer) or metastatic or locally advanced tumors (e.g., solid tumors) are refractory to standard therapy. In certain embodiments, cancer (e.g., cervical cancer) or metastatic or locally advanced tumors (e.g., solid tumors) recur after standard therapy. In certain embodiments, standard therapy comprises platinum-containing chemotherapy. In certain embodiments, standard therapy is platinum-containing doublet. In certain embodiments, cancer (e.g., cervical cancer) is a metastatic or locally advanced unresectable squamous cell carcinoma, adenosquamous carcinoma or cervical adenocarcinoma recurring after platinum-containing dual therapy administered for the treatment of advanced (recurrent, unresectable or metastatic) disease. In certain embodiments, cancer (e.g., cervical cancer) or metastatic or locally advanced tumors are HPV positive. In certain embodiments, cancer or metastatic or locally advanced solid tumors are head and neck cancer, melanoma, renal cell carcinoma, urothelial carcinoma or endometrial cancer. In certain embodiments, cancer (e.g., cervical cancer) or metastatic or locally advanced solid tumors are associated with microsatellite instability.

[0060] In certain embodiments, the subject has cervical cancer (e.g., metastatic or locally advanced unresectable squamous cell carcinoma, adenosquamous carcinoma, or cervical adenocarcinoma), 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 a pharmaceutical composition comprising such an anti-CTLA-4 antibody as a first cancer therapy after diagnosis of 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 a pharmaceutical composition comprising such an anti-CTLA-4 antibody is administered to the subject as a first cancer therapy. S239D / A330L / I332E)) or a pharmaceutical composition comprising such an anti-CTLA-4 antibody is administered at a dose 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., metastatic or locally advanced unresectable squamous cell carcinoma, adenosquamous carcinoma, or cervical adenocarcinoma), 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., AGEN1884.H3 (IgG1S239D / A330L / I332E)) or comprising such an anti-CTLA-4 antibody and pembrolizumab after diagnosis of 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). A pharmaceutical composition for a first cancer therapy, optionally wherein an anti-CTLA-4 antibody described herein (e.g., AGEN1884.H3 (IgG1S239D / A330L / I332E)) or a pharmaceutical composition comprising such an anti-CTLA-4 antibody is administered at a dose 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.

[0061] In certain embodiments, the cancer is non-small cell lung cancer (NSCLC). In certain embodiments, NSCLC is stage IV NSCLC. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in NSCLC samples 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 that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in NSCLC samples is at least 1%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in NSCLC samples is at least 5%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in NSCLC samples is at least 25%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in the NSCLC sample is at least 50%. In certain embodiments, the NSCLC has no EGFR or ALK genomic tumor aberrations. In certain embodiments, metastatic or locally advanced NSCLC has no EGFR sensitizing mutations (e.g., mutations suitable for treatment with tyrosine kinase inhibitors, including erlotinib, gefitinib, or afatanib) or ALK translocations. In certain embodiments, the subject has not previously received systemic chemotherapy for NSCLC.

[0062] In certain embodiments, metastatic or locally advanced solid tumors are metastatic or locally advanced non-small cell lung cancer (NSCLC). In certain embodiments, metastatic or locally advanced solid tumors are metastatic non-small cell lung cancer (NSCLC). In certain embodiments, metastatic or locally advanced solid tumors are stage IV metastatic or locally advanced NSCLC. In certain embodiments, metastatic or locally advanced solid tumors are stage IV metastatic NSCLC. In certain embodiments, the percentage of tumor cells showing detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced NSCLC samples 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 showing detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced NSCLC samples is at least 1%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced NSCLC samples is at least 5%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced NSCLC samples is at least 25%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced NSCLC samples is at least 50%. In certain embodiments, metastatic or locally advanced NSCLC has no EGFR or ALK genomic tumor aberrations. In certain embodiments, the subject has not previously received systemic chemotherapy treatment for metastatic or locally advanced NSCLC.

[0063] In certain embodiments, the subject has NSCLC (e.g., stage IV metastatic or locally advanced NSCLC), optionally wherein the percentage of tumor cells in the NSCLC sample that exhibit detectable PD-L1 expression (e.g., membrane expression, partial or complete membrane expression) 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 an effective amount of an anti-CTLA-4 antibody described herein (e.g., AGEN1884.H3 (IgG1 S239D / A330L / I332E)) or a pharmaceutical composition comprising such an anti-CTLA-4 antibody as a first cancer therapy, optionally wherein an anti-CTLA-4 antibody described herein (e.g., AGEN1884.H3 (IgG1 S239D / A330L / I332E)) or a pharmaceutical composition comprising such an anti-CTLA-4 antibody is administered at a dose 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 NSCLC (e.g., stage IV metastatic or locally advanced NSCLC), optionally wherein the percentage of tumor cells in the NSCLC sample that exhibit detectable PD-L1 expression (e.g., membrane expression, partial or complete membrane expression) 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 drug comprising an anti-CTLA-4 antibody described herein (e.g., AGEN1884.H3 (IgG1 S239D / A330L / I332E)) or a pharmaceutical composition comprising such an anti-CTLA-4 antibody and pembrolizumab as a first cancer therapy, optionally wherein an anti-CTLA-4 antibody described herein (e.g., AGEN1884.H3 (IgG1 S239D / A330L / I332E)) or a pharmaceutical composition comprising such an anti-CTLA-4 antibody and pembrolizumab is administered at a dose 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.

[0064] In certain embodiments, the cancer is cutaneous squamous cell carcinoma (cSCC). In certain embodiments, the metastatic or locally advanced solid tumor is stage IV cutaneous squamous cell carcinoma (cSCC). In certain embodiments, the cSCC is diagnosed histologically or cytologically according to the eighth edition of the American Joint Committee on Cancer Staging Manual. In certain embodiments, cSCC cannot be cured with radiation therapy. In certain embodiments, the subject suffers from 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 (IgG1S239D / A330L / I332E)) or a pharmaceutical composition comprising such an anti-CTLA-4 antibody as a first cancer therapy after diagnosis of cSCC (e.g., 1, 2, 3, 4, 5, or 6 days after diagnosis; 1, 2, 3, 4, 6, 8, or 12 weeks; or 1, 2, 3, 4, 6, 8, or 12 months, Optionally wherein an anti-CTLA-4 antibody described herein (e.g., AGEN1884.H3 (IgG1S239D / A330L / I332E)) or a pharmaceutical composition comprising such an anti-CTLA-4 antibody is administered at a dose 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 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., AGEN1884.H3 (IgG1 S239D / A330L / I332E)) or a pharmaceutical composition comprising such an anti-CTLA-4 antibody and pembrolizumab as a first cancer therapy after diagnosis of 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)) is administered to the subject as a first cancer therapy. S239D / A330L / I332E)) or a pharmaceutical composition comprising such an anti-CTLA-4 antibody is administered at a dose 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.

[0065] In certain embodiments, the anti-CTLA-4 antibodies or pharmaceutical compositions described herein are administered intravenously. In certain embodiments, the anti-CTLA-4 antibodies or pharmaceutical compositions described herein are optionally administered intravenously at intervals of once every two weeks 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. In certain embodiments, the anti-CTLA-4 antibodies or pharmaceutical compositions described herein are optionally administered intravenously at intervals of once every three weeks 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. In certain embodiments, the anti-CTLA-4 antibodies or pharmaceutical compositions described herein are optionally administered intravenously at a time interval of once every four weeks 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. In certain embodiments, the anti-CTLA-4 antibodies or pharmaceutical compositions described herein are optionally administered intravenously at a time interval of once every six weeks 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. In certain embodiments, the anti-CTLA-4 antibodies or pharmaceutical compositions described herein are optionally administered intravenously at a time interval of once every twelve weeks 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. 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 optionally administered intratumorally at a time interval of once every three weeks 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 optionally administered intratumorally at a time interval of once every three weeks at 0.03 mg / kg, 0.1 mg / kg or 0.3 mg / kg. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered intratumorally at a dose that is up to 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or 200 times lower than the dose administered by systemic administration. In certain embodiments, an anti-CTLA-4 antibody or pharmaceutical composition described herein is administered intratumorally at a dose that is up to 10-fold lower than the dose given by systemic administration.In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered intratumorally at a dose up to 100 times lower than the dose given by systemic administration. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered as a monotherapy (e.g., intratumorally or systemically). In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered intratumorally, and the method further comprises administering an additional therapeutic agent to the subject. In certain embodiments, the additional therapeutic agent is administered systemically. In certain embodiments, the subject suffers from a solid tumor, and the additional therapeutic agent is an anti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody is pembrolizumab or nivolumab. In certain embodiments, pembrolizumab is administered at a dose of 200 mg every three weeks. In certain embodiments, the subject suffers from 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 suffers from 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 systemically administered. In certain embodiments, the chemotherapeutic agent is gemcitabine. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered intratumorally, and the subject suffers from advanced or metastatic solid tumors. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered intratumorally, and the subject suffers from head and neck cancer (e.g., recurrent / refractory head and neck squamous cell carcinoma). In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered intratumorally, and the subject suffers from breast cancer (e.g., recurrent / refractory HER2+ breast cancer). In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is delivered to tumor-draining lymph nodes. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is delivered via local administration (e.g., subcutaneous administration). In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is delivered via local administration (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 local administration (e.g., subcutaneous administration) at a dose that is up to 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or 200 times lower than the dose given by systemic administration.In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is delivered via local administration (e.g., subcutaneous administration) at a dose up to 10 times lower than the dose administered by systemic administration. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is delivered via local administration (e.g., subcutaneous administration) at a dose up to 100 times lower than the dose administered by systemic administration. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is delivered via local administration (e.g., subcutaneous administration), and the method further comprises administering 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 peptide complex (HSPPC) comprising a heat shock protein complexed with an antigenic peptide. In one embodiment, the heat shock protein is a gp96 protein and is complexed with a tumor-associated antigenic peptide, wherein the HSPPC is derived from a tumor obtained from a subject. In certain embodiments, the heat shock protein is selected from the group consisting of: hsc70, hsp70, hsp90, hsp110, grp170, gp96, calreticulin, a mutant thereof, and a combination 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 subject suffers from cancer. In certain embodiments, the subject suffers from an infectious disease. In certain embodiments, the methods further comprise administering an additional therapeutic agent to the subject. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent or a checkpoint targeting agent. In certain embodiments, the checkpoint targeting agent is selected from the group consisting of: antagonist anti-PD-1 antibodies, antagonist anti-PD-L1 antibodies, antagonist anti-PD-L2 antibodies, antagonist anti-CTLA-4 antibodies, antagonist anti-TIM-3 antibodies, antagonist anti-LAG-3 antibodies, antagonist anti-CEACAM1 antibodies, agonist anti-GITR antibodies, agonist anti-OX40 antibodies and agonist anti-CD137 antibodies, agonist anti-DR3 antibodies, agonist anti-TNFSF14 antibodies and agonist anti-CD27 antibodies. In certain embodiments, the additional therapeutic agent is radiotherapy. In certain embodiments, the additional therapeutic agent is an indoleamine-2,3-dioxygenase (IDO) inhibitor. Suitable IDO inhibitors include, but are not limited to, epacadostat, F001287, 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) comprising a heat shock protein complexed with an antigenic peptide. In one embodiment, the heat shock protein is a gp96 protein and is complexed with a tumor-associated antigenic peptide, wherein the HSPPC is derived from a tumor obtained from a subject.

[0066] In another aspect, the present disclosure provides a method for 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 herein. In another aspect, the present disclosure provides a method for preventing 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 herein.

[0067] In one embodiment, the present invention relates to an antibody of the present invention, a polynucleotide of the present invention, a vector of the present invention and / or a recombinant host cell of the present invention for use as a medicament.

[0068] In one embodiment, the present invention relates to an antibody of the present invention, a polynucleotide of the present invention, a vector of the present invention and / or a recombinant host cell of the present invention for use as a diagnostic agent.

[0069] In one embodiment, the present invention relates to a use of the antibody of the present invention, the polynucleotide of the present invention, the vector of the present invention and / or the recombinant host cell of the present invention for detecting human CTLA-4 in a biological sample in vitro.

[0070] In one aspect, provided herein is a pharmaceutical composition comprising an anti-CTLA-4 antibody described herein and a pharmaceutically acceptable carrier or excipient for use as a medicament.

[0071] In one aspect, provided herein is a pharmaceutical composition comprising an anti-CTLA-4 antibody described herein and a pharmaceutically acceptable carrier or excipient for use as a diagnostic agent.

[0072] In one aspect, a pharmaceutical composition is provided herein, comprising an anti-CTLA-4 antibody as 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 used as a medicament and / or a diagnostic agent.

[0073] In one aspect, the invention relates to an antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the invention for use in a method for increasing T cell activation in response to an antigen.

[0074] In one aspect, the invention relates to an antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the invention for use in a method of increasing T cell activation in a subject in response to an antigen.

[0075] In one aspect, the present invention relates to an antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the present invention, which is used in a method for increasing T cell activation in a subject in response to an antigen, the method comprising administering to the subject an effective amount of the antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the present invention.

[0076] 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 treating cancer.

[0077] In one aspect, the invention relates to an antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the invention for use in a method of treating cancer in a subject.

[0078] In one aspect, the present invention relates to an antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the present invention, which is used in a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of the antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the present invention.

[0079] 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, preferably an anti-PD-1 antibody, for use as a medicament.

[0080] 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, preferably an anti-PD-1 antibody, for use in a method for treating cancer. In a preferred embodiment, the cancer is a solid tumor. In another preferred embodiment, the antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the present invention is administered intratumorally to a subject, preferably, optionally at a time interval of once every three weeks, at 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg or 3 mg / kg.

[0081] 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.

[0082] In one aspect, the invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the invention, and (b) an anti-EGFR antibody, and optionally (c) a chemotherapeutic agent, for use as a medicament.

[0083] 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-EGFR antibody, and optionally (c) a chemotherapeutic agent, for use in a method for treating 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 a subject, preferably, optionally at a time interval of once every three weeks, at 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg or 3 mg / kg.

[0084] In one aspect, the 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 invention, and (b) an anti-EGFR antibody, and optionally (c) a chemotherapeutic agent.

[0085] In one aspect, the invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the invention, and (b) an anti-HER2 antibody, and optionally (c) a chemotherapeutic agent, for use as a medicament.

[0086] In one aspect, the present invention relates to (a) antibodies, polynucleotides, vectors, recombinant host cells and / or pharmaceutical compositions of the present invention, and (b) anti-HER2 antibodies, and optionally (c) chemotherapeutic agents, for use in methods for treating HER2+ breast cancer. In another preferred embodiment, the antibodies, polynucleotides, vectors, recombinant host cells and / or pharmaceutical compositions of the present invention are administered intratumorally to a subject, preferably, optionally at intervals of once every three weeks, at 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg or 3 mg / kg intratumorally to a subject.

[0087] In one aspect, the 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 invention, and (b) an anti-HER2 antibody, and optionally (c) a chemotherapeutic agent.

[0088] In one aspect, the present invention relates to an antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the present invention, which is used in a method for treating cancer, wherein the antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the present invention is administered intratumorally to a subject, preferably, optionally at a time interval of once every three weeks, at 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg or 3 mg / kg.

[0089] 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 treating cancer, wherein the antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the present invention is administered subcutaneously or intravenously to a subject, preferably, optionally at an interval of once every three weeks, 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.

[0090] 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 embodiment, the additional therapeutic agent is a chemotherapeutic agent or a checkpoint targeting agent or an indoleamine-2,3-dioxygenase (IDO) inhibitor or a vaccine.

[0091] 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 in a method for treating cancer. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic agent or a checkpoint targeting agent or an indoleamine-2,3-dioxygenase (IDO) inhibitor or a vaccine.

[0092] In one aspect, the present invention relates to a pharmaceutical composition, a kit or a kit of parts, comprising (a) an antibody, a polynucleotide, a vector, a recombinant host cell and / or a pharmaceutical composition 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 indoleamine-2,3-dioxygenase (IDO) inhibitor or a vaccine.

[0093] In one aspect, the invention relates to an antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the invention for use in a method for treating cancer, and / or for use in a method for increasing T cell activation in response to an antigen, wherein the antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the invention is delivered to a tumor-draining lymph node.

[0094] 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 invention in a method for treating cancer, and / or in a method for increasing T cell activation in a subject in response to an antigen, wherein the antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the present invention is delivered to a tumor-draining lymph node.

[0095] 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 invention for preparing a medicament for immunotherapy (e.g., for increasing T cell activation in a subject in response to an antigen, treating cancer, or treating or preventing an infectious disease).

[0096] 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 invention for preparing a medicament for immunotherapy (e.g., for increasing T cell activation in response to an antigen in a subject, treating cancer, or treating or preventing an infectious disease), wherein the antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the present invention is delivered to tumor-draining lymph nodes.

[0097] In one aspect, the present invention relates to a 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, for preparing a medicament for immunotherapy (e.g., for increasing T cell activation in a subject in response to an antigen, treating cancer, or treating or preventing an infectious disease).

[0098] In one aspect, the present invention relates to a 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, for preparing a medicament for immunotherapy (e.g., for increasing T cell activation in a subject in response to an antigen, treating cancer, or treating or preventing an infectious disease), wherein the antibody, polynucleotide, vector, recombinant host cell and / or pharmaceutical composition of the present invention is delivered to tumor-draining lymph nodes.

[0099] In particular, the present application relates to the following:

[0100] 1. An isolated antibody that specifically 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:

[0101] (a) CDRH1 comprises the amino acid sequence SYSMN (SEQ ID NO: 10);

[0102] (b) CDRH2 comprises the amino acid sequence SISSSSSYIYYAXSVKG (SEQ ID NO: 18), wherein X is E or D;

[0103] (c) CDRH3 comprises the amino acid sequence VGLXGPFDI (SEQ ID NO: 19), wherein X is F or M;

[0104] (d) CDRL1 comprises the amino acid sequence RASQSVSRYLG (SEQ ID NO: 15);

[0105] (e) CDRL2 comprises the amino acid sequence GASTRAT (SEQ ID NO: 16); and

[0106] (f) CDRL3 comprises the amino acid sequence QQYGSSPWT (SEQ ID NO: 17),

[0107] And wherein the CDRH1, CDRH2 and CDRH3 sequences of the antibody are not SEQ ID NOs: 10, 11 and 13, respectively.

[0108] 2. The isolated antibody according to item 1, wherein CDRH2 comprises the amino acid sequence of SEQ ID NO:12.

[0109] 3. The isolated antibody according to item 1 or 2, wherein CDRH3 comprises the amino acid sequence of SEQ ID NO:14.

[0110] 4. An isolated antibody that specifically binds to human CTLA-4, the antibody comprising a heavy chain variable region comprising complementary determining regions CDRH1, CDRH2 and CDRH3 and a light chain variable region comprising complementary 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.

[0111] 5. The isolated antibody according to item 1, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20.

[0112] 6. The isolated antibody of item 1, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence that 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.

[0113] 7. The isolated antibody of item 6, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 4-8.

[0114] 8. The isolated antibody according to item 7, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:8.

[0115] 9. An isolated antibody according to any of the preceding items, wherein the antibody comprises a light chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:9.

[0116] 10. The isolated antibody of item 9, wherein the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:9.

[0117] 11. An isolated antibody that specifically binds to human CTLA-4, said antibody comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8.

[0118] 12. The isolated antibody according to item 11, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8.

[0119] 13. The isolated antibody of item 12, wherein the antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-26.

[0120] 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 sequences set forth in SEQ ID NO: 2 and 9; 3 and 9; 4 and 9; 5 and 9; 6 and 9; 7 and 9; or 8 and 9, respectively.

[0121] 15. The isolated antibody according to item 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.

[0122] 16. An isolated antibody that specifically binds to human CTLA-4, said antibody comprising a heavy chain variable region having an amino acid sequence derived from a human IGHV3-21 germline sequence, wherein said heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:14.

[0123] 17. The isolated antibody of item 16, wherein the antibody comprises a light chain variable region having an amino acid sequence derived from a human IGKV3-20 germline sequence.

[0124] 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.

[0125] 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.

[0126] 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:25 and a light chain comprising the amino acid sequence of SEQ ID NO:27.

[0127] 21. 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.

