EA044966B1 - ANTIBODIES AGAINST CTLA-4 AND METHODS OF THEIR APPLICATION - Google Patents

Description

Related applications

This application claims benefit from U.S. Provisional Patent Application No. 62/431,272, filed December 7, 2016, which is incorporated herein by reference in its entirety.

Field of technology

The present invention relates to antibodies that specifically bind to CTLA-4 (eg, human CTLA-4) and methods of using them.

State of the art

T lymphocytes play a key role in the adaptive immune response to antigen. At least two signals are required for full activation of naive T cells (Bretscher 1999, Proc Natl Acad Sci USA 96:185-90). The first antigen-specific signal mediates the interaction of the T-cell receptor (TCR) with the MHC/peptide complex on the antigen-presenting cell (APC). The second, costimulatory signal is provided by interactions between receptors on the T cell and their ligands on the antigen presenting cell (APC). Recruitment of both TCR/MHC and co-stimulatory interactions results in T cell activation through a number of intracellular pathways, including the calcium calcineurin and RAS mitogen-activated protein kinase pathways, followed by activation of transcription factors of a number of effector compounds, including cytokines such as ILs -2. These events lead to the proliferation of T cells, the production of a pool of CD4+ helper T cells (TH) and the expansion of activated CD8+ cytotoxic T cells. Costimulation is not only critical for full T cell activation, but its absence upon TCR/MHC recruitment leads to anergy and/or apoptosis.

Several positive and negative costimulatory pathways are involved in T cell regulation; however, the most important are those associated with CD28 on T cells and B7-1 (CD80) and B7-2 (CD86) on APCs. CD28 promotes the differentiation of T cells into cells with a TH1 phenotype and also enhances B cell antibody production and T cell activation. B7-1 and B7-2, expressed on APCs such as dendritic cells (DCs) and B cells, have overlapping but distinct functions. B7-2 is constitutively expressed and rapidly up-regulated on APCs concurrently with TCR/MHC recruitment (signal 1). B7-1 expression is very low in resting cells but is typically induced after prolonged stimulation of T cells. These differences suggest that although B7-2 may be important for initiating T cell activation, B7-1 may play a more significant role in prolonging the immune response.

Following T cell activation, the negative regulatory receptor, cytotoxic T lymphocyte antigen 4 (CTLA-4) is up-regulated 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 ligands, B7-1 and B7-2. CTLA-4 inhibits the immune response in several ways: it competes with CD28 for B7 ligands and, accordingly, blocks costimulation; it provides a negative signal to inhibit T cell activation; and it can take up CD80 and CD86 from oncoming cells by trans-endocytosis, resulting in impaired costimulation by CD28 (Krummel and Allison, 1995, J Exp Med 182:459465; Walunas et al., 1994, Immunity 1:405-413 ; Qureshi et al., 2011, Science 332:600-603).

Given the critical role of the B7 co-stimulatory pathway in stimulating and maintaining the immune response, therapeutic agents designed to antagonize this pathway are of potential interest for the treatment of autoimmune diseases and disorders.

Brief description of the invention

The present invention provides antibodies that specifically bind to human CTLA-4 and antagonize the function of CTLA-4, eg, CTLA-4-mediated immune suppression. Also provided are pharmaceutical compositions containing said antibodies, nucleic acids encoding said antibodies, expression vectors and host cells for producing said antibodies, as well as methods of treating a subject using said antibodies. The antibodies described herein are particularly suitable for enhancing T cell activation in response to an antigen (eg, tumor antigen or infectious disease antigen) and/or reducing Treg-mediated immune suppression; and, respectively, for treating cancer in a subject or for treating or preventing an infectious disease in a subject.

Accordingly, according to one aspect of the present invention, there is provided an isolated antibody comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein:

(a) CDRH1 contains the amino acid sequence SYSMN (SEQ ID NO: 10;

(b) CDRH2 contains the amino acid sequence SISSSSSYIYYAXSVKG (SEQ ID NO: 18), where X represents E or D;

(c) CDRH3 contains the amino acid sequence VGLXGPFDI (SEQ ID NO: 19), where X represents F or M;

(d) CDRL1 contains the amino acid sequence RASQSVSRYLG (SEQ ID NO: 15);

(e) CDRL2 contains the amino acid sequence GASTRAT (SEQ ID NO: 16) and

- 1 044966 (f) CDRL3 contains the amino acid sequence QQYGSSPWT (SEQ ID NO: 17), and the CDRH1, CDRH2 and CDRH3 sequences of the specified antibody are not the sequences of SEQ ID NO: 10, 11 and 13, respectively.