[0128] 22. The isolated antibody according to any one of items 1 to 12 or 14 to 17, wherein the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

[0129] 23. The isolated antibody according to any one of items 1 to 12 or 14 to 17, wherein the antibody comprises an IgG1 heavy chain constant region.

[0130] 24. The isolated antibody of item 23, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 28.

[0131] 25. The isolated antibody according to item 23, wherein the amino acid sequence of the IgG1 heavy chain constant region comprises the S239D / I332E mutation, numbered according to the EU numbering system.

[0132] 26. The isolated antibody of item 25, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 29.

[0133] 27. The isolated antibody according to item 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.

[0134] 28. The isolated antibody of item 27, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 30.

[0135] 29. The isolated antibody according to item 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 numbering system.

[0136] 30. The isolated antibody of item 29, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 31.

[0137] 31. The isolated antibody according to item 23, wherein the IgG1 heavy chain constant region is afucosylated IgG1.

[0138] 32. An isolated antibody according to any one of items 1 to 12 or 14 to 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 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 constant region binds to FcγRIIIA.

[0139] 33. The isolated antibody of item 32, wherein the variant human IgG heavy chain constant region is a variant human IgG1 heavy chain constant region.

[0140] 34. The isolated antibody of any one of items 1 to 17 or 22 to 33, wherein the antibody comprises a light chain constant region selected from the group consisting of human Igκ and Igλ.

[0141] 35. The isolated antibody of item 34, wherein the antibody comprises an Ig kappa light chain constant region.

[0142] 36. The isolated antibody of item 35, wherein the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO:32.

[0143] 37. The isolated antibody according to any of the preceding items, 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 sequence of SEQ ID NO:9.

[0144] 38. The isolated antibody of any preceding claim, wherein the antibody binds to an epitope located within a region of human CTLA-4 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-39.

[0145] 39. An isolated antibody according to any preceding claim, wherein the antibody is a human antibody.

[0146] 40. The isolated antibody according to any of the preceding items, wherein the antibody is a bispecific antibody.

[0147] 41. The isolated antibody of any preceding claim, wherein the antibody is antagonistic to human CTLA-4.

[0148] 42. An isolated antibody according to any of the preceding claims, wherein the antibody inactivates, reduces or inhibits the activity of human CTLA-4.

[0149] 43. The isolated antibody of item 42, wherein the antibody inhibits the binding of human CTLA-4 to human CD80 or human CD86.

[0150] 44. The isolated antibody of item 42, wherein the antibody induces IL-2 production in peripheral blood mononuclear cells (PBMCs) stimulated with Staphylococcal enterotoxin A (SEA).

[0151] 45. An isolated antibody according to any preceding claim, conjugated to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide or a detectable label.

[0152] 46. ​​An isolated antibody according to any preceding claim, 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 pyroglutamic acid.

[0153] 47. A pharmaceutical composition comprising the antibody according to any one of the preceding claims and a pharmaceutically acceptable carrier or excipient.

[0154] 48. An isolated polynucleotide encoding the heavy chain and / or light chain of the antibody according to any one of items 1 to 45.

[0155] 49. A vector comprising the polynucleotide according to item 48.

[0156] 50. A recombinant host cell comprising the polynucleotide according to item 48 or the vector according to item 49.

[0157] 51. A method for producing an antibody that specifically binds to human CTLA-4, the method comprising culturing the host cell according to item 50 so as to express the polynucleotide and produce the antibody.

[0158] 52. A method of increasing T cell activation in a subject in response to an antigen, the method comprising administering to the subject an effective amount of the antibody or pharmaceutical composition according to any one of items 1 to 47.

[0159] 53. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the antibody or pharmaceutical composition according to any one of items 1 to 47.

[0160] 54. The method of item 53, wherein the cancer is a metastatic or locally advanced tumor.

[0161] 55. The method of item 53 or 54, wherein the cancer is a solid tumor.

[0162] 56. The method of any one of items 53 to 55, wherein the cancer is metastatic or locally advanced unresectable squamous cell carcinoma, adenosquamous carcinoma, or cervical adenocarcinoma.

[0163] 57. The method of any one of items 53 to 56, wherein no standard therapy is available for the cancer.

[0164] 58. The method of any one of items 53 to 56, wherein the cancer is refractory to standard therapy.

[0165] 59. The method of any one of items 53 to 56, wherein the cancer relapses after standard therapy.

[0166] 60. The method of item 58 or 59, wherein the standard therapy comprises platinum-containing chemotherapy.

[0167] 61. The method of item 60, wherein the standard therapy comprises platinum-containing doublet therapy.

[0168] 62. The method of any one of items 53 to 61, wherein the cancer is HPV positive.

[0169] 63. A method according to any one of items 53 to 55, wherein the cancer is non-small cell lung cancer (NSCLC).

[0170] 64. The method of item 63, wherein the cancer is metastatic NSCLC.

[0171] 65. The method of item 63 or 64, wherein the NSCLC does not have EGFR or ALK genomic tumor aberrations.

[0172] 66. The method according to any one of items 63 to 65, wherein the NSCLC does not have an EGFR sensitizing mutation or ALK translocation.

[0173] 67. The method of any one of items 63 to 66, wherein the subject has not previously been treated with systemic chemotherapy for the NSCLC.

[0174] 68. The method of any one of items 53 to 55, wherein the cancer is cutaneous squamous cell carcinoma (cSCC).

[0175] 69. The method of item 68, wherein the cancer is stage IV cSCC.

[0176] 70. The method according to item 69, wherein the cSCC is diagnosed histologically or cytologically according to the 8th edition of the American Joint Committee on Cancer Staging Manual.

[0177] 71. The method according to any one of items 68 to 70, wherein the cSCC is not curable with radiotherapy.

[0178] 72. The method of any one of items 53 to 71, wherein the percentage of tumor cells in the 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 90%.

[0179] 73. The method of any one of items 53 to 72, wherein the antibody is administered as a first cancer therapy following diagnosis of the cancer.

[0180] 74. The method of any one of items 53 to 73, wherein the antibody is administered as the first cancer therapy after:

[0181] (a) diagnosis of tumor progression that has occurred despite previous treatment of said cancer with a different cancer therapy; or

[0182] (b) diagnosis of toxicity of different cancer therapies,

[0183] Optionally wherein the antibody is a second cancer therapy administered to the subject.

[0184] 75. The method according to any one of items 52 to 75, wherein the antibody or pharmaceutical composition is administered intravenously.

[0185] 76. The method of item 75, wherein the antibody or pharmaceutical composition is optionally 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 at intervals of once every two weeks.

[0186] 77. The method of item 75, wherein the antibody or pharmaceutical composition is optionally 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 at intervals once every three weeks.

[0187] 78. The method of item 75, wherein the antibody or pharmaceutical composition is optionally 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 at intervals of once every four weeks.

[0188] 79. The method of item 75, wherein the antibody or pharmaceutical composition is optionally 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 at intervals of once every six weeks.

[0189] 80. The method of item 75, wherein the antibody or pharmaceutical composition is optionally 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 at intervals of once every twelve weeks.

[0190] 81. The method of any one of items 52 to 75, wherein the antibody or pharmaceutical composition is administered subcutaneously.

[0191] 82. The method of any one of items 53 to 75, wherein the antibody or pharmaceutical composition is administered intratumorally.

[0192] 83. The method of item 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 intervals of once every three weeks.

[0193] 84. The method of any one of items 52 to 75, wherein the antibody or pharmaceutical composition is delivered to tumor-draining lymph nodes.

[0194] 85. The method according to any one of items 81 to 84, wherein the antibody is the antibody according to any one of items 25 to 33.

[0195] 86. The method of any one of items 52 to 85, wherein the antibody is administered as a monotherapy.

[0196] 87. The method of any one of items 52 to 85, further comprising administering to the subject an additional therapeutic agent.

[0197] 88. The method of item 87, wherein the additional therapeutic agent is administered systemically.

[0198] 89. The method of item 87 or 88, wherein the additional therapeutic agent is an anti-PD-1 antibody, optionally wherein the anti-PD-1 antibody is pembrolizumab or nivolumab.

[0199] 90. The method of item 89, wherein the additional therapeutic agent is pembrolizumab administered at 200 mg every three weeks.

[0200] 91. The method of clause 87 or 88, wherein the subject has head and neck squamous cell carcinoma, and wherein the additional therapeutic agent is an anti-EGFR antibody, optionally wherein the anti-EGFR antibody is cetuximab,

[0201] Optionally wherein the method further comprises administering to said subject a chemotherapeutic agent, optionally wherein said chemotherapeutic agent is administered systemically, optionally wherein said chemotherapeutic agent is gemcitabine.

[0202] 92. The method of clause 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,

[0203] Optionally wherein the method further comprises administering to the subject a chemotherapeutic agent, optionally wherein the chemotherapeutic agent is administered systemically, optionally wherein the chemotherapeutic agent is gemcitabine.

[0204] 93. The method of item 87 or 88, wherein the additional therapeutic agent is a chemotherapeutic agent or a checkpoint targeting agent.

[0205] 94. The method of item 93, wherein the checkpoint targeting agent is selected from the group consisting of: antagonist anti-PD-1 antibody, antagonist anti-PD-L1 antibody, antagonist anti-PD-L2 antibody, antagonist anti-CTLA-4 antibody, antagonist anti-TIM-3 antibody, antagonist anti-LAG-3 antibody, antagonist anti-CEACAM1 antibody, agonist anti-GITR antibody, agonist anti-OX40 antibody and agonist anti-CD137 antibody.

[0206] 95. The method of item 87 or 88, wherein the additional therapeutic agent is an indoleamine-2,3-dioxygenase (IDO) inhibitor.

[0207] 96. The method of item 95, wherein the inhibitor is selected from the group consisting of epacadostat, F001287, indoximod, and NLG919.

[0208] 97. The method of item 87 or 88, wherein the additional therapeutic agent is a vaccine.

[0209] 98. The method of item 97, wherein the vaccine comprises a heat shock protein peptide complex (HSPPC) comprising a heat shock protein complexed with an antigenic peptide.

[0210] 99. The method of item 98, wherein the heat shock protein is hsc70 and is complexed with a tumor-associated antigenic peptide.

[0211] 100. The method of item 98, wherein the heat shock protein is gp96 protein and is complexed with a tumor-associated antigenic peptide, wherein the HSPPC is derived from a tumor obtained from a subject.

[0212] 101. A method of treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of the antibody or pharmaceutical composition according to any one of items 1 to 47. BRIEF DESCRIPTION OF THE DRAWINGS

[0213] Figure 1A , 1B , 1C, 1D, 1E, 1F, and 1G are flow cytometry histograms showing binding of anti-CTLA-4 antibodies or IgG1 isotype control antibodies to Jurkat cells engineered to express human CTLA-4 on the cell surface. The anti-CTLA-4 antibodies tested were: AGEN1884.H1.1 (IgG1), AGEN1884.H1.2 (IgG1), AGEN1884.H1.3 (IgG1), AGEN1884.H2.1 (IgG1), AGEN1884.H2.2 (IgG1), AGEN1884.H2.3 (IgG1), and AGEN1884.H3 (IgG1).

[0214] Figure 2Is a graph showing IL-2 production by primary human PBMCs after incubation with Staphylococcal enterotoxin A (SEA) superantigen suboptimal stimulation in the absence or presence of anti-CTLA-4 antibody AGEN1884.H3 (IgG1) or isotype control antibody (IgG1). Eight replicates were performed for each group, and the average of the eight replicates is indicated with a black bar.

[0215] Figure 3 is a graph showing the results of an IL-2-luciferase reporter assay demonstrating that blocking CTLA-4 results in T cell activation. The fold response of luciferase expression (a surrogate marker of IL-2 gene activation) is plotted relative to a range of antibody concentrations of AGEN1884.H3 (IgG1) or an isotype control antibody (IgG1).

[0216] Figure 4 is a graph showing the results of a reporter assay, in which simultaneous engagement of AGEN1884.H3 (IgG1) with target cells (via CTLA-4 binding) and effector cells (via FcγRIIIA binding) triggers expression of luciferase by an effector cell line. Luciferase activity is a surrogate marker for FcγRIIIA signaling. Multiple responses of RLU values ​​are plotted relative to a range of antibody concentrations of AGEN1884.H3 (IgG1) and isotype control antibody (IgG1).

[0217] Figure 5A , 5B , 5C and 5D are flow cytometry histograms showing Jurkat cells expressing CTLA-4 incubated with anti-CTLA-4 antibodies AGEN1884.H3(IgG1), AGEN1884.H3(IgG1S239D / I332E), AGEN1884.H3(IgG1S239D / A330L / I332E) or AGEN1884.H3(IgG1L235V / F243L / R292P / Y300L / P396L) or isotype control antibodies. Antibody binding was detected using a fluorescent dye-conjugated secondary antibody.

[0218] Fig. 6A and 6BFigures showing the blocking of binding between human CTLA-4 and its ligands CD80 and CD86 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), reference anti-CTLA-4 antibody or isotype control antibody (IgG1), and then stained with designated fluorescently labeled ligands. Ligand binding was then analyzed by flow cytometry.

[0219] Fig. 7A , 7B and 7C are graphs showing IL-2 production by primary human PBMCs cultured in the absence or presence of isotype control antibody (IgG1) or anti-CTLA-4 antibody under suboptimal stimulation with SEA superantigen. Fig. 7A and 7B is a graph showing IL-2 production stimulated by a single dose or dose titration of isotype control antibody (IgG1) or anti-CTLA-4 antibodies AGEN1884.H3(IgG1), AGEN1884.H3(IgG1 S239D / I332E), AGEN1884.H3(IgG1S239D / A330L / I332E), and AGEN1884.H3(IgG1L235V / F243L / R292P / Y300L / P396L). Figure 7B In the studies shown in , cells in each sample were incubated with an IgG4 S228P isotype control antibody in addition to an isotype control antibody (IgG1) or an anti-CTLA-4 antibody. Figure 7C is a graph showing IL-2 production stimulated by dose-titrated isotype control antibody (IgG1) or anti-CTLA-4 antibodies AGEN1884(IgG1), AGEN1884(IgG1 S239D / I332E), AGEN1884(IgG1 S239D / A330L / I332E), and afucosylated AGEN1884(IgG1).

[0220] Fig. 8A Immunoblot analysis of phosphorylated ZAP70 (Y493) in human PBMCs after stimulation with 50 ng / ml SEA superantigen and 10 μg / ml isotype control antibody (IgG1) or anti-CTLA-4 antibodies AGEN1884.H3(IgG1), AGEN1884.H3(IgG1 S239D / A330L / I332E) or AGEN1884.H3(IgG1 N297A). Figure 8B It is a display Fig. 8AGraph of normalized densitometric analysis of the data shown in .

[0221] Fig.9A , 9B , 9C and 9D are graphs showing the anti-tumor efficacy and intratumoral regulatory T cell (Treg) depletion induced by the Fc variant of the murine anti-CTLA-4 antibody 9D9.

[0222] Fig.9A Tumor growth in CT26 mice after single-dose treatment with murine anti-CTLA-4 antibody 9D9 (mIgG2a), Fc-static variant of anti-CTLA-4 antibody 9D9 (mIgG2a-N297A), Fc variant of anti-CTLA-4 antibody 9D9 (mIgG2a-S239D / A330L / I332E), or isotype control antibody (mIgG2a) is shown. The upper figure shows the median tumor volume over time for each treatment group. The remaining figures show the tumor volume over time for individual animals in each treatment group. Fig. 9B The effect of anti-CTLA-4 antibody treatment on T cell populations from tumor infiltrates collected from mice treated with a single dose of anti-CTLA-4 antibody 9D9 (mIgG2a), anti-CTLA-4 antibody 9D9 (mIgG2a-N297A), anti-CTLA-4 antibody 9D9 (mIgG2a-S239D / A330L / I332E) or isotype control antibody (mIgG2a) is shown. Tumor infiltrates were harvested at designated time points after injection of the antibody and analyzed by flow cytometry. The cell populations analyzed included: FoxP3+Treg (upper left), CD45+ leukocytes (upper right), and CD4+ non-Treg (lower left). The lower right figure shows the ratio of CD8+T cells to Treg observed in tumor infiltrates. Fig. 9C Shows how Fig. 9B FoxP3+ Treg populations over time in tumor-draining lymph nodes harvested from mice treated as described. Fig.9D Shows how Fig. 9B Fold changes in splenic FoxP3+ Tregs at 72 hours following the described treatments.

[0223] Fig.10It is a series of figures showing the anti-tumor efficacy of murine anti-CTLA-4 antibodies when combined with tumor vaccines (viral antigens E6 / E7) derived from HPV+ tumors. Tumor volumes over time for individual mice that received no treatment, an isotype control antibody (mIgG2a), anti-CTLA-4 antibody 9D9 (mIgG2a), or an Fc variant of anti-CTLA-4 antibody 9D9 (mIgG2a-S239D / A330L / I332E) are shown. The top row of figures shows the results of animals treated with the specified antibody alone. The bottom row of figures shows the results of animals treated with a combination of the specified antibody and tumor vaccine.

[0224] Fig.11A and 11B A graph showing gene expression and CpG methylation of a human T cell population. + CD25 + / - FOXP3 - Non-regulatory T cells (Teff) and CD4 + CD25 + FOXP3 + Regulatory T cells (Tregs) were isolated from the peripheral blood of healthy donors, expanded and activated. Fig.11A Shown are the levels of FOXP3, intracellular CTLA-4, and membrane CTLA-4 in each activated T cell population as determined by flow cytometry. Fig. 11B Shown are the levels of CpG methylation in CpG regions within the FOXP3 (upper panel) and CTLA4 (lower panel) loci in naive T cells, activated effector T cells, and activated regulatory T cells, each from the same donor.

[0225] Fig. 12A and 12B It is a graph showing the time course of antibody-dependent cellular cytotoxicity (ADCC) of human CTLA-4+ target cells after incubation with anti-CTLA-4 antibody AGEN1884.H3 (IgG1) or its Fc variants. NK-92 cells (expressing FcγRIIIA V158) were co-cultured with CTLA-4+ target cells, which were incubated with different Fc variants of anti-CTLA-4 antibodies or IgG1 isotype control (10 μg / ml). High-content microscopy of caspase 3 / 7 activation was then used to quantify ADCC activity. Fig. 12AADCC activity is demonstrated in Jurkat cells engineered to express CTLA-4 on the cell surface when incubated with 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). Fig. 12B ADCC activity in primary human activated effector T cells (left panel) or regulatory T cells (right panel) when incubated with these antibodies is shown.

[0226] Fig.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 anti-PD-1 antibodies. Human PBMCs were isolated from two donors and incubated under stimulating culture conditions with anti-CTLA-4 antibody AGEN1884.H3 (IgG1), Fc variant anti-CTLA-4 antibody AGEN1884.H3 (IgG1S239D / A330L / I332E) or a combination of isotype control antibody (IgG1) and reference anti-PD-1 antibody or isotype control antibody (IgG4), as indicated. For each treatment condition listed, dose 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, with 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 Fig.13A (repeat 1) and Fig. 13B (Replicate 2). The levels of IL-2 production induced by each antibody combination on PBMCs collected from the second donor are shown in Fig. 13C (repeat 1) and Fig.13D (Repeat 2).

[0227] Fig.14A , 14B and 14C is a series of sequence alignments. Fig.14A : is a sequence alignment of human CTLA-4 (SEQ ID NO: 33), cynomolgus macaque CTLA-4 (SEQ ID NO: 40), mouse CTLA-4 (SEQ ID NO: 41), and rat CTLA-4 (SEQ ID NO: 42). Dots indicate residues that are identical to the corresponding human residues. "*" (asterisk) indicates positions with a single completely conserved residue. ":" (colon) indicates conservation between groups with strongly similar properties. "." (period) indicates conservation between groups with weakly similar properties. Fig. 14B and 14C is a sequence alignment of human CTLA-4 (residues 1-144 and residues 145-223, respectively, of SEQ ID NO:33), cynomolgus macaque CTLA-4 (residues 1-144 and residues 145-223, respectively, of SEQ ID NO:40), human CD28 (residues 1-127 and residues 128-220, respectively, of SEQ ID NO:43), human ICOS (residues 1-124 and residues 125-199, respectively, of SEQ ID NO:44), human BTLA (residues 1-125 and residues 126-289, respectively, of SEQ ID NO:45), and human PD-1 (residues 1-143 and residues 144-288, respectively, of SEQ ID NO:46). Two regions showing a strong reduction in deuterium uptake when human CTLA-4 is bound to AGEN1884-Fab are Figures 14A-14C Underlined: residues 80-82 (QVT, SEQ ID NO:39) and residues 135-149 (YPPPYYLGIGNGTQI, SEQ ID NO:37), numbered according to SEQ ID NO:33. DETAILED DESCRIPTION

[0228] The present disclosure provides antibodies that specifically bind to CTLA-4 (e.g., human CTLA-4) and antagonize CTLA-4 function (e.g., CTLA-4-mediated immunosuppression). Pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for producing these antibodies, and methods of treating subjects using these antibodies are also provided. The antibodies described herein are particularly useful for increasing T cell activation in response to antigens (e.g., tumor antigens or infectious disease antigens), and are therefore useful for treating cancer in subjects or treating or preventing infectious diseases in subjects. All instances of "isolated antibodies" described herein are additionally encompassed as antibodies that may but need not be isolated. All instances of "isolated polynucleotides" described herein are additionally encompassed as polynucleotides that may but need not be isolated. All instances of "antibodies" described herein are additionally encompassed as antibodies that may but need not be isolated. All instances of "polynucleotides" described herein are additionally encompassed as polynucleotides that may but need not be isolated.

[0229] Skilled workers will appreciate that under certain conditions, the glutamic acid (E) or glutamine (Q) amino acid residue at the N-terminus of the heavy chain variable region and / or light chain variable region of any of the antibodies described herein (e.g., anti-CTLA4 antibodies) can be spontaneously converted to pyroglutamic acid by post-translational cyclization of the free amino group to form a lactam. Thus, in certain embodiments of each of the methods, uses, pharmaceutical compositions, or kits described herein, the N-terminal amino acid residue of one or more heavy chain variable regions and / or light chain variable regions of the antibody has been converted to pyroglutamic acid (e.g., as a result of post-translational cyclization of the free amino group of the N-terminal E or Q residue).

[0230] 1.1 Definition

[0231] As used herein, the terms "about" and "approximately" when used to modify a numerical value or numerical range, indicate that deviations of 5% to 10% above (e.g., as much as 5% to 10% above) and 5% to 10% below (e.g., as much as 5% to 10% below) the stated value or range remain within the intended meaning of the stated value or range.

[0232] As used herein, the term "CTLA-4" refers to cytotoxic T lymphocyte-associated protein 4. As used herein, the term "human CTLA-4" refers to the human CTLA-4 protein encoded by the wild-type human CTLA-4 gene, such as GenBank TM 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.

[0233] 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 include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heterologous conjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In certain embodiments, the antibodies described herein refer to polyclonal antibody populations. The antibodies can be of any type of immunoglobulin molecule (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., IgG 2a or IgG 2b ). In certain embodiments, the antibodies described herein are IgG antibodies, or their class (e.g., human IgG1 or IgG4) or subclasses. In a specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody.

[0234] As used herein, the terms "VH region" and "VL region" refer to a single antibody heavy chain and light chain variable region comprising FR (framework regions) 1, 2, 3 and 4 and CDR (complementarity determining regions) 1, 2 and 3, respectively (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is incorporated herein by reference in its entirety).

[0235] As used herein, the term "CDR" or "complementarity determining region" means the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977); and Kabat et al., Sequences of Proteins of Immunological Interest. (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); and MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), all of which are incorporated herein by reference in their entirety, wherein definitions include overlaps or subgroups of amino acid residues when compared relative to each other. In certain embodiments, the term "CDR" is a CDR as defined by Kabat et al., Journal of Biological Chemistry 252, 6609-6616 (1977) and Kabat et al., Sequences of Proteins of Immunological Interest. (1991). In certain embodiments, the term "CDR" is a CDR as defined by Chothia et al., Journal of Mol. Biol. 196: 901-917 (1987). In certain embodiments, the term "CDR" is a CDR as defined by MacCallum et al., J. Molecular Biology 262:732-745 (1996) and Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains", in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

[0236] As used herein, the term "framework (FR) amino acid residues" refers to those amino acids in the framework region of an immunoglobulin chain. As used herein, the term "framework region" or "FR region" includes amino acid residues that are part of the variable region but not part of the CDR (e.g., using the Kabat or Chothia definition of CDR).

[0237] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a part of an antibody, usually a part of a light chain or a heavy chain, typically about 110 to 125 amino acids at the amino terminus in a mature heavy chain and about 90 to 115 amino acids in a mature light chain, which are very different in sequence among antibodies and are used for the binding and specificity of a specific antibody to its specific antigen. Sequence variability is concentrated in those regions referred to as complementary determining regions (CDRs), while the more highly conserved regions in the variable domains are referred to as framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of light and heavy chains are primarily responsible for the interaction and specificity of antibodies to antigens. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In a specific embodiment, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable regions comprise rodent or murine CDRs and primate (eg, non-human primate) framework regions (FRs).

[0238] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0239] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0240] As used herein, the terms "constant region" and "constant domain" are interchangeable and common in the art. The constant region is the portion of an antibody that is not directly involved in the binding of an antibody to an antigen but can exhibit various effector functions (such as interactions with Fc receptors (e.g., Fcγ receptors), e.g., the carboxyl terminal portion of a light chain and / or a heavy chain. The constant region of an immunoglobulin molecule typically has an amino acid sequence that is more conservative relative to an immunoglobulin variable domain.

[0241] As used herein, the term "heavy chain" when used in reference to an antibody can refer to any unique type based on the amino acid sequence of the constant domains, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including the subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0242] As used herein, the term "light chain" when used with respect to an antibody can refer to any unique type based on the amino acid sequence of the constant domain, for example, kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art.

[0243] As used herein, the term "EU numbering system" refers to the EU numbering convention for constant regions of antibodies as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 5th Edition, 1991, each of which is incorporated herein by reference in its entirety.

[0244] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (K D Affinity can be measured and / or expressed in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant (K D ) and equilibrium association constant (K A ). D From k off / k on The quotient of K A From k on / k off Calculate the quotient of k on refers to, for example, the rate constant for the association of an antibody with an antigen, and k off Refers to, for example, the dissociation rate constant of an antibody from an antigen. on and k off It can be done by techniques known to those skilled in the art, such as As used herein, "lower affinity" refers to a greater K D .

[0245] As used herein, the terms "specific binding", "specific recognition", "immunospecific binding", and "immunospecific recognition" are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope or an immune complex) as such binding is understood by those skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, typically with lower affinity, as determined by, for example, immunoassays, The K of a molecule that specifically binds to an antigen is determined by a KinEx A3000 instrument (Sapidyne Instruments, Boise, ID) or other analytical methods known in the art. In a specific embodiment, the K of a molecule that specifically binds to an antigen is calculated as the K of a molecule that nonspecifically binds to another antigen. A At least 2 logs (i.e., a factor of 10), 2.5 logs, 3 logs, 4 logs, or more of K A Binds to antigen.

[0246] In another specific embodiment, the molecule that specifically binds to the antigen does not cross-react with other proteins under similar binding conditions. In another specific embodiment, the molecule that specifically binds to CTLA-4 does not cross-react with other non-CTLA-4 proteins. In a specific embodiment, there is provided herein an antibody that binds to CTLA-4 (e.g., human CTLA-4) with a higher affinity than binding to another unrelated antigen. In certain embodiments, there is provided herein an antibody that binds to CTLA-4 (e.g., human CTLA-4) with an affinity higher than binding to another unrelated antigen of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, as measured according to, for example, radioimmunoassay, surface plasmon resonance or dynamic exclusion analysis. 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 the CTLA-4 protein as measured by, e.g., radioimmunoassay.

[0247] As used herein, the term "afucosylation" or "afucosylated" in the context of Fc refers to substantially no fucose covalently attached, directly or indirectly, to residue 297 (numbered according to the EU numbering system) of the human IgG1 Fc region, or the corresponding residue in a non-IgG1 or non-human IgG1 immunoglobulin. Thus, in a composition comprising a plurality of afucosylated antibodies, at least 70% of the antibodies are not fucosylated, directly or indirectly (e.g., via intercalated sugars), at residue 297 of the Fc region of the antibody, and in some embodiments, at least 80%, 85%, 90%, 95%, or 99% are not fucosylated, directly or indirectly, at residue 297 of the Fc region.

[0248] As used herein, "epitope" is a term in the art and refers to a local antigenic region that can be specifically bound to an antibody. An epitope can be, for example, a continuous amino acid (linear or continuous epitope) of a polypeptide, or an epitope can be, for example, assembled from two or more discontinuous regions of one or more polypeptides (conformation, nonlinear, discontinuous or discontinuous epitopes). In certain embodiments, the epitope to which an antibody is combined can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA analysis, hydrogen / deuterium exchange coupled mass spectrometry (for example, liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning analysis (for example, using CLIPS (peptide chemistry is bonded to the skeleton) to locate the restricted peptides of discontinuous or conformational epitopes) and / or mutagenesis location (for example, site-directed mutagenesis location). In X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al., (1994) Acta Crystallographica Sin. D: Biological Crystallography (Acta Crystallogr DBiol Crystallogr)", 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) Journal of Biological Chemistry 251:6300-6303, all of which are incorporated herein by reference in their entirety). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) Vols. 114 & 115, Wyckoff HW et al., eds.; US2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallographica Sin. D: Biological Crystallography 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol. 276A:361-423, Carter CW, ed.; Roversi P et al., (2000) Acta Crystallographica Sin. D: Biological Crystallography 56(Pt 10):1316-1323), all of which are incorporated herein by reference in their entirety. Mutagenesis site-directed studies can be accomplished using any method known to those skilled in the art.For a description of mutagenesis techniques (including alanine scanning mutagenesis techniques), see, for example, Champe M et al., (1995) Journal of Biological Chemistry 270: 1388-1394 and Cunningham BC and Wells JA (1989) Science 244: 1081-1085, each of which is incorporated herein by reference in its entirety. CLIPS (peptide chemical bonding to the backbone) is a technique for presenting one or more peptides in a structurally constrained configuration to be expressed as a functional mimetic of a complex protein domain. See, for example, U.S. Publication No. US2008 / 0139407 A1 and No. US2007 / 099240 A1 and U.S. Patent No. 7,972,993, each of which is incorporated herein by reference in its entirety. In a specific embodiment, the epitope of the antibody is determined using alanine scanning mutagenesis studies. In a specific embodiment, the epitope of the antibody is determined using hydrogen / deuterium exchange coupled mass spectrometry. In a specific embodiment, the epitope of the antibody is determined using CLIPS epitope mapping technology from Pepscan Therapeutics.

[0249] As used herein, the term "epitope located within a region of human CTLA-4" consisting of a specific amino acid sequence or a group of amino acid residues refers to an epitope that includes one or more of the amino acid residues in the specified region, wherein the specified region includes the first specified amino acid residue and the last specified amino acid residue of the region of human CTLA-4. In certain embodiments, the epitope includes each 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 are bound to the antibody together with the epitope located within the specified region.

[0250] As used herein, the terms "T cell receptor" and "TCR" are used interchangeably and refer to full-length heterodimeric αβ or γδ TCR, antigen binding fragments of full-length TCR, and molecules comprising TCR CDR or variable regions. Examples of TCRs include, but are not limited to, full-length TCRs, antigen binding fragments of full-length TCRs, soluble TCRs without transmembrane and cytoplasmic regions, single-chain TCRs containing variable regions of TCRs connected by flexible linkers, TCR chains connected by engineered disulfide bonds, monospecific TCRs, multispecific TCRs (including bispecific TCRs), TCR fusions, human TCRs, humanized TCRs, chimeric TCRs, TCRs produced recombinantly, and synthetic TCRs. The term encompasses wild-type TCRs and genetically engineered TCRs (e.g., chimeric TCRs comprising chimeric TCR chains, which include a first portion of a TCR from a first species and a second portion of a TCR from a second species).

[0251] As used herein, the terms "major histocompatibility complex" and "MHC" are used interchangeably and refer to MHC class I molecules and / or MHC class II molecules.

[0252] As used herein, the term "peptide-MHC complex" refers to an MHC molecule (MHC class I or MHC class II) with a peptide bound to the art-recognized peptide binding pocket of the MHC.

[0253] As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or preventive measures as described herein. "Therapeutic" methods employ the administration of an antibody to a subject having a disease or condition or susceptible to such a disease or condition in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or condition or a recurring disease or condition, or to prolong the subject's survival beyond what would be expected in the absence of such treatment.

[0254] As used herein, the term "effective amount," in the context of administering a therapy to a subject, refers to that amount of the therapy that achieves the desired prophylactic or therapeutic effect.

[0255] As used herein with respect to the response of a cancer to therapy, the terms "refractory" and "resistant" have their art-recognized meanings and are used interchangeably.

[0256] As used herein, the term "subject" includes any human or non-human animal. In one embodiment, the subject is a human or non-human mammal. In one embodiment, the subject is a human.

[0257] The determination of "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be achieved using a mathematical algorithm. A specific non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin S and Altschul SF (1990) PNAS 87:2264-2268, as modified in Karlin S and Altschul SF (1993) PNAS 90:5873-5877, each of which is incorporated herein by reference in its entirety. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) Journal of Molecular Biology 215:403, which are incorporated herein by reference in their entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameter set, e.g., score = 100, word length = 12, to obtain nucleotide sequences homologous to nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameter set, e.g., score 50, word length = 3, to obtain amino acid sequences homologous to protein molecules described herein. In order 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 incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterative search, which detects long-range relationships between molecules (ibid). When utilizing BLAST, gapped BLAST, and PSI Blast programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the World Wide Web ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4: 11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 may be used.

[0258] The percent identity between two sequences can be determined using techniques similar to those described above with or without allowing gaps. In calculating the percent identity, typically only exact matches are counted.

[0259] 1.2 Anti-CTLA-4 Antibodies

[0260] In one aspect, the disclosure provides antibodies that specifically bind to CTLA-4 (e.g., human CTLA-4) and antagonize CTLA-4 function. The amino acid sequences of exemplary antibodies are set forth in Tables 1-4 herein.

[0261] Table 1. Amino acid sequences of exemplary anti-CTLA-4 antibodies.*

[0262]

[0263]

[0264]

[0265]

[0266]

[0267] *CDRs are defined according to the Kabat numbering system.

[0268] Table 2. Heavy chain CDR amino acid sequences of exemplary anti-CTLA-4 antibodies.*

[0269]

[0270] *Defined according to the Kabat numbering system.

[0271] Table 3. Light chain CDR amino acid sequences of exemplary anti-CTLA-4 antibodies.*

[0272]

[0273] *Defined according to the Kabat numbering system.

[0274] Table 4. Exemplary anti-CTLA-4 antibodies.

[0275]

[0276] Table 5. Closest germline genes.

[0277]

[0278] Table 6. Exemplary sequences of CTLA-4 and family members.

[0279]

[0280]

[0281] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a VH domain comprising one, two, or all three of the CDRs of the VH domains described herein in Table 1. In certain embodiments, the antibody comprises a CDRH1 of one of the VH domains described in Table 1. In certain embodiments, the antibody comprises a CDRH2 of one of the VH domains described in Table 1. In certain embodiments, the antibody comprises a CDRH3 of one of the VH domains described in Table 1.

[0282] In certain embodiments, the 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 the VL domains disclosed herein in Table 1. In certain embodiments, the antibody comprises CDRL1 of one of the VL domains described in Table 1. In certain embodiments, the antibody comprises CDRL2 of one of the VL domains described in Table 1. In certain embodiments, the antibody comprises CDRL3 of one of the VL domains described in Table 1.

[0283] In certain embodiments, the CDRs of an antibody can be determined according to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of Proteins of Immunological Interest (1991), each of which is incorporated herein by reference in its entirety.

[0284] In certain embodiments, the CDRs of an antibody can be identified according to the Chothia numbering scheme, which refers to the positions of immunoglobulin structural loops (see, e.g., Chothia C and Lesk AM, (1987), J. Mol. Biol. 196:901-917; Al-Lazikani B et al., (1997) J. Mol. Biol. 273:927-948; Chothia C et al., (1992) J. Mol. Biol. 227:799-817; Tramontano A et al., (1990) J. Mol. Biol. 215(1):175-82; and U.S. Pat. No. 7,709,226, all of which are incorporated herein by reference in their entirety). Typically, when using the Kabat numbering convention, the Chothia CDRH1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDRH2 loop is present at heavy chain amino acids 52 to 56, and the Chothia CDRH3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDRL1 loop is present at light chain amino acids 24 to 34, the Chothia CDRL2 loop is present at light chain amino acids 50 to 56, and the Chothia CDRL3 loop is present at light chain amino acids 89 to 97. When numbered using the Kabat numbering convention, the end of the Chothia CDRH1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, 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).

[0285] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a Chothia VH CDR of a VH disclosed in Table 1 herein. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a Chothia VL CDR of a VL disclosed in Table 1 herein. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a Chothia VH CDR and a Chothia VL CDR of an antibody disclosed in Table 1 herein. In certain embodiments, the antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) comprises one or more CDRs in which the Chothia and Kabat CDRs have the same amino acid sequence. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) and comprises a combination of Kabat CDRs and Chothia CDRs.

[0286] In certain embodiments, the CDRs of an antibody may be identified according to the IMGT numbering system as described in Lefranc MP, (1999) The Immunologist 7: 132-136 and Lefranc MP et al., (1999) Nucleic Acids Res 27: 209-212, each of which is incorporated herein by reference in its entirety.

[0287] In certain embodiments, the present disclosure provides antibodies that specifically bind to CTLA-4 (e.g., human CTLA-4) and comprise the CDRs of the antibodies disclosed in Table 1 herein, as determined by the IMGT numbering system as described, for example, in Lefranc MP (1999) supra and Lefranc MP et al., (1999) supra.

[0288] In certain embodiments, the CDRs of an antibody can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable region, which represents a compromise between the Kabat CDRs and the Chothia structural loops, and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), which is incorporated herein by reference in its entirety. In a specific embodiment, the present disclosure provides antibodies that specifically bind to CTLA-4 (e.g., human CTLA-4) and comprise the CDRs of the antibodies disclosed in Table 1 herein, as determined by the AbM numbering scheme.

[0289] In certain embodiments, the present disclosure provides 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 amino acid sequence of the CDRH1, CDRH2, and CDRH3 regions of the VH domain set forth in SEQ ID NO:2, 4, 5, 6, 7 or 8, and a light chain variable region comprising the amino acid sequence of the CDRL1, CDRL2, and CDRL3 regions of the VL domain set forth in SEQ ID NO:9, wherein each CDR is defined according to the MacCallum definition, the Kabat definition, the Chothia definition, a combination of the Kabat definition and the Chothia definition, the IMGT numbering system, or the AbM definition of a CDR.

[0290] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising:

[0291] (a) CDRH1 comprises the amino acid sequence SYSMN (SEQ ID NO: 10); and / or

[0292] (b) CDRH2 comprises the amino acid sequence SISSSSSYIYYAXSVKG (SEQ ID NO: 18), wherein X is E or D; and / or

[0293] (c) the CDRH3 comprises the amino acid sequence VGLXGPFDI (SEQ ID NO: 19), wherein X is F or M; and / or

[0294] (d) CDRL1 comprises the amino acid sequence RASQSVSRYLG (SEQ ID NO: 15); and / or

[0295] (e) CDRL2 comprises the amino acid sequence GASTRAT (SEQ ID NO: 16); and / or

[0296] (f) CDRL3 comprises the amino acid sequence QQYGSSPWT (SEQ ID NO: 17),

[0297] And wherein the CDRH1, CDRH2 and CDRH3 sequences of the antibody are not SEQ ID NOs: 10, 11 and 13, respectively.

[0298] 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 certain embodiments, CDRH3 comprises the amino acid sequence of SEQ ID NO: 14.

[0299] In certain embodiments, the present disclosure provides 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, 11, and 14; 10, 12, and 13; or 10, 12, and 14, respectively. In certain embodiments, the present disclosure provides 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.