In some embodiments, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, said CDRH3 comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, said CDRH3 comprises the amino acid sequence of SEQ ID NO: 13. In embodiments, said CDRH3 comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, CDRH1, CDRH2, and CDRH3 comprise the amino acid sequences of CDRH1, CDRH2, and CDRH3 presented in SEQ ID NO: 10, 11, and 14; 10, 12 and 13; or 10, 12 and 14, respectively. In some embodiments, CDRH1, CDRH2, and CDRH3 comprise the amino acid sequences CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NOs: 10, 12, and 14, respectively. In some 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 some 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.

According to another aspect of the present invention, there is provided an isolated antibody that specifically binds to human CTLA-4, comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, Where:

(a) CDRH1 contains the amino acid sequence SYSMN (SEQ ID NO: 10);

(b) CDRH2 contains the amino acid sequence SISSSSSYIYYAXSVKG (SEQ ID NO: 18), where X represents E or D;

(c) CDRH3 contains the amino acid sequence VGLXGPFDI (SEQ ID NO: 19), where X represents F or M;

(d) CDRL1 contains the amino acid sequence RASQSVSRYLG (SEQ ID NO: 15);

(e) CDRL2 contains the amino acid sequence GASTRAT (SEQ ID NO: 16) and (f) CDRL3 contains the amino acid sequence QQYGSSPWT (SEQ ID NO: 17), and the CDRH1, CDRH2 and CDRH3 sequences of the specified antibody are not the sequences of SEQ ID NO: 10, 11 and 13, respectively.

In some embodiments, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, said CDRH3 comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, said CDRH3 comprises the amino acid sequence of SEQ ID NO: 13. In embodiments, said CDRH3 comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, CDRH1, CDRH2, and CDRH3 comprise the amino acid sequences of CDRH1, CDRH2, and CDRH3 presented in SEQ ID NO: 10, 11, and 14; 10, 12 and 13; or 10, 12 and 14, respectively. In some embodiments, CDRH1, CDRH2, and CDRH3 comprise the amino acid sequences CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NOs: 10, 12, and 14, respectively. In some 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 some 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.

According to another aspect of the present invention, there is provided an isolated antibody that specifically binds to human CTLA-4, comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 contain the amino acid sequences shown in SEQ ID NO: 10, 12, 14, 15, 16 and 17, respectively.

In some embodiments, the antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 20. In some 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 NOs: 2 and 4-8. In some 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 from SEQ ID NO: 3. In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4-8. In some embodiments, said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, said antibody comprises a heavy chain. a chain comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24. 25. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence derived from the human germline IGHV3-21 sequence (e.g., IGHV3-21*01, for example, with the amino acid sequence of SEQ ID NO: 21).

In some 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 some embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the antibody comprises a light chain variable region having an amino acid sequence derived from the human germline IGHV3-20 sequence (eg, IGKV3-20*01, eg, with the amino acid sequence of SEQ ID NO: 22).

In some embodiments, the present invention provides an isolated antibody that specifically binds to human CTLA-4, comprising a heavy chain variable region having an amino acid sequence derived from the human germline IGHV3-21 sequence (e.g., IGHV3-21*01, e.g., with sequence of amino acids from SEQ ID NO: 21), wherein the specified heavy chain variable region contains the amino acid sequence presented in SEQ ID NO: 14. In some embodiments, the specified antibody contains a light chain variable region having an amino acid sequence derived from the sequence of IGHV3- 20 human germline (eg, IGKV320*01, eg, with the amino acid sequence of SEQ ID NO: 22).

According to another aspect of the present invention, there is provided an isolated antibody that specifically binds to human CTLA-4 comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 23-26. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 26.