[0300] In certain embodiments, the present disclosure provides an isolated 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 regions and a light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences set forth in SEQ ID NOs: 10, 11, 14, 15, 16, and 17; 10, 12, 13, 15, 16, and 17; or 10, 12, 14, 15, 16, and 17, respectively. In certain embodiments, the present disclosure provides an isolated 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 regions and a light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences set forth in SEQ ID NOs: 10, 12, 14, 15, 16, and 17, respectively.

[0301] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the present 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. In certain embodiments, the present 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 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: 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 certain 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 certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 8.

[0302] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), comprising 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 sequence set forth in SEQ ID NO: 9. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 9.

[0303] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9. In certain embodiments, the present 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 sequence set forth in SEQ ID NO:9. In certain embodiments, the present 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, 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 sequence set forth in SEQ ID NO:9. In certain embodiments, the antibodies comprise heavy chain variable regions and light chain variable regions having the 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 antibodies comprise heavy chain variable regions and light chain variable regions having the amino acid sequences set forth in SEQ ID NOs: 2 and 9, respectively. In certain embodiments, the antibodies comprise heavy chain variable regions and light chain variable regions having the amino acid sequences set forth in SEQ ID NOs: 3 and 9, respectively. In certain embodiments, the antibodies comprise heavy chain variable regions and light chain variable regions having the amino acid sequences set forth in SEQ ID NOs: 4 and 9, respectively. In certain embodiments, the antibodies comprise heavy chain variable regions and light chain variable regions having the amino acid sequences set forth in SEQ ID NOs: 5 and 9, respectively.In certain embodiments, the antibodies comprise a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 6 and 9, respectively. In certain embodiments, the antibodies comprise a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 7 and 9, respectively. In certain embodiments, the antibodies comprise a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 8 and 9, respectively.

[0304] In certain embodiments, the present 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 an 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 an 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 an amino acid sequence of SEQ ID NO: 21).

[0305] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), comprising 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 SEQ ID NO: 22). One or more regions selected from framework 1, framework 2, framework 3, CDRL1, and CDRL2 (e.g., two, three, four, or five of these regions) can be derived from a human germline sequence selected from the group consisting of IGKV3-20 (e.g., IGKV3-20*01, e.g., having the amino acid sequence SEQ ID NO: 22). In one embodiment, framework 1, framework 2, framework 3, CDRL1, and CDRL2 are all derived from a human germline sequence selected from the group consisting of IGKV3-20 (e.g., IGKV3-20*01, e.g., having the amino acid sequence SEQ ID NO: 22).

[0306] In certain embodiments, the present 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 sequence 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).

[0307] In certain embodiments, the present 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: 2 and 9; 4 and 9; 5 and 9; 6 and 9; 7 and 9; or 8 and 9, respectively. In certain embodiments, the present 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.

[0308] In certain embodiments, the present disclosure provides an isolated antibody that binds to the same or 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 chain 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 present disclosure provides an isolated antibody that binds to the same or 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 chain and light chain variable region amino acid sequences set forth in SEQ ID NOs: 3 and 9, respectively. In certain embodiments, the epitope of the antibody can be determined by, for example, NMR spectroscopy, surface plasmon resonance X-ray diffraction crystallographic studies, ELISA analysis, hydrogen / deuterium exchange coupled mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning analysis and / or mutagenesis localization (e.g., site-directed mutagenesis localization) determination. In X-ray crystallography, crystallization can be achieved using any of the known methods in the art (e.g., Giegé R et al., (1994) Acta Crystallogica Sinica D: Biological Crystallography 50 (Pt 4): 339-350; McPherson A (1990) European Journal of Biochemistry 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) Journal of Biological Chemistry 251: 6300-6303, all of which are incorporated herein by reference in their entirety). Antibody: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Methods in Enzymology (1985) Vols. 114 and 115, Wyckoff HW et al., eds.; U.S. Patent Application No. 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallographica Sinica, Series D: Biological Crystallography 49 (Part 1): 37-60; Bricogne G (1997) Methods in Enzymology 276A: 361-423, Carter CW ed.; Roversi P et al., (2000) Acta Crystallographica Sinica, Series D: Biological Crystallography 56 (Part 10): 1316-1323), all of which are incorporated herein by reference in their entirety. Mutagenesis localization studies can be achieved using any method known to those skilled in the art. For a description of mutagenesis techniques (including alanine scanning mutagenesis techniques), see, for example, Champe M et al., (1995) supra and Cunningham BC and Wells JA (1989) supra. In a specific embodiment, the epitope of the antibody is determined using alanine scanning mutagenesis studies. In addition, antibodies that recognize and bind to the same or overlapping epitopes of CTLA-4 (e.g., human CTLA-4) can be identified using conventional techniques, such as immunoassays, for example, by demonstrating the ability of one antibody to block the binding of another antibody to the target antigen, i.e., competitive binding assays. Competitive binding assays can also be used to determine whether two antibodies have similar binding specificities for an epitope. Competitive binding can be determined in an assay in which the immunoglobulin being tested inhibits specific binding of a reference antibody to a common antigen, such as CTLA-4 (e.g., human CTLA-4).Many 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 competition assay (see Stahli C et al., (1983) Methods in Enzymology 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 and Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeled RIA using I-125 labeling (see Morel GA et al., (1988) Molecular Immunology 137:3614-9); solid phase direct labeled RIA using I-125 labeling (see Morel GA et al., (1988) Molecular Immunology 137:3614-9). Immunol 25(1):7-15); solid phase direct biotin-avidin EIA (see Cheung RC et al., (1990) Virology 176:546-52); and direct label RIA (see Moldenhauer G et al., (1990) Scand J Immunol 32:77-82), all of which are incorporated herein by reference in their entirety. Typically, such assays involve the use of purified antigen (e.g., CTLA-4, such as human CTLA-4) bound to a solid surface or cells carrying either, an unlabeled test immunoglobulin, and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. The test immunoglobulin is typically present in excess. Typically, when the competing antibody is present in excess, it inhibits specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. Competitive binding assays can be configured using labeled antigens or labeled antibodies in many different formats. In a common version of this assay, the antigen is immobilized on a 96-well plate. The ability of the unlabeled antibody to block the labeled antibody from binding to the antigen is then measured using a radioactive or enzyme label.For additional details, see, e.g., Wagener C et al., (1983) J. Immunol. 130:2308-2315; Wagener C et al., (1984) J. Immunol. Methods 68:269-274; Kuroki M et al., (1990) Cancer Res 50:4872-4879; Kuroki M et al., (1992) Immunol. Invest. 21:523-538; Kuroki M et al., (1992) Hybridoma 11:391-407; and Antibodies: A Laboratory Manual, Harlow E and Lane D, eds., supra, pp. 386-389, all of which are incorporated herein by reference in their entirety.

[0309] In certain embodiments, the present 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 set forth in SEQ ID NO:23, 24, 25, or 26. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:23. In certain embodiments, 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.

[0310] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO:27.

[0311] In certain embodiments, the present 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: 23 and a light chain comprising the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the present 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 present 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: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the present 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.

[0312] Any Ig constant region can be used in the antibodies described herein. In certain embodiments, the Ig region is a human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any class of immunoglobulin molecules (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass (e.g., IgG 2a and IgG 2b ).

[0313] In certain embodiments, the present disclosure provides an isolated 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 present disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a light chain constant region comprising the amino acid sequence of SEQ ID NO: 32.

[0314] In certain embodiments, the present 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 a fragment thereof comprising a mutation selected from the group consisting of: S239D, 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 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 SEQ ID NO: 29.

[0315] In certain embodiments, the present 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 a fragment thereof comprising a mutation 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 constant region comprising the amino acid sequence SEQ ID NO: 30.

[0316] In certain embodiments, the present 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 a fragment thereof comprising a mutation selected from the group consisting of: L235V, F243L, R292P, Y300L, P396L, and combinations thereof, numbered according to the EU numbering system. In certain embodiments, the antibody comprises an IgG1 heavy chain constant region comprising L235V, F243L, R292P, Y300L, and P396L mutations, numbered according to the EU numbering system. In certain embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence SEQ ID NO: 31.

[0317] In certain embodiments, for example, the IgG region of the antibodies described herein has an increased affinity for FcγRIIIA compared to an antibody with a wild-type Fc region (e.g., IgG1 Fc). Sequence changes that result in increased affinity for FcγRIIIA are known in the art, e.g., Kellner et al., Methods 65: 105-113 (2014), Lazar et al., Proc. Natl. Acad. Sci. 103: 4005-4010 (2006), Shields et al., J. Biol. Chem. 276 (9): 6591-6604 (2001), each of which is incorporated herein by reference in its entirety. In certain embodiments, the antibody comprises a heavy chain constant region (e.g., an IgG1 constant region) or a fragment 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 a 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 a fragment thereof comprising T256A, 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 a fragment thereof comprising K290A, 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 a fragment thereof comprising S298A, 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 a fragment thereof comprising 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 a 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 a 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 a fragment 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 a fragment thereof comprising S239D and I332E, numbered according to the EU numbering system. In certain embodiments, the antibody comprises a heavy chain constant region (eg, an IgG1 constant region) or a fragment thereof comprising S239D, A330L, 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 a 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 a fragment thereof comprising G236A, 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 a fragment thereof comprising F243L, R292P, Y300L, V305I and P396L, numbered according to the EU numbering system.

[0318] In certain embodiments, the antibodies described herein exhibit antibody-dependent cellular cytotoxicity (ADCC) activity. In certain embodiments, the antibodies described herein trigger natural killer cell-mediated cell depletion. In certain embodiments, the antibodies described herein are used to treat tumors infiltrated by natural killer cells. In certain embodiments, the antibodies described herein exhibit antibody-dependent cellular phagocytosis (ADCP) activity. In certain embodiments, the antibodies described herein trigger macrophage-mediated cell depletion. In certain embodiments, the antibodies described herein are used to treat tumors infiltrated by macrophages. In certain embodiments, the antibodies described herein selectively deplete regulatory T cells in tumors.

[0319] In certain embodiments, the antibodies described herein are activatable antibodies that bind to human CTLA-4 proteins in an activated state. In certain embodiments, the activatable antibody comprises a masking portion that inhibits the binding of the uncleaved antibody to the human CTLA-4 protein, and at least one cleavable portion coupled to the antibody, such as a polypeptide that serves as a substrate for a protease that is abundant in the tumor microenvironment. Exemplary activatable antibodies are described in, for example, U.S. Patent Nos. 8,513,390 and 8,518,404 and U.S. Patent Application Publication Nos. US2014 / 0255313, US2014 / 0010810, and US2014 / 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 a higher affinity than the wild-type human IgG heavy chain constant region.

[0320] In certain embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., CH2 domain (residues 231-340 of human IgG1) and / or CH3 domain (residues 341-447 of human IgG1) and / or hinge region, numbered according to the EU numbering system) of an antibody described herein to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cellular cytotoxicity.

[0321] In certain embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), as described, for example, in U.S. Pat. No. 5,677,425, which is incorporated herein by reference in its entirety. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, for example, to facilitate assembly of the light chain and the heavy chain, or to alter (e.g., to increase or decrease) the stability of the antibody.

[0322] In a specific embodiment, one, two or more amino acid mutations (e.g., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably Fc or hinge-Fc domain fragment) to change (e.g., shorten or prolong) the half-life of the antibody in vivo. For examples of mutations that change (e.g., shorten or prolong) the half-life of an antibody in vivo, see, e.g., International Publication Nos. WO 02 / 060919, WO 98 / 23289 and WO 97 / 34631 and U.S. Pat. Nos. 5,869,046, 6,121,022, 6,277,375 and 6,165,745, all of which are incorporated herein by reference in their entirety. In some embodiments, one, two or more amino acid mutations (e.g., substitutions, insertions or deletions) are introduced into an IgG constant domain or its FcRn binding fragment (preferably an Fc or hinge-Fc domain fragment) to shorten the half-life of the antibody in vivo. In other embodiments, one, two or more amino acid mutations (e.g., substitutions, insertions or deletions) are introduced into an IgG constant domain or its FcRn binding fragment (preferably an Fc hinge-Fc domain fragment) to extend the half-life of the antibody in vivo. In a specific embodiment, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 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 the antibodies described herein comprises a substitution of methionine (M) to tyrosine (Y) at position 252, a substitution of serine (S) to threonine (T) at position 254, and a substitution of threonine (T) to glutamate (E) at position 256, numbered according to the EU numbering system. See U.S. Patent No. 7,658,921, which is incorporated herein by reference in its entirety. It has been shown that this type of mutant IgG (referred to as a "YTE mutant") exhibits a four-fold extension of half-life compared to the wild-type form of the same antibody (see Dall'Acqua WF et al., (2006) Journal of Biol. Chem. 281: 23514-24, which is incorporated herein by reference in its entirety). In certain embodiments, the antibody comprises an IgG constant domain comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389 and 428-436, as numbered according to the EU numbering system.

[0323] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., CH2 domain (residues 231-340 of human IgG1) and / or CH3 domain (residues 341-447 of human IgG1) and / or hinge region, numbered according to the EU numbering system) of an antibody described herein to increase or decrease the affinity of the antibody for an Fc receptor on the surface of an effector cell (e.g., an activated Fc receptor). Mutations in the Fc region of an antibody that increase or decrease the affinity of the antibody for an Fc receptor and techniques for introducing such mutations into an Fc receptor or fragment thereof are known to those skilled in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody for the Fc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, all of which are incorporated herein by reference in their entirety.

[0324] In another embodiment, one, two or more amino acid substitutions are introduced into the IgG constant domain Fc region to change the (one or more) effector functions 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 different amino acid residues, so that the affinity of the antibody to the effector ligand changes but retains the antigen binding ability of the parent antibody. The effector ligand with changed affinity can be, for example, an Fc receptor or C1 component of complement. This method is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260, each of which is incorporated herein by reference in its entirety. In some embodiments, the deletion or inactivation of the constant region domain (by point mutation or other means) may reduce the Fc receptor binding of circulating antibodies, thereby enhancing tumor localization. For a description of mutations that delete or inactivate constant domains and thereby enhance tumor localization, see, e.g., U.S. Pat. Nos. 5,585,097 and 8,591,886, each of which is incorporated herein by reference in its entirety. In certain embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on the Fc region that may reduce Fc receptor binding (see, e.g., Shields RL et al., (2001) J. Biol. Chem. 276: 6591-604, which is incorporated herein by reference in its entirety). In various embodiments, one or more of the following mutations in the constant region of an antibody described herein may be made: N297A substitution; N297Q substitution; L235A substitution and L237A substitution; L234A substitution and L235A substitution; E233P substitution; L234V substitution; L235A substitution; C236 deletion; P238A substitution; D265A substitution; A327Q substitution; or P329A substitution, numbered according to the EU numbering system. In certain embodiments, a mutation selected from the group consisting of: D265A, P329A, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein.

[0325] In a specific embodiment, the antibodies described herein comprise a constant domain of an IgG1 having an N297Q or N297A amino acid substitution, numbered according to the EU numbering system. In one embodiment, the antibodies described herein comprise a constant domain of an IgG1 having a mutation selected from a group consisting of: D265A, P329A, and combinations thereof, numbered according to the EU numbering system. In another embodiment, the antibodies described herein comprise a constant domain of an IgG1 having a mutation selected from a group consisting of: L234A, L235A, and combinations thereof, numbered according to the EU numbering system. In certain embodiments, the amino acid residues in the constant region of the antibodies described herein corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are not L, L, and D, respectively. This method is described in detail in International Publication No. WO 14 / 108483, which is incorporated herein by reference in its entirety. In a specific 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, as numbered according to the EU numbering system.

[0326] In certain embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 in the constant region of an antibody described herein (numbered according to the EU numbering system) can be replaced with a different amino acid residue such that the antibody's C1q binding is altered and / or complement-dependent cytotoxicity (CDC) is reduced or eliminated. This approach is described in more detail in U.S. Pat. No. 6,194,551 (Idusogie et al.), which is incorporated herein 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 altered, thereby altering the ability of the antibody to fix complement, numbered according to the EU numbering system. This approach is further described in International Publication No. WO 94 / 29351, which is incorporated herein by reference in its entirety. In certain embodiments, the Fc region of an antibody described herein is modified to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or enhance the affinity of the antibody for an Fcγ receptor by mutating (e.g., introducing an amino acid substitution) one or more amino acids 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, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315 435, 437, 438 or 439, numbered according to the EU numbering system. This method is further described in International Publication No. WO 00 / 42072, which is incorporated herein by reference in its entirety.

[0327] In certain embodiments, the antibodies described herein comprise the constant region of an IgG4 antibody, and a serine at amino acid residue 228 (numbered according to the EU numbering system) of the heavy chain is substituted for a proline.

[0328] 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.

[0329] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CTLA-4 (eg, human CTLA-4) and acts as an antagonist.

[0330] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) and reduces 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% relative to CTLA-4 (e.g., human CTLA-4) activity in the absence of any antibody or in the presence of an irrelevant antibody (e.g., an antibody that does not specifically bind to CTLA-4 (e.g., human CTLA-4)) as assessed by methods described herein and / or known to those of skill in the art. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) and reduces 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, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold relative to CTLA-4 (e.g., human CTLA-4) activity in the absence of any antibody or in the presence of an irrelevant antibody (e.g., an antibody that does not specifically bind to CTLA-4 (e.g., human CTLA-4)) as assessed by methods described herein and / or known to those of skill in the art. Non-limiting examples of CTLA-4 (e.g., human CTLA-4) activity can include CTLA-4 (e.g., human CTLA-4) signaling, CTLA-4 (e.g., human CTLA-4) binding to CTLA-4 (e.g., human CTLA-4) ligands (e.g., CD80 or CD86), and inhibition of cytokine production (e.g., IL-2, IFN-γ or TNF-α). In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) and inactivates, reduces or inhibits CTLA-4 (e.g., human CTLA-4) activity. In specific embodiments, the reduction in CTLA-4 (e.g., human CTLA-4) activity is assessed as described in the Examples below.

[0331] In specific embodiments, the disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) and reduces the binding of CTLA-4 (e.g., human CTLA-4) to its ligand (e.g., CD80 or CD86) 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% relative to the binding of CTLA-4 (e.g., human CTLA-4) to its ligand (e.g., CD80 or CD86) in the absence of any antibody or in the presence of an irrelevant antibody (e.g., an antibody that does not specifically bind to CTLA-4 (e.g., human CTLA-4)) as assessed by methods known to those of skill in the art. In specific embodiments, the present disclosure provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) and reduces the binding of CTLA-4 (e.g., human CTLA-4) 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, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold relative to the binding of CTLA-4 (e.g., human CTLA-4) to its ligand (e.g., CD80 or CD86) in the absence of any antibody or in the presence of an irrelevant antibody (e.g., an antibody that does not specifically bind to CTLA-4 (e.g., human CTLA-4)) as assessed by methods known to those of skill in the art.

[0332] In specific embodiments, the disclosure provides an isolated antibody 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 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% relative to cytokine production in the absence of any antibody or in the presence of an irrelevant antibody (e.g., an antibody that does not specifically bind to CTLA-4 (e.g., human CTLA-4)), as assessed by methods described herein (see Examples below) or known to those of skill in the art. In specific embodiments, the present disclosure provides an isolated antibody 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 relative to cytokine production in the absence of any antibody or in the presence of an irrelevant antibody (e.g., an antibody that does not specifically bind to CTLA-4 (e.g., human CTLA-4)) as assessed by methods described herein (see Examples below) or known to those of skill in the art.

[0333] In certain embodiments, human peripheral blood mononuclear cells (PBMCs) stimulated with Staphylococcal enterotoxin A (SEA) in the presence of an antibody described herein that specifically binds to CTLA-4 (e.g., human CTLA-4) increases 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 stimulated with SEA alone in the absence of any antibody or in the presence of an irrelevant antibody (e.g., an antibody that does not specifically bind to CTLA-4 (e.g., human CTLA-4)) as assessed by methods described herein (see Examples below) or known to those of skill in the art.

[0334] 1.3 Pharmaceutical Composition

[0335] Provided herein are compositions comprising an anti-CTLA-4 antibody described herein (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA) having a desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexahydroxyquaternium chloride; benzalkonium chloride, benzethonium chloride, chloride); phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0336] In a specific embodiment, the pharmaceutical composition comprises 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, the pharmaceutical composition comprises an effective amount of an antibody or antigen-binding fragment thereof described herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. In some embodiments, the antibody is the only active ingredient included in the pharmaceutical composition. The pharmaceutical compositions described herein can be used to inhibit CTLA-4 activity and treat conditions such as cancer or infectious diseases.

[0337] In one aspect, provided herein is a pharmaceutical composition comprising an anti-CTLA-4 antibody of the present invention and a pharmaceutically acceptable carrier or excipient for use as a medicament.

[0338] In one aspect, provided herein is a pharmaceutical composition comprising an anti-CTLA-4 antibody of the present invention and a pharmaceutically acceptable carrier or excipient for use in a method for treating cancer.

[0339] Pharmaceutically acceptable carriers for parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending agents and dispersants, emulsifiers, chelating or chelating agents (sequestering or chelating agent) and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringers Injection, isotonic dextrose injection, sterile water for injection, dextrose and lactated Ringers Injection. Non-aqueous parenteral vehicles include fixed oils, cottonseed oil, corn oil, sesame oil and peanut oil of plant origin. Antimicrobial agents that inhibit bacteria or inhibit fungal concentrations can be added to parenteral preparations packaged in multi-dose containers, and the multi-dose containers include phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl parahydroxybenzoates, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinyl pyrrolidone. Emulsifiers include polysorbate 80 ( 80). Chelating agents for metal ions include EDTA. Pharmaceutical carriers also include ethanol, polyethylene glycol and propylene glycol for water-miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0340] The pharmaceutical composition can be formulated for any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, pulmonary, transdermal, intradermal and parenteral. Parenteral administration is also contemplated herein, characterized in that it is injected subcutaneously, intramuscularly or intravenously. Injectables can be prepared in conventional forms, in the form of liquid solutions or suspensions, in the form of solids suitable for forming solutions or suspensions in liquids before injection, or in the form of emulsions. Injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if necessary, the pharmaceutical composition to be administered may also contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrin.

[0341] Preparations for parenteral administration of antibodies include sterile solutions that can be used immediately for injection; sterile dry soluble products that are ready to be combined with solvents before use, such as lyophilized powders, including subcutaneous tablets; sterile suspensions that can be used immediately for injection; sterile dry insoluble products that are ready to be combined with vehicles before use; and sterile emulsions. Solutions can be aqueous or non-aqueous.

[0342] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

[0343] Topical mixtures containing the antibody are prepared as described for topical and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc. and may be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, rinse, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.

[0344] The anti-CTLA-4 antibodies described herein can be formulated as aerosols for topical administration, such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivering steroids useful for treating inflammatory diseases, particularly asthma, and are incorporated herein by reference in their entirety). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or in the form of a finely divided powder for insufflation, alone or in combination with an inert carrier such as lactose. In this case, the particles of the formulation will have a diameter of less than 50 microns, in one embodiment less than 10 microns, in one embodiment.

[0345] The anti-CTLA-4 antibodies described herein can be formulated for local or topical administration, such as topical administration to the skin and mucous membranes, such as in the eye, in the form of gels, creams and lotions, and formulated for administration to the eye or for intracisternal or intraspinal administration. Topical administration is contemplated for transdermal delivery as well as for administration to the eye or mucous membranes, or for inhalation therapy. Nasal solutions of the antibodies, alone or in combination with other pharmaceutically acceptable excipients, can also be administered.

[0346] Transdermal patches, including iontophoresis and electrophoresis devices, are well known to those skilled in the art and can be used to administer antibodies. For example, such patches are disclosed in U.S. Patents Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433 and 5,860,957, all of which are incorporated herein by reference in their entirety.

[0347] In certain embodiments, the pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein is a lyophilized powder, which can be reconstituted for administration in the form of solutions, emulsions, and other mixtures. It can also be reconstituted and formulated as a solid or gel. The lyophilized powder is prepared by dissolving the 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 excipients that can improve the stability of the powder or the reconstituted solution prepared from the powder or other pharmacological components. Excipients that can be used include (but are not limited to) dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, such as citrate, sodium phosphate or potassium phosphate, or other such buffers known to those skilled in the art, and in one embodiment, the buffer is approximately neutral pH. The solution is then sterile filtered and then freeze-dried under standard conditions known to those skilled in the art to obtain the desired formulation. In one embodiment, the resulting solution is distributed into vials for freeze-drying. Each vial will contain a single dose or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature. This lyophilized powder is reconstituted with water for injection to obtain a formulation for parenteral administration. During reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the selected compound. This amount can be determined empirically.

[0348] The anti-CTLA-4 antibodies described herein and other compositions provided herein can also be formulated to target specific tissues, receptors or other areas of the body of the subject to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in the compositions of the present invention. For non-limiting examples of targeted methods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874, all of which are incorporated herein by reference in their entirety. In a specific embodiment, the antibodies or antigen-binding fragments thereof described herein are targeted to a tumor.

[0349] Compositions to be used for in vivo administration may be sterile. This is readily achieved, for example, by filtration through sterile filtration membranes.

[0350] 1.4 Usage and Purpose

[0351] In another aspect, the present disclosure provides a method of treating a subject using an anti-CTLA-4 antibody as described herein. Any disease or condition of 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 suppressing the immune system's tolerance to tumors, and therefore can be used as an immunotherapy for subjects with cancer. For example, in certain embodiments, the present disclosure provides a method of increasing T cell activation in response to an antigen in a subject, the method comprising administering to the subject an effective amount of an anti-CTLA-4 antibody as described herein, or a pharmaceutical composition thereof. In certain embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody or pharmaceutical composition as described herein.

[0352] Cancers that can be treated with the antibodies, therapeutic combinations, or pharmaceutical compositions described herein include, but are not limited to, solid cancers (e.g., recurrent or refractory solid cancers and advanced or metastatic solid cancers), carcinomas, sarcomas, melanomas (e.g., stage III or IV melanomas), small cell lung cancer, non-small cell lung cancer, urothelial carcinoma, ovarian cancer, prostate cancer (e.g., metastatic hormone-refractory prostate cancer and advanced metastatic prostate cancer), pancreatic cancer, breast cancer, e.g., HER2 +Breast cancer (eg, relapsed / refractory HER2 + Breast cancer)), head and neck cancer (e.g., recurrent / refractory head and neck squamous cell carcinoma (HNSCC)), glioma, malignant glioma, glioblastoma multiforme, brain metastasis, Merck cancer, gastric cancer, gastroesophageal cancer, renal cell carcinoma, uveal melanoma, colon cancer, cervical cancer, lymphoma (e.g., recurrent or refractory lymphoma), non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, and multiple myeloma. In certain embodiments, cancer is treated by intratumoral administration of an antibody, therapeutic combination, or pharmaceutical composition described herein. Cancers that can be treated by intratumoral administration of an antibody, therapeutic combination, or pharmaceutical composition described herein include, but are not limited to, solid tumors (e.g., advanced or metastatic solid tumors), head and neck cancer (e.g., recurrent / refractory head and neck squamous cell carcinoma (HNSCC)), and breast cancer (e.g., HER2 + Breast cancer (eg, relapsed / refractory HER2 + Breast cancer)).

[0353] In certain embodiments, the cancer treated according to the methods described herein is a solid tumor. In certain embodiments, the cancer treated according to the methods described herein is metastatic or locally advanced cancer (e.g., metastatic or locally advanced solid tumor). In certain embodiments, after the diagnosis of metastatic or locally advanced tumors (e.g., 1, 2, 3, 4, 5 or 6 days after diagnosis; 1, 2, 3, 4, 6, 8 or 12 weeks; or within 1, 2, 3, 4, 6, 8 or 12 months), cancer is treated as the first cancer therapy according to the methods described herein. In certain embodiments, after the diagnosis of tumor progression that occurred despite the previous treatment of tumors with different cancer therapies (e.g., 1, 2, 3, 4, 5 or 6 days after the diagnosis of tumor progression; 1, 2, 3, 4, 6, 8 or 12 weeks; or within 1, 2, 3, 4, 6, 8 or 12 months), cancer is treated as the first cancer therapy according to the methods described herein, optionally wherein the methods described herein are provided as the second cancer therapy administered. In certain embodiments, after the diagnosis of toxicity of different cancer therapies (e.g., within 1, 2, 3, 4, 5, or 6 days after the diagnosis of toxicity of different cancer therapies; 1, 2, 3, 4, 6, 8, or 12 weeks; or within 1, 2, 3, 4, 6, 8, or 12 months), cancer is treated according to the methods described herein as a first cancer therapy, optionally wherein the methods described herein are provided as a second cancer therapy administered. In certain embodiments, the cancer treated according to the methods described herein is a metastatic or locally advanced cancer (e.g., a solid tumor) for which no standard therapy is available. In other embodiments, the cancer treated according to the methods described herein is a metastatic or locally advanced cancer (e.g., a solid tumor) for which standard therapy has failed (i.e., after standard therapy, the cancer has progressed). In certain embodiments, if the cancer is refractory to therapy, then the therapy fails. In certain embodiments, if the cancer recurs after a complete or partial response to therapy, then the therapy fails. In certain embodiments, metastatic or locally advanced cancer (e.g., a solid tumor) has been diagnosed histologically or cytologically.

[0354] In certain embodiments, cancer is a solid tumor. In certain embodiments, cancer (e.g., solid tumor) expresses PD-L1. In certain embodiments, the percentage of tumor cells expressing detectable PD-L1 (e.g., partial or complete expression) in cancer (e.g., solid tumor) samples 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 expressing detectable PD-L1 membrane (e.g., partial or complete membrane expression) in cancer (e.g., solid tumor) samples 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 cancer (e.g., solid tumor) sample that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) is at least 1%. In certain embodiments, the percentage of tumor cells in a cancer (e.g., solid tumor) sample that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) is at least 5%. In certain embodiments, the percentage of tumor cells in a cancer (e.g., solid tumor) sample that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) is at least 25%. In certain embodiments, the percentage of tumor cells in a cancer (e.g., solid tumor) sample that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) is at least 50%.

[0355] In certain embodiments, metastatic or locally advanced cancer (e.g., solid tumor) expresses PD-L1. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 expression (e.g., partial or complete expression) in metastatic or locally advanced cancer (e.g., solid tumor) samples 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 that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced cancer (e.g., solid tumor) samples 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 that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in a metastatic or locally advanced cancer (e.g., solid tumor) sample is at least 1%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in a metastatic or locally advanced cancer (e.g., solid tumor) sample is at least 5%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in a metastatic or locally advanced cancer (e.g., solid tumor) sample is at least 25%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in a metastatic or locally advanced cancer (e.g., solid tumor) sample is at least 50%.

[0356] In each of the methods described herein that require a certain percentage of cells in a sample to exhibit detectable PD-L1 expression (e.g., membrane expression, partial or complete membrane expression), the expression of PD-L1 can be detected by any method well known in the art, including, but not limited to, immunohistochemistry. Exemplary immunohistochemical 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 herein by reference in their entirety.

[0357] In certain embodiments, the cancer treated according to the methods described herein is metastatic or locally advanced non-small cell lung cancer (NSCLC). In certain embodiments, the cancer treated according to the methods described herein is metastatic non-small cell lung cancer (NSCLC). In certain embodiments, the cancer treated according to the methods described herein is stage IV metastatic or locally advanced NSCLC. In certain embodiments, the cancer treated according to the methods described herein is stage IV metastatic NSCLC. In certain embodiments, the percentage of tumor cells expressing detectable PD-L1 (e.g., partial or complete expression) in metastatic or locally advanced NSCLC samples 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 that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced NSCLC samples 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 that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced NSCLC samples is at least 1%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced NSCLC samples is at least 5%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced NSCLC samples is at least 25%. In certain embodiments, the percentage of tumor cells that exhibit detectable PD-L1 membrane expression (e.g., partial or complete membrane expression) in metastatic or locally advanced NSCLC samples is at least 50%. In certain embodiments, metastatic or locally advanced NSCLC has no EGFR or ALK genomic tumor aberrations. In certain embodiments, metastatic or locally advanced NSCLC has no EGFR sensitizing mutations (e.g., mutations suitable for treatment with tyrosine kinase inhibitors, tyrosine kinase inhibitors include erlotinib, gefitinib or afatinib) or ALK translocations. In certain embodiments, subjects with metastatic or locally advanced NSCLC have not previously received systemic chemotherapy for metastatic or locally advanced NSCLC. In certain embodiments, after the diagnosis of metastatic or locally advanced NSCLC (e.g., 1, 2, 3, 4, 5 or 6 days after diagnosis; 1, 2, 3, 4, 6, 8 or 12 weeks; or 1, 2, 3, 4, 6, 8 or 12 months), metastatic or locally advanced NSCLC is treated as the first cancer therapy according to the methods described herein.In certain embodiments, the method comprises using an anti-CTLA-4 antibody described herein (e.g., AGEN1884.H3 (IgG1 S239D / A330L / I332E)) or a pharmaceutical composition comprising such an anti-CTLA-4 antibody to treat a subject with NSCLC (e.g., stage IV metastatic or locally advanced NSCLC), wherein the percentage of tumor cells in the NSCLC sample that exhibit detectable membrane expression of PD-L1 (e.g., partial or complete membrane expression) 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 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).

[0358] In certain embodiments, the cancer treated according to the methods described herein is cervical cancer. In certain embodiments, the cancer treated according to the methods described herein is metastatic or locally advanced unresectable squamous cell carcinoma, adenosquamous carcinoma or cervical adenocarcinoma. In certain embodiments, the cancer treated according to the methods described herein is unresectable or metastatic cervical cancer. In certain embodiments, cervical cancer progresses after standard therapy (e.g., has recurred after standard therapy, or is refractory to standard therapy). In certain embodiments, standard therapy comprises platinum-containing chemotherapy. In certain embodiments, platinum-containing chemotherapy is selected from the group consisting of: cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, picoplatin, triplatin, phenanthriplatin, iproplatin, lobapatin, heptaplatin, lipoplatin and combinations thereof. In certain embodiments, standard therapy further comprises a second chemotherapy. In certain embodiments, the second chemotherapy is selected from the group consisting of: nucleotide analogs (e.g., gemcitabine), folic acid antimetabolites (e.g., pemetrexed) and taxanes (e.g., paclitaxel). In certain embodiments, the standard therapy is any platinum-based dual chemotherapy (PT-DC) known in the art (also referred to as platinum-containing dual therapy). In certain embodiments, PT-DC comprises cisplatin and gemcitabine, cisplatin and pemetrexed, cisplatin and paclitaxel, carboplatin and paclitaxel, or cisplatin and topotecan. Standard therapy (e.g., a standard therapy comprising PT-DC) may optionally further comprise one or more additional therapies, such as bevacizumab. In certain embodiments, the standard therapy comprises paclitaxel and topotecan. In certain embodiments, cervical cancer is HPV positive. In certain embodiments, cervical cancer is associated with microsatellite instability. In certain embodiments, the cancer treated according to the methods described herein is a metastatic or locally advanced unresectable squamous cell carcinoma, adenosquamous carcinoma, or cervical adenocarcinoma that relapsed after platinum-containing double therapy administered for the treatment of advanced (recurrent, unresectable or metastatic) disease. In certain embodiments, cervical cancer is treated as a first cancer therapy according to the methods described herein after diagnosis of cervical cancer (e.g., 1, 2, 3, 4, 5, or 6 days after diagnosis; 1, 2, 3, 4, 6, 8, or 12 weeks; or 1, 2, 3, 4, 6, 8, or 12 months).In certain embodiments, cervical cancer is treated according to the methods described herein as a first cancer therapy, optionally wherein the methods described herein are provided as a second cancer therapy administered, following a diagnosis of tumor progression that occurred despite prior treatment of the cervical cancer with 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 the diagnosis of tumor progression). In certain embodiments, cervical cancer is treated according to the methods described herein as a first cancer therapy, optionally wherein the methods described herein are provided as a second cancer therapy administered, following a 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 the diagnosis of toxicity of a different cancer therapy). In certain embodiments, the method comprises treating a subject having cervical cancer (e.g., metastatic or locally advanced unresectable squamous cell carcinoma, adenosquamous carcinoma, or cervical adenocarcinoma) with an anti-CTLA-4 antibody described herein (e.g., AGEN1884.H3 (IgG1 S239D / A330L / I332E)) or a pharmaceutical composition comprising such an anti-CTLA-4 antibody, wherein the method is provided as a first cancer therapy after diagnosis of cervical cancer (e.g., 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 method comprises treating a subject having cervical cancer (e.g., metastatic or locally advanced unresectable squamous cell carcinoma, adenosquamous carcinoma, or cervical adenocarcinoma) with an anti-CTLA-4 antibody described herein (e.g., AGEN1884.H3 (IgG1 S239D / A330L / I332E)) or a pharmaceutical composition comprising such an anti-CTLA-4 antibody S239D / A330L / I332E)) or a pharmaceutical composition comprising such an anti-CTLA-4 antibody to treat a subject having cervical cancer (e.g., metastatic or locally advanced unresectable squamous cell carcinoma, adenosquamous carcinoma, or cervical adenocarcinoma), wherein the method is provided after diagnosis of tumor progression that occurs despite prior treatment of cervical cancer with 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 tumor progression), or 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 a different cancer therapy), and wherein the method described herein is provided as a second cancer therapy administered.

[0359] In certain embodiments, the cancer treated according to the methods described herein is squamous cell carcinoma of the skin (cSCC). In certain embodiments, the cancer treated according to the methods described herein is stage IV squamous cell carcinoma of the skin (cSCC). In certain embodiments, cSCC (e.g., stage IV cSCC) cannot be cured with radiotherapy. In certain embodiments, stage IV cSCC is diagnosed histologically or cytologically according to the eighth edition of the American Joint Committee on Cancer Staging Manual (AJCC-8). In certain embodiments, after the diagnosis of cSCC (e.g., stage IV cSCC) (e.g., 1, 2, 3, 4, 5 or 6 days after diagnosis; 1, 2, 3, 4, 6, 8 or 12 weeks; or within 1, 2, 3, 4, 6, 8 or 12 months), cSCC (e.g., stage IV cSCC) is treated as the first cancer therapy according to the methods described herein. In certain embodiments, after diagnosis of tumor progression that occurred despite prior treatment of the cSCC (e.g., stage IV cSCC) with 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 tumor progression), cSCC (e.g., stage IV cSCC) is treated according to the methods described herein as a first cancer therapy, optionally wherein the methods described herein are provided as a second cancer therapy administered. In certain embodiments, 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), cSCC (e.g., stage IV cSCC) is treated according to the methods described herein as a first cancer therapy, optionally wherein the methods described herein are provided as a second cancer therapy administered.

[0360] In certain embodiments, the cancer treated according to the methods described herein is a B-cell lymphoma (e.g., B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin lymphoma, cutaneous B-cell lymphoma, diffuse large B-cell lymphoma), basal cell carcinoma, bladder cancer, blastoma, brain metastasis, breast cancer, Burkitt lymphoma, carcinoma (e.g., gastroesophageal junction adenocarcinoma), cervical cancer, colon cancer, colorectal cancer (colon and rectum cancer), endometrial cancer, esophageal cancer, Ewing's sarcoma (Ewing's sarcoma), or adenocarcinoma of the pancreas. sarcoma), follicular lymphoma, gastric cancer, gastroesophageal junction cancer, gastrointestinal cancer, glioblastoma (e.g., glioblastoma multiforme, such as newly diagnosed or recurrent), glioma, head and neck cancer (e.g., head and neck squamous cell carcinoma), liver metastasis, Hodgkin's and non-Hodgkin's lymphoma, kidney cancer (e.g., renal cell carcinoma and Wilms' tumor), laryngeal cancer, leukemia (e.g., chronic myeloid leukemia, hairy cell leukemia), liver cancer (e.g., hepatocarcinoma and liver tumor), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), lymphoblastic lymphoma, lymphoma, mantle cell lymphoma, metastatic brain tumor, metastatic cancer, myeloma (e.g., multiple myeloma) , neuroblastoma, ocular melanoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer (e.g., pancreatitis ductal adenocarcinoma), prostate cancer (e.g., hormone refractory (e.g., castration resistant), metastatic, metastatic hormone refractory (e.g., castration resistant, androgen independent)), renal cell carcinoma (e.g., metastatic), salivary gland cancer, sarcoma (e.g., rhabdomyosarcoma), skin cancer (e.g., melanoma (e.g., metastatic melanoma)), soft tissue sarcoma, solid tumors, squamous cell carcinoma, synovial sarcoma, testicular cancer, thyroid cancer, transitional cell carcinoma (urothelial cell carcinoma), uveal melanoma (e.g., metastatic), verrucous carcinoma, vulvar cancer, and Waldenstrom macroglobulinemia.

[0361] In certain embodiments, the cancer treated according to the methods described herein is a human sarcoma or carcinoma, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphoendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma , bronchogenic carcinoma, renal cell carcinoma (e.g., metastatic), liver tumor, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma or retinoblastoma.

[0362] In certain embodiments, the cancer treated in accordance with the methods described herein is angiosarcoma.

[0363] In certain embodiments, the cancer treated according to the methods described herein is acute lymphocytic leukemia or acute myeloid leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia or chronic lymphocytic leukemia); Hodgkin's disease; non-Hodgkin's disease; acute myeloid leukemia; B-cell lymphoma; T-cell lymphoma; anaplastic large cell lymphoma; intraocular lymphoma; follicular lymphoma; small intestinal lymphoma; or splenic marginal zone lymphoma.

[0364] In certain embodiments, the cancer treated according to the methods described herein is multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, gastrointestinal stromal tumor, head and / or neck cancer (e.g., squamous cell carcinoma of the tongue, squamous cell carcinoma of the larynx, cell carcinoma of the oropharynx, or verrucous carcinoma of the larynx), endometrial stromal sarcoma, mast cell sarcoma, adult soft tissue sarcoma, uterine sarcoma, merkel cell carcinoma, urothelial carcinoma, melanoma with brain metastasis, uveal melanoma, uveal melanoma with liver metastasis, non-small cell lung cancer, rectal cancer, or myelodysplastic syndrome. In some embodiments, the cancer treated according to the methods is metastatic.

[0365] In certain embodiments, the cancer treated according to 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, oral cancer or pharyngeal cancer, non-Hodgkin's lymphoma, thyroid cancer, kidney cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, squamous cell carcinoma, mesothelioma, bone cancer, thymus cancer (thyoma / thymic carcinoma), glioblastoma, myelodysplastic syndrome, soft tissue sarcoma, DIPG, adenocarcinoma, osteosarcoma, chondrosarcoma, leukemia or pancreatic cancer. In some embodiments, the cancer treated according to the methods described herein includes a carcinoma (e.g., adenocarcinoma), a lymphoma, a blastoma, a melanoma, a sarcoma or a leukemia.

[0366] In certain embodiments, the cancer treated according to the methods described herein is squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer (e.g., hepatoma and liver tumor), bladder cancer, breast cancer, inflammatory breast cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, bladder cancer, endometrial cancer, myeloma (e.g., multiple myeloma), salivary gland cancer, kidney cancer (e.g., renal cell carcinoma and Wilms' tumor), basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, serous adenocarcinoma or various types of head and neck cancer. In certain embodiments, the cancer treated according to the methods described herein includes desmoplastic melanoma, inflammatory breast cancer, thymoma, rectal cancer, anal cancer or surgically treatable or non-surgically treatable brainstem glioma. In a specific embodiment, the cancer is a solid tumor. In another specific embodiment, the cancer is glioblastoma multiforme. In some embodiments, glioblastoma multiforme is recurrent. In some embodiments, glioblastoma multiforme is newly diagnosed. In some embodiments, glioblastoma multiforme is in a subject with a non-methylated MGMT promoter. In some embodiments, glioblastoma multiforme is refractory to bevacizumab therapy. In some embodiments, glioblastoma multiforme is in a subject who has not yet received bevacizumab therapy.

[0367] In certain embodiments, the cancer treated according to the methods described herein is metastatic melanoma (e.g., resistant metastatic melanoma), metastatic ovarian cancer, or metastatic renal cell carcinoma. In certain embodiments, the cancer treated according to the methods described herein is a melanoma resistant to Ipilimumab. In some embodiments, the cancer treated according to the methods described herein is a melanoma resistant to Nivolumab or Pembrolizumab. In some embodiments, the cancer treated according to the methods described herein is a melanoma resistant to Ipilimumab and Nivolumab or Pembrolizumab.

[0368] In certain embodiments, the cancer treated according to the methods described herein is breast cancer (e.g., herceptin-resistant breast cancer and trastuzumab-DM1 (T-DM1)-resistant breast cancer), prostate cancer, glioblastoma multiforme, colorectal cancer, sarcoma, bladder cancer, cervical cancer, HPV-related cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer, rectal cancer, oropharyngeal cancer, multiple myeloma, renal cell carcinoma, ovarian cancer, hepatocellular carcinoma, endometrial cancer, pancreatic cancer, lymphoma, and leukemia (e.g., senile leukemia, acute myeloid leukemia (AML) and senile AML).

[0369] In certain embodiments, the cancer treated according to the methods described herein is metastatic malignant melanoma (e.g., cutaneous or intraocular malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), bone cancer, pancreatic cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, cancer of the endocrine system, Thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney cancer or ureteral cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including asbestos induced cancers), esophageal cancer, liver cancer, refractory or recurrent malignancies, metastatic cancers, cancers expressing PD-L1, and combinations of the above cancers.

[0370] In certain embodiments, the subject has previously received immunotherapy. In certain embodiments, the subject has not previously received any immunotherapy. In certain embodiments, the cancer is advanced or metastatic cancer.

[0371] In certain embodiments, the present disclosure provides a method for preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of an anti-CTLA-4 antibody or a pharmaceutical composition thereof as described herein. In one embodiment, provided herein is a method for preventing and / or treating an infection (e.g., a viral infection, a bacterial infection, a fungal infection, a protozoan infection, or a parasitic infection). The infection prevented and / or treated according to the method may be caused by an infectious agent identified herein. In a specific embodiment, the anti-CTLA-4 antibody or a composition thereof described herein is the only active agent administered to the subject. In some embodiments, the anti-CTLA-4 antibody or a composition thereof described herein is used in combination with an anti-infective intervention (e.g., an antiviral agent, an antibacterial agent, an antifungal agent, or an antihelminthic agent) for treating an infectious disease.

[0372] Infectious diseases that can be treated and / or prevented by the anti-CTLA-4 antibodies or pharmaceutical compositions described herein are caused by infectious agents including, but not limited to, bacteria, parasites, fungi, protozoa, and viruses. In a specific embodiment, the infectious disease treated and / or prevented by the 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 according to the methods described herein include, but are not limited to, those caused by hepatitis A, hepatitis B, hepatitis C, influenza (e.g., influenza A or influenza B), varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, papovavirus, cytomegalovirus, spinovirus, arbovirus, hantavirus, coxsackie virus, mumps virus, measles virus, rubella virus, poliovirus, smallpox, Epstein Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), and the agents of viral diseases such as viral meningitis, encephalitis, dengue fever, or smallpox.

[0373] Bacterial infections that may be prevented and / or treated include infections caused by Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris, Staphylococcus viridans, and Pseudomonas aeruginosa. Bacterial diseases caused by bacteria (e.g., Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris, Staphylococcus viridans, and Pseudomonas aeruginosa) that can be prevented and / or treated according to the methods described herein include, but are not limited to, Mycobacteria rickettsia, Mycoplasma, Neisseria, S. pneumoniae, Borrelia burgdorferi (Lyme disease), Bacillus antracis (anthrax), tetanus, Streptococcus, Staphylococcus, Mycobacterium, pertussis, cholera, plague, diphtheria, Chlamydia, Staphylococcus aureus, and Legionella.

[0374] Protozoan diseases or protozoan infections caused by protozoa that can be prevented and / or treated according to the methods described herein include, but are not limited to, leishmania, coccidiosis, trypanosoma schistosomiasis, or malaria. Parasitic diseases or parasitic infections caused by parasites that can be prevented and / or treated according to the methods described herein include, but are not limited to, chlamydia and rickettsia.

[0375] Fungal diseases or infections that can be prevented and / or treated according to the methods described herein include, but are not limited to, those caused by Candida infection, zygomycosis, Candida mastitis, progressive disseminated trichosporonosis with latent trichosporonemia, disseminated candidiasis, pulmonary paracoccidioidomycosis, pulmonary aspergillosis, Pneumocystis carinii pneumonia, cryptococcal meningitis, coccidioidal meningoencephalitis and cerebrospinal vasculitis, Aspergillus niger infection, Fusarium keratitis, sinusomycosis, Aspergillus fumigatus endocarditis, tibial dyschondroplasia, Candida glabrata, glabrata vaginitis, oropharyngeal candidiasis, X-linked chronic granulomatous disease, tinea pedis, cutaneous candidiasis, mycotic placenta, disseminated trichosporonosis, allergic bronchopulmonary aspergillosis, mycotic keratitis, Cryptococcus neoformans infection, fungal peritonitis, Curvularia geniculata infection, staphylococcal endophthalmitis, sporotrichosis, and dermatophytosis.

[0376] In certain embodiments, the infectious disease is acute. In certain embodiments, the infectious disease is chronic. In certain embodiments, the infectious disease is caused by a flavivirus, such as West Nile virus, Saint Louis encephalitis 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 certain embodiments, the infectious disease is caused by human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV). In certain embodiments, an anti-CTLA-4 antibody or a pharmaceutical composition thereof as described herein promotes viral control. In certain embodiments, an anti-CTLA-4 antibody or a pharmaceutical composition thereof as described herein eliminates viral reservoirs.

[0377] In one aspect, the invention relates to 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 for use as a medicament.

[0378] In one aspect, the present invention relates to an anti-CTLA-4 antibody of the present invention and / or its use in combination with a pharmaceutically acceptable carrier or excipient for the preparation of a pharmaceutical composition or medicament for immunotherapy (e.g., immunotherapy for increasing T cell activation in response to an antigen in a subject, treating cancer, or treating or preventing an infectious disease).

[0379] In one aspect, the invention relates to 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 for use in a method for treating cancer.

[0380] In one aspect, the present invention relates to an anti-CTLA-4 antibody of the present invention and / or a pharmaceutical composition of the present invention comprising an anti-CTLA-4 antibody of the present invention and a pharmaceutically acceptable carrier or excipient, for use in a method of suppressing immune system tolerance to tumors and / or for immunotherapy of a subject suffering from cancer.

[0381] In one aspect, the invention relates to 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 for use in a method for treating an infectious disease.

[0382] In certain embodiments, an anti-CTLA-4 antibody or pharmaceutical composition described herein is administered as a monotherapy.

[0383] In certain embodiments, the methods further comprise administering an additional therapeutic agent to the subject. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent or a checkpoint targeting agent. In certain embodiments, the checkpoint targeting agent is selected from the group consisting of: antagonist anti-PD-1 antibodies, antagonist anti-PD-L1 antibodies, antagonist anti-PD-L2 antibodies, antagonist anti-CTLA-4 antibodies, antagonist anti-TIM-3 antibodies, antagonist anti-LAG-3 antibodies, antagonist anti-CEACAM1 antibodies, agonist anti-GITR antibodies, agonist anti-OX40 antibodies, agonist anti-CD137 antibodies, agonist anti-DR3 antibodies, agonist anti-TNFSF14 antibodies, and agonist anti-CD27 antibodies. In certain embodiments, the checkpoint targeting agent is an antagonist anti-PD-1 antibody. In certain embodiments, the checkpoint targeting agent is an antagonist anti-PD-L1 antibody. In certain embodiments, the checkpoint targeting agent is an antagonist anti-LAG-3 antibody. In certain embodiments, the additional therapeutic agent is a tumor necrosis factor receptor superfamily member or an agonist of a tumor necrosis factor superfamily member.

[0384] In certain embodiments, the 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 as a medicament. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic agent or a checkpoint targeting agent.

[0385] In certain embodiments, the 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 of treating cancer.

[0386] In certain embodiments, the 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 of treating an infectious disease.

[0387] In certain embodiments, the anti-CTLA-4 antibodies described herein are administered to a subject in combination with a compound that targets an immunomodulatory enzyme, such as IDO (indoleamine-(2,3)-dioxygenase) and / or TDO (tryptophan 2,3-dioxygenase). In certain embodiments, such compounds are selected from the group consisting of: icaridostat (Incyte Corp; see, e.g., WO 2010 / 005958, which is incorporated herein by reference in its entirety), F001287 (Flexus Biosciences), indolemod (NewLink Genetics), and NLG919 (NewLink Genetics). In one embodiment, the compound is icaridostat. In another embodiment, the compound is F001287. In another embodiment, the compound is indolemod. In another embodiment, the compound is NLG919.

[0388] In certain embodiments, the present invention relates to (a) an anti-CTLA-4 antibody of the present invention and / or a compound comprising an anti-CTLA-4 antibody of the present invention and a pharmaceutically acceptable carrier or excipient and (b) a compound targeting an immunomodulatory enzyme for use as a medicament. In a preferred embodiment, the compound targets IDO and / or TDO.

[0389] In certain embodiments, the present invention relates to (a) an anti-CTLA-4 antibody of the present invention and / or a compound comprising an anti-CTLA-4 antibody of the present invention and a pharmaceutically acceptable carrier or excipient and (b) a compound targeting an immunomodulatory enzyme for use in a method for treating cancer. In a preferred embodiment, the compound targets IDO and / or TDO.

[0390] In certain embodiments, the anti-CTLA-4 antibodies described herein are administered to a subject in combination with a vaccine. In certain embodiments, the vaccine is a tumor vaccine based on heat shock proteins or a pathogen vaccine based on heat shock proteins. In a specific embodiment, the anti-CTLA-4 antibodies described herein are administered to a subject in combination with a tumor vaccine based on heat shock proteins. Heat shock proteins (HSPs) are a highly conserved protein family that is ubiquitous in all species. Due to heat shock or other forms of stress, including exposure to toxins, oxidative stress, or glucose deprivation, their expression can be strongly induced to much higher levels. Five families have been classified according to molecular weight: HSP-110, -90, -70, -60, and -28. HSPs deliver immunogenic peptides through cross-presentation pathways in antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs), causing T cell activation. HSPs act as chaperone carriers for tumor-associated antigenic peptides, forming complexes that can induce tumor-specific immunity. After release from dying tumor cells, HSP-antigen complexes are taken up by antigen presenting cells (APCs), where the antigens are processed into peptides that bind to MHC class I and class II molecules, leading to the activation of anti-tumor CD8+ and CD4+ T cells. The immunity elicited by HSP complexes derived from tumor preparations is specific to the unique spectrum of antigenic peptides expressed by each subject's cancer.

[0391] Heat shock protein peptide complex (HSPPC) is a protein peptide complex composed of heat shock proteins non-covalently complexed with antigenic peptides. HSPPC triggers innate and adaptive immune responses. In a specific embodiment, the antigenic peptide presents antigenicity to the cancer being treated. HSPPC is efficiently captured by APC via membrane receptors (mainly CD91) or by binding to Toll-like receptors. HSPPC internalization leads to functional maturation of APC, accompanied by the production of chemokines and cytokines, leading to the activation of natural killer cells (NK), monocytes, and Th1 and Th-2 mediated immune responses. In certain embodiments, the HSPPC used in the methods described herein comprises one or more heat shock proteins from the hsp60, hsp70 or hsp90 stress protein family complexed with antigenic peptides. In certain embodiments, HSPPC comprises hsc70, hsp70, hsp90, hsp110, grp170, gp96, calreticulin or a combination of two or more thereof.

[0392] In a specific embodiment, the anti-CTLA-4 antibodies described herein are administered to a subject in combination with a heat shock protein peptide complex (HSPPC), e.g., heat shock protein peptide complex-96 (HSPPC-96), to treat cancer. HSPPC-96 comprises a 96 kDa heat shock protein (Hsp), gp96, complexed with an antigenic peptide. HSPPC-96 is a cancer immunotherapy made from a subject's tumor and contains an antigenic "fingerprint" of the cancer. In certain embodiments, this fingerprint contains unique antigens that are present only in specific cancer cells of the specific subject, and the injected vaccine is intended to stimulate the subject's immune system to recognize and attack any cells with a specific cancer fingerprint.

[0393] In certain embodiments, HSPPC (e.g., HSPPC-96) is produced by tumor tissue of a subject. In a specific embodiment, HSPPC (e.g., HSPPC-96) is produced by a tumor of the type of cancer being treated or its metastasis. In another specific embodiment, HSPPC (e.g., HSPPC-96) is the subject being treated itself. In certain embodiments, the tumor tissue is non-necrotic tumor tissue. In certain embodiments, at least 1 gram (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 grams) of non-necrotic tumor tissue is used to produce a vaccine regimen. In certain embodiments, after surgical resection, non-necrotic tumor tissue is frozen before being used for vaccine preparation. In certain embodiments, HSPPC (e.g., HSPPC-96) is separated, filtered and prepared for injectable vaccines from tumor tissue by purification technology. In certain embodiments, 6-12 doses of HSPPC, e.g., HSPCC-96, are administered to a subject. In such embodiments, the first 4 of the HSPPC (eg, HSPPC-96) doses may be administered weekly, and then 2-8 additional doses administered every two weeks.

[0394] Other examples of HSPPCs that can be used in accordance with the methods described herein are disclosed in the following patents and patent applications, which are incorporated herein by reference in their entireties: U.S. Pat. Nos. 6,391,306, 6,383,492, 6,403,095, 6,410,026, 6,436,404, 6,447,780, 6,447,781, and 6,610,659, all of which are incorporated herein by reference in their entireties.

[0395] In certain embodiments, the present invention relates to (a) an anti-CTLA-4 antibody of the present invention and / or a composition comprising an anti-CTLA-4 antibody of the present invention and a pharmaceutically acceptable carrier or excipient and (b) a vaccine for use as a medicament. In a preferred embodiment, the vaccine is a tumor vaccine based on heat shock protein or a pathogen vaccine based on heat shock protein. In a preferred embodiment, the vaccine is a viral vaccine based on heat shock protein.

[0396] In certain embodiments, the present invention relates to (a) an anti-CTLA-4 antibody of the present invention and / or a method comprising an anti-CTLA-4 antibody of the present invention and a pharmaceutically acceptable carrier or excipient and (b) a vaccine for use in a method for treating cancer. In a preferred embodiment, the vaccine is a tumor vaccine based on heat shock protein.

[0397] The anti-CTLA-4 antibody and the additional therapeutic agent (e.g., a chemotherapeutic agent, a checkpoint targeting agent, an IDO inhibitor, and / or a vaccine) can be administered separately, sequentially, or simultaneously in separate dosage forms. In one embodiment, the anti-CTLA-4 antibody is administered parenterally, and the IDO inhibitor is administered orally.

[0398] In certain embodiments, the anti-CTLA-4 antibodies described herein are administered intratumorally to a subject. In certain embodiments, the anti-CTLA-4 antibodies described herein are administered intratumorally to a subject in combination with an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is administered systemically. In certain embodiments, the subject has a solid tumor. In certain embodiments, the subject has head and neck squamous cell carcinoma (HNSCC). In certain embodiments, the subject has HER2 + Breast cancer. In certain embodiments, the additional therapeutic agent administered systemically is an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab). In certain embodiments, the additional therapeutic agent administered systemically is an anti-EGFR antibody (e.g., cetuximab). In certain embodiments, the additional therapeutic agent administered systemically is an anti-HER2 antibody (e.g., trastuzumab). In certain embodiments, the additional therapeutic agent administered systemically is a chemotherapeutic agent (e.g., gemcitabine). In certain embodiments, the subject has a solid tumor, and the additional therapeutic agent administered systemically is an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab). In certain 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 administered systemically is an anti-EGFR antibody (e.g., cetuximab). In certain embodiments, the subject has HER2 +Breast cancer, and the additional therapeutic agent administered systemically is an anti-HER2 antibody (e.g., trastuzumab). In certain embodiments, the subject further receives a chemotherapeutic agent (e.g., gemcitabine). 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 treating cancer, wherein the anti-CTLA-4 antibody and / or pharmaceutical composition of the present invention is administered intratumorally to the subject. In a preferred embodiment, an additional therapeutic agent is administered to the subject, more preferably, an additional therapeutic agent is administered systemically to the subject.

[0399] In certain embodiments, anti-PD-1 antibodies are used in the methods described herein. In certain embodiments, anti-PD-1 antibodies are nivolumab developed by Bristol-Myers Squibb, also known as BMS-936558 or MDX1106. In certain embodiments, anti-PD-1 antibodies are pembrolizumab developed by Merck & Co, also known as Lambrolizumab or MK-3475. In certain embodiments, anti-PD-1 antibodies are pidilizumab developed by CureTech, also known as CT-011. In certain embodiments, anti-PD-1 antibodies are MEDI0680 developed by Medimmune, also known as AMP-514. In certain embodiments, anti-PD-1 antibodies are PDR001 developed by Novartis Pharmaceuticals. In certain embodiments, anti-PD-1 antibodies are REGN2810 developed by Regeneron Pharmaceuticals. In certain embodiments, the anti-PD-1 antibody is PF-06801591 developed by Pfizer. In certain embodiments, the anti-PD-1 antibody is BGB-A317 developed by BeiGene. In certain embodiments, the anti-PD-1 antibody is TSR-042 developed by AnaptysBio and Tesaro. In certain embodiments, the anti-PD-1 antibody is SHR-1210 developed by Hengrui.

[0400] Other non-limiting examples of anti-PD-1 antibodies that can be used in the treatment methods described herein are disclosed in the following patents and patent applications, which are incorporated herein by reference in their entirety 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 ...14,845, U.S. Patent No. 8,168,757, US Patent No. 8,354,509, US Patent No. 8,686,119, US Patent No. 8,735,553, US Patent No. 8,747,847, US Patent No. 8,779,105, US Patent No. 8,927,697, US Patent No. 8,993,731, US Patent No. 9,102,727, US Patent No. 9,205,148; US Publication No. US2013 / 0202623 A1, U.S. Publication No. US 2013 / 0291136A1, U.S. Publication No. US2014 / 0044738 A1, U.S. Publication No. US2014 / 0356363 A1, U.S. Publication No. US2016 / 0075783 A1; and PCT Publication No. WO 2013 / 033091 A1, PCT Publication No. WO 2015 / 036394A1, PCT Publication No. WO2014 / 179664 A2, PCT Publication No. WO 2014 / 209804A1, PCT Publication No. WO 2014 / 206107 A1, PCT Publication No. WO 2015 / 058573 A1, PCT Publication No. WO 2015 / 085847 A1, PCT Publication No. WO 2015 / 200119 A1, PCT Publication No. WO 2016 / 015685 A1, and PCT Publication No. WO 2016 / 020856 A1.

[0401] In certain embodiments, an anti-PD-L1 antibody is used in the methods 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 Medical Immunity. In certain embodiments, the anti-PD-L1 antibody is avelumab, also known as MSB0010718C, developed by Merck Serono and Pfizer. In certain embodiments, the anti-PD-L1 antibody is MDX-1105 developed by Bristol-Myers Squibb. In certain embodiments, the anti-PD-L1 antibody is AMP-224 developed by Amplimmune and GSK.

[0402] Non-limiting examples of anti-PD-L1 antibodies that can be used in the treatment methods described herein are disclosed in the following patents and patent applications, which are incorporated herein by reference in their entirety for all purposes: U.S. Patent No. 7,943,743, U.S. Patent No. 8,168,179, U.S. 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. US2010 / 0203056 A1, U.S. Publication No. US2003 / 0232323 A1, U.S. Publication No. US2013 / 0323249 A1, U.S. Publication No. US 2014 / 0341917 A1, U.S. Publication No. US2014 / 0044738 A1, U.S. Publication No. US2015 / 0203580 A1, U.S. Publication No. US2015 / 0225483 A1, U.S. Publication No. US2015 / 0346208 A1, U.S. Publication No. US2015 / 0355184 A1; and PCT Publication No. WO 2014 / 100079 A1, PCT Publication No. WO 2014 / 022758 A1, PCT Publication No. WO 2014 / 055897 A2, PCT Publication No. WO 2015 / 061668 A1, PCT Publication No. WO 2015 / 109124 A1, PCT Publication No. WO 2015 / 195163 A1, PCT Publication No. WO 2016 / 000619 A1 and PCT Publication No. WO 2016 / 030350 A1.

[0403] In certain embodiments, an anti-LAG-3 antibody is used in the methods described herein. In certain embodiments, the anti-LAG-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.

[0404] Non-limiting examples of anti-LAG-3 antibodies that can be used in the treatment methods described herein are disclosed in the following patents and patent applications, which are incorporated herein by reference in their entirety for all purposes: U.S. Patent No. 9,244,059; U.S. Publication No. US2011 / 0150892A1, U.S. Publication No. US2014 / 0093511 A1, U.S. Publication No. US 2014 / 0286935A1, U.S. Publication No. US2015 / 0259420 A1; and PCT Publication No. WO 2015 / 042246 A1, PCT Publication No. WO 2015 / 116539 A1, PCT Publication No. WO 2015 / 200119 A1, and PCT Publication No. WO 2016 / 028672 A1.

[0405] In certain embodiments, anti-EGFR antibodies are used in the methods described herein. In certain embodiments, anti-EGFR antibodies are cetuximab developed by Bristol-Myers Squibb and ImClone, panitumumab developed by Abgenix and Amgen, and levofloxacin developed by CMI Cuba and YM Biotechnology. nimotuzumab developed by BioSciences, necitumumab developed by Imclone, zalutumumab developed by Genmab, matuzumab developed by Takeda, Sym004 developed by Merck Serono and Symphogen, imgatuzumab developed by Glycart 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 developed by Amgen and ImmunoGen 595, CetuGEX developed by Glycotope, laprituximab emtansine developed by Ingenics, 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.

[0406] In certain embodiments, an anti-HER2 antibody is used in the methods described herein. In certain embodiments, an anti-HER2 antibody is trastuzumab developed by Genentech and Roche, trastuzumab emtacin developed by Genentech and Roche, pertuzumab developed by Genentech, ertumaxomab developed by Fresenius, margetuximab developed by MacroGenics, MM-111 developed by Merrimack, CT-P06 developed by Celltrion, PF-05280014 developed by Pfizer, MM-302 developed by Merrimack, SB3 developed by Merck, CMAB302 developed by Shanghai CP Guojian, TrasGEX developed by Glycotope, ERTUMAX developed by Ambrx and Zhejiang Medicine 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.

[0407] The antibodies or pharmaceutical compositions described herein can be delivered to the subject by a variety of routes. These routes include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, and subcutaneous routes. Pulmonary administration can also be used, for example, by using an inhaler or nebulizer, and a formulation containing an atomizer suitable for use as a spray. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered subcutaneously or intravenously. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered intratumorally. In certain embodiments, the anti-CTLA-4 antibodies or pharmaceutical compositions described herein are delivered to tumor-draining lymph nodes. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered via local administration (e.g., subcutaneous administration). In certain embodiments, the anti-CTLA-4 antibodies or pharmaceutical compositions described herein are delivered systemically. In certain embodiments, the anti-CTLA-4 antibodies or pharmaceutical compositions described herein are delivered locally.

[0408] In one aspect, the invention relates to an anti-CTLA-4 antibody and / or pharmaceutical composition of the invention, and optionally an additional therapeutic agent, for use in a method of treating cancer, wherein the anti-CTLA-4 antibody and / or pharmaceutical composition of the invention is delivered intratumorally to a subject, is delivered to a tumor-draining lymph node of a subject, or is delivered to a subject via local administration (e.g., subcutaneous administration).

[0409] The amount of the antibody or composition effective to treat and / or prevent a condition will depend on the nature of the disease, and can be determined by standard clinical techniques.

[0410] The precise dose used in the composition will also depend on the route of administration and the severity of the infection or disease caused thereby, and should be determined according to the judgment of the practitioner and the circumstances of each subject. For example, the effective dose may also vary depending on the mode of administration, the target site, the physiological state of the patient (including age, weight and health), whether the patient is human or animal, other agents administered, or whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, may also be treated. The therapeutic dose is optimally titrated to optimize safety and efficacy.

[0411] In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a 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, 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 (e.g., via intravenous injection). In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject weekly, 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, for example, at the doses described above (e.g., via intravenous injection). In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject at the doses described above (e.g., via intravenous injection) every three weeks.

[0412] In one aspect, the invention relates to an anti-CTLA-4 antibody and / or pharmaceutical composition of the invention, and optionally an additional therapeutic agent, for use in a method of treating cancer, wherein the anti-CTLA-4 antibody and / or pharmaceutical composition of the invention is administered to a 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, 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.

[0413] In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to the subject at 0.1 mg / kg (e.g., via intravenous injection) every two weeks, every three weeks, every four weeks, every six weeks, or every twelve weeks. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to the subject at 0.3 mg / kg (e.g., via intravenous injection) every two weeks, every three weeks, every four weeks, every six weeks, or every twelve weeks. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to the subject at 1 mg / kg (e.g., via intravenous injection) every two weeks, every three weeks, every four weeks, every six weeks, or every twelve weeks. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to the subject at 3 mg / kg (e.g., via intravenous injection) 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 at 6 mg / kg (e.g., via intravenous injection) 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 at 10 mg / kg (e.g., via intravenous injection) every two weeks, every three weeks, every four weeks, every six weeks, or every twelve weeks.

[0414] In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to the subject at 0.1 mg / kg or about 0.1 mg / kg (e.g., via intravenous injection) every three weeks. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to the subject at 0.3 mg / kg or about 0.3 mg / kg (e.g., via intravenous injection) every three weeks. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to the subject at 1 mg / kg or about 1 mg / kg (e.g., via intravenous injection) every three weeks. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to the subject at 3 mg / kg or about 3 mg / kg (e.g., via intravenous injection) every three weeks. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to the subject at 6 mg / kg or about 6 mg / kg (e.g., via intravenous injection) every three weeks. In certain embodiments, an anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject at or about 10 mg / kg (eg, via intravenous injection) every three weeks.

[0415] In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject via intratumoral 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, the anti-CTLA-4 antibody or pharmaceutical composition described 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 months, every six months, every eight months, and every year, for example, at the doses described above. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject via intratumoral injection at the doses described above every three weeks.

[0416] In certain embodiments, the 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 embodiments, the anti-CTLA-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, the anti-CTLA-4 antibody or pharmaceutical composition 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, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject via intratumoral injection at 0.3 mg / kg or about 0.3 mg / kg every three weeks. In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject via intratumoral injection at 1 mg / kg or about 1 mg / kg every three weeks. In certain embodiments, the 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.

[0417] In certain embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject via topical administration (e.g., subcutaneous 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, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject via topical 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, for example, at the doses described above.

[0418] 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.

[0419] In certain embodiments, the present disclosure provides a method for treating a subject with angiosarcoma, the method comprising administering to the subject an effective amount of an anti-CTLA-4 antibody or pharmaceutical composition described herein (e.g., intravenously, intratumorally or subcutaneously). In certain embodiments, the present disclosure provides a method for treating a subject with angiosarcoma, the method comprising optionally administering to the subject 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 intravenously every week, every two weeks or every three weeks. In certain embodiments, the present disclosure provides a method for treating a subject with angiosarcoma, the method comprising administering to the subject an antibody that specifically binds to human CTLA-4 at 0.1 mg / kg intravenously once every three weeks. In certain embodiments, the antibody comprises 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 sequence of SEQ ID NO:9. In certain embodiments, 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 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 antibody comprises 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.

[0420] The anti-CTLA-4 antibodies described herein can also be used to analyze CTLA-4 protein levels in biological samples using classical immunohistological methods known to those skilled in the art, including immunoassays such as 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; radioactive isotopes such as iodine ( 125 I. 121 I), carbon ( 14 C) Sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In) 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 the antibodies or antigen-binding fragments thereof described herein. Alternatively, a second antibody that recognizes the anti-CTLA-4 antibody or antigen-binding fragment thereof described herein can be labeled and combined with the anti-CTLA-4 antibody or antigen-binding fragment thereof for detecting CTLA-4 protein levels. In one embodiment, the present invention relates to a use of an anti-CTLA-4 antibody of the present invention for analyzing and / or detecting CTLA-4 protein levels in a biological sample in vitro.

[0421] Analysis of the expression level of CTLA-4 protein is intended to include measuring or estimating the level of CTLA-4 protein in a first biological sample, either directly (e.g., by determining or estimating absolute protein levels) or relatively (e.g., by comparing to disease-associated protein levels in a second biological sample), either qualitatively or quantitatively. The level of CTLA-4 polypeptide expression in a first biological sample can be measured or estimated and compared to a standard CTLA-4 protein level, the standard being obtained from a second biological sample obtained from an individual not suffering from the disorder or determined by averaging the levels of a population of individuals not suffering from the disorder. As is understood in the art, when a "standard" CTLA-4 polypeptide level is known, it can be repeatedly used as a standard for comparison.

[0422] As used herein, the term "biological sample" refers to any biological sample obtained from a subject, cell line, tissue, or other source of cells that may express 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 mononuclear blood cells.

[0423] The anti-CTLA-4 antibodies or antigen-binding fragments thereof described herein can be used for prognostic, diagnostic, monitoring and screening applications that are well known to the skilled artisan and standard and based on the description of the present invention, including in vitro and in vivo applications. Prognostic, diagnostic, monitoring and screening assays and kits for in vitro evaluation and assessment of immune system status and / or immune response can be used to predict, judge and monitor to evaluate patient samples, including those known to have or suspected of having immune system dysfunction or about expected or desired immune system response, antigen response or vaccine response. Evaluation and assessment of immune system status and / or immune response can also be used to determine the suitability of a patient for a drug clinical trial or for administration of a specific chemotherapeutic agent or antibody or antigen-binding fragment thereof (including combinations thereof) to compare different agents or antibodies or antigen-binding fragments thereof. This type of prognostic and diagnostic monitoring and assessment has been used in practice using antibodies to HER2 protein in breast cancer (HercepTest TM , Dako), where the analytical method is also used Evaluating patients for antibody therapy. In vivo applications include directed cell therapy and immune system modulation and radiological imaging of immune responses.

[0424] In one aspect, the invention relates to an anti-CTLA-4 antibody and / or pharmaceutical composition of the invention for use as a diagnostic agent.

[0425] In one aspect, the invention relates to an anti-CTLA-4 antibody and / or pharmaceutical composition of the invention for use in a method for predicting, diagnosing and / or monitoring immune system dysfunction and / or cancer.

[0426] In one embodiment, the present invention relates to a use of an anti-CTLA-4 antibody of the present invention for predicting, diagnosing and / or monitoring a subject's immune system dysfunction and / or cancer by analyzing and / or detecting the level of CTLA-4 protein in a biological sample outside the body of the subject.

[0427] In one embodiment, the anti-CTLA-4 antibody or its antigen-binding fragment can be used in immunohistochemistry of biopsy samples. In another embodiment, the anti-CTLA-4 antibody or its antigen-binding fragment can be used to detect the level of CTLA-4 or the level of cells containing CTLA-4 on its membrane surface, which level can be associated with certain disease symptoms. The anti-CTLA-4 antibody or its antigen-binding fragment described herein can carry a detectable or functional marker. When a fluorescent marker is used, currently available microscopy and fluorescence activated cell sorter analysis (FACS) or a combination of two methods and procedures known in the art can be used to identify and quantify specific binding members. The anti-CTLA-4 antibody or its antigen-binding fragment described herein can carry a fluorescent marker. Exemplary fluorescent markers include, for example, reactive and conjugated probes, such as aminocoumarin, fluorescein and Texas red, Alexa Fluor dyes, Cy dyes and DyLight dyes. The anti-CTLA-4 antibody or its antigen-binding fragment can carry a radioactive marker, such as an isotope. 3 H. 14 C. 32 P. 35 S. 36 Cl, 51 Cr, 57 Co. 58 Co. 59 Fe, 67 Cu, 90 Y. 99 Tc, 111 In, 117 Lu, 121 I. 124 I. 125I. 131 I. 198 Au, 211 At 213 Bi, 225 Ac and 186 Re. When a radioactive label is used, currently available counting procedures known in the art can be used to identify and quantify the specific binding of anti-CTLA-4 antibodies or antigen-binding fragments thereof to CTLA-4 (e.g., human CTLA-4). In the case where the label is an enzyme, detection can be achieved by any of the currently used colorimetric, spectrophotometric, fluorescence spectrophotometric, amperometric or gas quantitative techniques as known in the art. This can be achieved by contacting a sample or a control sample with an anti-CTLA-4 antibody or antigen-binding fragment thereof under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and CTLA-4. Any complex formed between the antibody or antigen-binding fragment thereof and CTLA-4 is detected and compared in the sample and the control. In view of the specific binding of the antibodies described herein to CTLA-4, the antibodies or antigen-binding fragments thereof can be used to specifically detect CTLA-4 expression on the cell surface. 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 analytical system that can be prepared in the form of a test kit for quantitatively analyzing, for example, the presence of CTLA-4 or a CTLA-4 / CTLA-4 ligand complex. A system or test kit can include a labeled component, eg, a labeled antibody, and one or more additional immunochemical reagents.

[0428] In one embodiment, the invention relates to an in vitro method for analyzing and / or detecting CTLA-4 protein levels in a biological sample, comprising (1) contacting the sample and optionally a control sample with an anti-CTLA-4 antibody or antigen-binding fragment thereof of the invention under conditions that allow formation of a complex between the antibody or antigen-binding fragment thereof and CTLA-4, and (2) detecting and comparing the complex formed in the sample and optionally the control.

[0429] 1.5 Polynucleotides, vectors and methods for producing anti-CTLA-4 antibodies

[0430] In another aspect, polynucleotides are provided herein, comprising nucleotide sequences encoding antibodies described herein or fragments thereof (e.g., light chain variable regions and / or heavy chain variable regions) that specifically bind to CTLA-4 (e.g., human CTLA-4) antigens; and vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells). Provided herein are polynucleotides comprising nucleotide sequences encoding any of the antibodies provided herein; and vectors comprising such polynucleotide sequences, e.g., expression vectors for their efficient expression in host cells (e.g., mammalian cells).

[0431] As used herein, an "isolated" polynucleotide or nucleic acid molecule is a polynucleotide or nucleic acid molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule (e.g., in a mouse or a human). In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the phrase "substantially free" includes polynucleotide or nucleic acid molecule preparations having less than about 15%, 10%, 5%, 2%, 1%, 0.5% or 0.1% (specifically, less than about 10%) of other substances (e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors and / or other chemicals). In a specific embodiment, the nucleic acid molecules encoding the antibodies described herein are isolated or purified.

[0432] In a specific aspect, provided herein are polynucleotides comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to a CTLA-4 polypeptide (e.g., human CTLA-4) and comprises an amino acid sequence as described herein, as well as antibodies that compete with such antibodies for binding to a CTLA-4 polypeptide (e.g., in a dose-dependent manner) or bind to the same epitope as such antibodies.

[0433] In certain aspects, polynucleotides are provided herein, comprising a nucleotide sequence encoding a light chain or heavy chain of an antibody as described herein. Polynucleotides may comprise a nucleotide sequence encoding a light chain comprising VL FR and CDR of an antibody as described herein (see, e.g., Table 1).

[0434] Also provided herein are polynucleotides encoding anti-CTLA-4 antibodies that are optimized, for example, by codon / RNA optimization, replacement by heterologous signal sequences, and elimination of mRNA unstable elements. Methods for generating optimized nucleic acids encoding anti-CTLA-4 antibodies or fragments 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 mRNA can be performed by adapting, for example, the optimization methods described in U.S. Pat. Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498, and accordingly, all of the patents are incorporated herein by reference in their entirety. For example, potential splice sites and unstable elements (e.g., A / T or A / U-rich elements) within RNA can be mutated to improve the stability of RNA for recombinant expression without changing the amino acid encoded by the nucleic acid sequence. The changes utilize the degeneracy of the genetic code, for example, using alternative codons for consistent amino acids. In some embodiments, it may be desirable to change one or more codons to encode a conservative mutation, e.g., an analogous amino acid having a 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, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more relative to expression of an anti-CTLA-4 antibody encoded by a non-optimized polynucleotide.

[0435] In certain embodiments, an optimized polynucleotide sequence encoding an anti-CTLA-4 antibody or fragment thereof (e.g., a VL domain and / or a VH domain) described herein can be hybridized with an antisense (e.g., complementary) polynucleotide of a non-optimized polynucleotide sequence encoding an anti-CTLA-4 antibody or fragment thereof (e.g., a VL domain and / or a VH domain) described herein. In a specific embodiment, an optimized nucleotide sequence encoding an anti-CTLA-4 antibody or fragment described herein hybridizes with an antisense polynucleotide of a non-optimized polynucleotide sequence encoding an anti-CTLA-4 antibody or fragment thereof described herein under high stringency conditions. In a specific embodiment, an optimized nucleotide sequence encoding an anti-CTLA-4 antibody or fragment thereof described herein hybridizes with an antisense polynucleotide of a non-optimized nucleotide sequence encoding an anti-CTLA-4 antibody or fragment thereof described herein under high stringency, medium, or low stringency hybridization conditions. Information about hybridization conditions has been described, see, for example, U.S. Patent Application Publication No. US2005 / 0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference.

[0436] By any method known in the art, polynucleotides can be obtained, and the nucleotide sequence of polynucleotides is measured. The nucleotide sequence encoding antibodies described herein (for example, antibodies described in Table 1) and the modified forms of these antibodies can be measured using methods well known in the art, that is, the nucleotide codons of known encoding specific amino acids are assembled in a manner to produce nucleic acids encoding antibodies. Such polynucleotides encoding antibodies can be assembled by chemically synthesized oligonucleotides (for example, such as Kutmeier G et al., (1994), " Biotechnology (BioTechniques) " 17: described in 242-6), which simply relate to the synthesis of overlapping oligonucleotides containing the part of the sequence encoding the antibody, annealing and connecting those oligonucleotides, and then amplifying the connected oligonucleotides by PCR.

[0437] Alternatively, polynucleotides encoding antibodies described herein can be produced from nucleic acids from a suitable source (e.g., hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers that can hybridize with the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells that produce the target antibody. Such PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding light and / or heavy chains of antibodies. Such PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding variable light and / or variable heavy chain regions of antibodies. Amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to produce chimeric and humanized antibodies.

[0438] If a clone containing nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, then nucleic acid encoding the immunoglobulin can be chemically synthesized or by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence or by cloning using oligonucleotide probes specific for a particular gene sequence to identify, for example, cDNA clones from a cDNA library encoding the antibody, from a suitable source (e.g., an antibody cDNA library or a cDNA library produced by any tissue or cell expressing the antibody (e.g., a hybridoma cell selected to express an antibody described herein), or nucleic acid isolated therefrom (preferably poly A+RNA). The amplified nucleic acid produced by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0439] DNA encoding the anti-CTLA-4 antibodies described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the anti-CTLA-4 antibodies). Hybridoma cells can serve as a source of such DNA. After isolation, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., from CHO GS System TM Synthesis of anti-CTLA-4 antibodies in recombinant host cells can be obtained by incubating the cells in CHO cells (Lonza) or in myeloma cells that do not otherwise produce immunoglobulin.

[0440] In order to produce a full antibody, PCR primers including VH or VL nucleotide sequences, restriction sites, and flanking sequences protecting restriction sites can be used to amplify the VH or VL sequences in scFv clones. Using cloning techniques known to those skilled in the art, the VH domain amplified by PCR can be cloned into a vector expressing a heavy chain constant region (e.g., a human γ4 constant region), and the VL domain amplified by PCR can be cloned into a vector expressing a light chain constant region (e.g., a human κ or λ constant region). In certain embodiments, the vector for expressing the VH or VL domain comprises an EF-1α promoter, a secretion signal, a cloning site for the variable region, a constant domain, and a selection marker (e.g., neomycin). The VH and VL domains can also be cloned into a vector expressing the necessary constant region. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line using techniques known to those skilled in the art to produce a stable or transient cell line expressing a full-length antibody (e.g., IgG).

[0441] The DNA also may be modified, for example, by substituting coding sequences for human heavy and light chain constant domains in place of the murine sequences or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0442] Also provided are polynucleotides that hybridize to polynucleotides encoding antibodies described herein under high stringency, medium or low stringency hybridization conditions. In specific embodiments, polynucleotides described herein hybridize to polynucleotides encoding VH domains and / or VL domains provided herein under high stringency, medium or low stringency hybridization conditions.

[0443] Hybridization conditions have been described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions may include hybridization of DNA bound to the filter in 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC / 0.1% SDS at about 50-65° C.; hybridization under highly stringent conditions may include hybridization of nucleic acids bound to the filter in 6×SSC at about 45° C., followed by one or more washes in 0.1×SSC / 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, e.g., Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pp. 6.3.1-6.3.6 and 2.10.3, incorporated herein by reference in its entirety.

[0444] In certain aspects, cells (e.g., host cells) that express (e.g., recombinantly) an antibody (or antigen-binding fragment thereof) described herein that specifically binds to CTLA-4 (e.g., human CTLA-4) are provided herein, and related polynucleotides and expression vectors. Vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding anti-CTLA-4 antibodies or fragments for recombinant expression in host cells, preferably mammalian cells, are provided herein. Host cells comprising such vectors for recombinant expression of anti-CTLA-4 antibodies (e.g., human or humanized antibodies) described herein are also provided herein. In a specific aspect, methods for producing antibodies described herein are provided herein, comprising expressing such antibodies from host cells.

[0445] Recombinant expression of an antibody described herein (e.g., a full-length antibody, a heavy chain and / or a light chain of an antibody, or a single-chain antibody) that specifically binds to CTLA-4 (e.g., human CTLA-4) includes constructing an expression vector containing a polynucleotide encoding the antibody. After obtaining a polynucleotide encoding an antibody molecule, an antibody heavy chain and / or a light chain, or a fragment thereof (e.g., a heavy chain and / or a light chain variable region) as described herein, a vector for producing an antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Thus, a method for preparing a protein by expressing a polynucleotide containing an antibody or antibody fragment (e.g., a light chain or a heavy chain) encoding nucleotide sequence is described herein. Methods well known to those skilled in the art can be used to construct an expression vector containing an antibody or antibody fragment (e.g., a light chain or a heavy chain) encoding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided is a replicable vector comprising a nucleotide sequence encoding an antibody molecule described herein, an antibody heavy chain or a light chain, an antibody heavy chain or a light chain variable region, or a heavy chain or a light chain CDR operably linked to a promoter. Such vectors may, for example, include nucleotide sequences encoding the constant regions of antibody molecules (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Pat. No. 5,122,464, which are incorporated herein by reference in their entireties), and the variable regions of the antibodies may be cloned into such vectors for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

[0446] The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques and the resulting cell can then be cultured by conventional techniques to produce an antibody as described herein or its fragment. Therefore, there is provided herein a host cell containing polynucleotides encoding an antibody as described herein or its fragment, or its heavy chain or light chain or its fragment, or a single-chain antibody as described herein, the polynucleotides being operably connected to a promoter for expressing such sequences in a host cell. In certain embodiments, in order to express a double-chain antibody, a vector encoding both heavy and light chains individually can be co-expressed in a host cell to express the entire immunoglobulin molecule, as described in detail below. In certain embodiments, the host cell contains a vector comprising a polynucleotide encoding both heavy and light chains of an antibody as described herein or its fragment. In a specific embodiment, the 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 as described herein or its fragment, and a second vector comprising a polynucleotide encoding a light chain or a light chain variable region of an antibody as described herein or its fragment. In other embodiments, the first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain or heavy chain variable region of an antibody or fragment thereof described herein, and the second host cell comprises a second vector comprising a polynucleotide encoding a light chain or light chain variable region of an antibody described herein. In specific embodiments, the heavy chain / heavy chain variable region expressed by the first cell combines with the light chain / light chain variable region of the second cell to form an anti-CTLA-4 antibody or antigen-binding fragment thereof described herein. In certain embodiments, provided herein is a host cell population comprising such a first host cell and such a second host cell.

[0447] In a specific embodiment, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding a light chain / light chain variable region of an anti-CTLA-4 antibody described herein, and a second vector comprising a polynucleotide encoding a heavy chain / heavy chain variable region of an anti-CTLA-4 antibody described herein.

[0448] A variety of host-expression vector systems can be used to express the 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 subsequently purified, and represent cells that can in situ express the antibody molecules described herein when transformed or transfected with the appropriate nucleotide coding sequences. It includes, but is not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; 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 vectors (e.g., CaMV; TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences. reinhardtii)); or a mammalian cell system (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7030, HsS78Bst, HeLa and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20 and BMT10 cells) carrying a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). In a specific embodiment, the cell used to express the antibody or antigen-binding fragment thereof described herein is a CHO cell, such as a CHO GS System TM In a specific embodiment, the cells used to express the antibodies described herein are human cells, e.g., human cell lines. In a specific embodiment, the mammalian expression vector is pOptiVEC TMOr pcDNA3.3. In a specific embodiment, bacterial cells such as Escherichia coli or eukaryotic cells (e.g., mammalian cells) particularly used to express complete recombinant antibody molecules are used for the expression of recombinant antibody molecules. For example, mammalian cells (e.g., Chinese hamster ovary (CHO) cells) combined with vectors (e.g., major mid-early gene promoter elements from human cytomegalovirus) are effective expression systems for antibodies (Foecking MK and Hofstetter H (1986) Gene 45: 101-5; and Cockett MI et al., (1990) Biotechnology 8 (7): 662-7, which are incorporated herein by reference in their entirety). In certain embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In a specific embodiment, the expression of the nucleotide sequence encoding the antibodies described herein that specifically bind to CTLA-4 (e.g., human CTLA-4) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0449] In bacterial systems, a variety of expression vectors can be advantageously selected depending on the intended use of the expressed antibody molecule. For example, when large quantities of such antibodies are to be produced for use in the generation of pharmaceutical compositions of antibody molecules, vectors that direct the expression of high levels of easily purified fusion protein products may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U and Mueller-Hill B (1983) EMBO J 2: 1791-1794, which is incorporated herein by reference in its entirety), in which the antibody coding sequence can be ligated separately into the vector in frame with the lac Z coding region to produce a fusion protein; pIN vectors (Inouye S and Inouye M (1985) Nucleic Acids Res. 13: 3101-3109; VanHeeke G and Schuster SM (1989) J. Biol. Chem. 24: 5503-5509, which are incorporated herein by reference in their entirety); and the like. For example, pGEX vectors can also be used to express exogenous polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can be purified from cracked cells by adsorption and binding matrix glutathione agarose beads, followed by elution in the presence of free glutathione. 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 portion.

[0450] In insect systems, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector for expressing foreign genes. The virus is grown in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into a non-essential region of the virus (e.g., a polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., a polyhedrin promoter).

[0451] In mammalian host cells, a variety of virus-based expression systems can be used. In the case where adenovirus is used as an expression vector, the target antibody coding sequence can be connected to an adenovirus transcription / translation control complex, for example, a late promoter and a triplet leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in the non-essential region of the viral genome (for example, E1 or E3 region) will produce a recombinant virus that is viable and can express antibody molecules in the infected host (for example, referring to Logan J and Shenk T (1984) PNAS 81 (12): 3655-9, which is incorporated herein by reference in its entirety). Specific initiation signals may also be required for the efficient translation of the inserted antibody coding sequence. These signals include an ATG initiation codon and adjacent sequences. In addition, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure the translation of the entire insert. These exogenous translation control signals and initiation codons can be from a variety of sources, natural and synthetic. The efficiency of expression can be enhanced by including appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol. 153:516-544, which is incorporated herein by reference in its entirety).

[0452] In addition, host cell lines that regulate the expression of inserted sequences or modify and process gene products in a desired specific manner can be selected. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure the correct modification and processing of the expressed foreign proteins. For this purpose, eukaryotic host cells with cellular mechanisms for the appropriate processing of primary transcripts, glycosylation of gene products and phosphorylation 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, BT20 and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In certain embodiments, the anti-CTLA-4 antibodies described herein are produced in mammalian cells, such as CHO cells.

[0453] In a specific embodiment, the antibodies or antigen-binding fragments thereof described herein have reduced or no fucose content. Such antibodies can be produced using techniques known to those skilled in the art. For example, the antibodies can be expressed in cells that are defective or lacking in fucosylation ability. In a specific example, a cell line in which both alleles of α1,6-fucosyltransferase are knocked out can be used to produce antibodies or antigen-binding fragments thereof with reduced fucose content. The ANTIBODYNAMIC® system (Lonza) is an example of such a system that can be used to generate antibodies or antigen-binding fragments thereof with reduced fucose content.

[0454] For long-term, high-yield production of recombinant proteins, stably expressing cells can be generated. For example, a cell line that stably expresses an anti-CTLA-4 antibody or antigen-binding fragment thereof described herein can be engineered. In a specific embodiment, the cells provided herein stably express a light chain / light chain variable region and a heavy chain / heavy chain variable region that combine to form an antibody or antigen-binding fragment thereof described herein.

[0455] In certain aspects, instead of using expression vectors containing viral origins of replication, host cells can be transformed with DNA and selectable markers controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). After the introduction of exogenous DNA / polynucleotides, the engineered cells can be allowed to grow in an enriched medium for 1-2 days and then switched to a selective medium. The selectable markers in the recombinant plasmid confer resistance to selection and allow the cells to stably integrate the plasmid into their chromosomes and grow to form a locus, which can then be cloned and amplified into a cell line. This method can be advantageously used to engineer cell lines expressing anti-CTLA-4 antibodies or fragments thereof described herein. Such engineered cell lines can be particularly useful for screening and evaluating compositions that interact directly or indirectly with antibody molecules.

[0456] A variety of selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler M et al. (1977) Cell 11(1):223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH and Szybalski W (1962) PNAS 48(12):2026-2034, which are incorporated herein by reference in their entirety), and adenine phosphoribosyltransferase (Lowy I et al. (1980) Cell 22(3):817-23, which are incorporated herein by reference in their entirety) genes in tk-, hgprt-, or aprt- cells, respectively. In addition, antimetabolite resistance can be used as the basis for 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 and Berg P (1981) PNAS 78(4):2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY and Wu CH (1991) Biotherapy 3:87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32:573-596; Mulligan R et al., (1994) PNAS 78(6):3567-70; O'Hare K et al., (1981) PNAS 78(7):1527-31); RC (1993) Science 260:926-932; and Morgan RA and Anderson WF (1993) Ann Rev Biochem 62:191-217; Nabel GJ and Felgner PL (1993) Trends Biotechnol 11(5):211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3):147-56), all of which are incorporated herein by reference in their entirety.Methods generally known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clones and such methods are described, for example, in Ausubel FM et al., (eds.), Modern Techniques in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbère-Garapin F et al., (1981) Journal of Molecular Biology 150: 1-14, which are incorporated herein by reference in their entirety.

[0457] The expression level of antibody molecules can be increased by vector amplification (for review, see Bebbington CR and Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Volume 3 (Academic Press, New York, 1987), which is incorporated herein by reference in its entirety). When the marker in the vector system expressing the antibody is amplifiable, the increase in the level of inhibitors present in the host cell culture will increase the copy number of the marker gene. Because the amplified region is associated with the antibody gene, the production of the antibody is also increased (Crouse GF et al., (1983) Mol Cell Biol 3:257-66, which is incorporated herein by reference in its entirety).

[0458] Host cells can be co-transfected with two or more expression vectors as described herein, the first vector encoding a polypeptide derived from a heavy chain and the second vector encoding a polypeptide derived from a light chain. Two vectors can contain a consistent selectable marker, which enables the expression of heavy and light chain polypeptides equally. Host cells can be co-transfected with two or more expression vectors of varying amounts. Host cells can be transfected with the first expression vector and the second expression vector in any of the following ratios: 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.

[0459] Alternatively, a single vector encoding and capable of expressing both heavy and light chain polypeptides may be used. In such cases, the light chain should be placed before the heavy chain to avoid excess toxic free heavy chain (Proudfoot NJ (1986) Nature 322: 562-565; and G (1980) PNAS 77: 2197-2199, which is incorporated herein by reference in its entirety). The coding sequences of the heavy and light chains may comprise cDNA or genomic DNA. The expression vector may be monocistronic or polycistronic. The polycistronic nucleic acid construct may encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more or 2-5, 5-10 or 10-20 gene / nucleotide sequences. For example, a bicistronic nucleic acid construct may comprise a promoter, a first gene (e.g., a heavy chain of an antibody described herein) and a second gene (e.g., a light chain of an antibody described herein) in the following order. In such expression vectors, transcription of the two genes may be driven by a promoter, while translation of the mRNA from the first gene may be by a cap-dependent scanning mechanism and translation of the mRNA from the second gene may be by a cap-independent mechanism, e.g., by IRES.

[0460] When the antibody molecules described herein have been produced by recombinant expression, they can be purified by any method known in the art for purification of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly by affinity for a specific antigen after protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. In addition, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0461] In a specific embodiment, the antibodies or antigen-binding fragments thereof described herein are separated or purified. In general, an isolated antibody is an antibody that is substantially free of other antibodies having antigenic specificities different from the isolated antibody. For example, in a specific embodiment, the preparation of the antibodies described herein is substantially free of cellular material and / or chemical precursors. The wording "substantially free of cellular material" includes antibody preparations in which the antibody is separated from the cellular components of the cells from which it is separated or recombinantly produced. Therefore, antibodies substantially free of cellular material include antibody preparations with less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5% or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or antibody variants, for example, different post-translational modified forms of antibodies or other different forms of antibodies (e.g., antibody fragments). When the antibody is recombinantly produced, it is also generally substantially free of culture medium, that is, culture medium accounts for less than about 20%, 10%, 2%, 1%, 0.5% or 0.1% of the volume of the protein preparation. When an antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in protein synthesis. Thus, such antibody preparations 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, the antibodies described herein are separated or purified.

[0462] 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 antibody synthesis, for example, by chemical synthesis or by recombinant expression techniques. Unless otherwise indicated, the methods described herein utilize conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields of the art. These techniques are described, for example, in the references cited herein and are fully described 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, 2nd ed., Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Modern Laboratory Techniques in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press. Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, all of which are incorporated herein by reference in their entirety.

[0463] In a specific embodiment, the antibodies described herein are antibodies (e.g., recombinant antibodies) prepared, expressed, produced, or isolated by any means involving the generation of DNA sequences (e.g., via synthesis), genetic engineering, etc. In certain embodiments, such antibodies comprise sequences (e.g., DNA sequences or amino acid sequences) that do not naturally occur within the antibody germline repertoire in animals or mammals (e.g., humans).

[0464] In one aspect, a method for producing an antibody or antigen-binding fragment thereof that specifically binds to CTLA-4 (e.g., human CTLA-4) is provided herein, comprising culturing a cell or host cell as described herein. In a certain aspect, a method for producing an antibody or antigen-binding fragment thereof that specifically binds to CTLA-4 (e.g., human CTLA-4) is provided herein, comprising expressing (e.g., recombinantly expressing) an antibody or antigen-binding fragment thereof using a cell or host cell as described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody as described herein). In a specific embodiment, the cell is an isolated cell. In a specific embodiment, an exogenous polynucleotide has been introduced into the cell. In a specific embodiment, the method further comprises a step of purifying the antibody or antigen-binding fragment thereof obtained from the cell or host cell. Preferably, the method is performed in vitro.

[0465] Methods for producing polyclonal antibodies are known in the art (see, e.g., Chapter 11 in Short Prot...

Claims

1. An isolated antibody that specifically 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 SYSMN (SEQ ID NO: 10); (b) CDRH2 comprises the amino acid sequence SISSSSSYIYYAXSVKG (SEQ ID NO: 18), wherein X is E or D; (c) CDRH3 comprises the amino acid sequence VGLXGPFDI (SEQ ID NO: 19), wherein X is F or M; (d) CDRL1 comprises the amino acid sequence RASQSVSRYLG (SEQ ID NO: 15); (e) CDRL2 comprises the amino acid sequence GASTRAT (SEQ ID NO: 16); and (f) CDRL3 comprises the amino acid sequence 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 claim 1, wherein CDRH2 comprises 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 antibody that specifically binds to human CTLA-4, the antibody comprising a heavy chain variable region comprising complementary determining regions CDRH1, CDRH2 and CDRH3 and a light chain variable region comprising complementary 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.

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 that 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 comprises 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 of the preceding claims, wherein the antibody comprises a light chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

9.

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.

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