According to another aspect of the present invention, there is provided an isolated antibody that specifically binds to human CTLA-4, comprising a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region and 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. In some 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 some embodiments, the antibody comprises a heavy chain comprising the sequence amino acids from SEQ ID NO: 23; and a light chain comprising the amino acid sequence of SEQ ID NO: 27. In some 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 some 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 some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 26; and a light chain containing the amino acid sequence of SEQ ID NO: 27.

According to another aspect of the present invention, there is provided an isolated antibody that specifically binds to human CTLA-4, comprising a heavy chain variable region having an amino acid sequence derived from the IGHV3-21*01 germline sequence

- 3 044966 human line (for example, IGHV3-21*01, for example, with the amino acid sequence from SEQ ID NO:

21); and a light chain variable region having an amino acid sequence derived from the human germline sequence IGKV3-20*01 (eg, IGKV3-20*01, eg, with the amino acid sequence of SEQ ID NO: 22).

In some embodiments, the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, _IgG2 , _IgG3 , _IgG4 , IgA1, and human _IgA2 . In some embodiments, said heavy chain constant region is the corresponding region of IgG1. In some embodiments, said heavy chain constant region is the corresponding IgG2 region. In some embodiments, the antibody comprises a light chain constant region selected from the group consisting of human IgK and Igλ.

In some embodiments, the antibody comprises an IgG1 heavy chain constant region. In some embodiments, said antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 28. In certain embodiments, said IgG1 heavy chain constant region amino acid sequence contains mutations S239D/I332E in accordance with the EU numbering system. In some embodiments, said antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, said IgG1 heavy chain constant region amino acid sequence contains mutations S239D/A330L/I332E in accordance with the EU numbering system. In some embodiments, said antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, said IgG1 heavy chain constant region amino acid sequence comprises mutations L235V/F243L/R292P/Y300L/P396L according to the numbering system EU. In some embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, said IgG1 heavy chain constant region is an afucosylated region of IgG1.

In some embodiments, the antibody comprises a human IgG heavy chain constant region that is a wild-type human IgG heavy chain constant region variant, wherein the human IgG heavy chain constant region variant binds to FcyRIIIA with a higher affinity than the IgG heavy chain constant region wild-type human binds to FcyRIIIA. In some embodiments, the human IgG heavy chain constant region variant is a human IgG1 heavy chain constant region variant.

In some embodiments, the antibody comprises a light chain constant region selected from the group consisting of human IgK and Igλ. In some embodiments, the antibody comprises an IgK light chain constant region. In some embodiments, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 32.

According to another aspect of the present invention, an isolated antibody is provided that cross-competes for binding to human CTLA-4 with an antibody described herein. According to another aspect of the present invention, there is provided an isolated antibody that cross-competes for binding to human CTLA-4 with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 8 and 9, respectively.

According to another aspect of the present invention, an isolated antibody is provided that binds to the same epitope on human CTLA-4 as the antibody described herein. According to another aspect of the present invention, there is provided an isolated antibody that binds to the same epitope on human CTLA-4 as the antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 8 and 9, respectively.

According to another aspect of the present invention, an isolated antibody is provided that binds, for example, specifically binds, to an epitope of human CTLA-4. In some embodiments, the antibody binds to an epitope located in the human CTLA-4 region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-39. In some embodiments, the antibody binds to an epitope located in a region of human CTLA-4 consisting of the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody binds to an epitope located in a region of human CTLA-4 consisting of the sequence amino acids from SEQ ID NO: 36. In some embodiments, the antibody binds to an epitope located in a region of human CTLA-4 consisting of the amino acid sequence from SEQ ID NO: 35. In some embodiments, the antibody binds to an epitope located in

- 4 044966 region of human CTLA-4 consisting of the amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody binds to an epitope located in the region of human CTLA-4 consisting of the amino acid sequence of SEQ ID NO: 38. According to In some embodiments, said antibody binds to an epitope located in the region

Human CTLA-4, consisting of the amino acid sequence from SEQ ID NO: 39.

According to another aspect of the present invention, an antibody or isolated antibody is provided that specifically binds to the same epitope of human CTLA-4 as any antibody of the present invention. In some embodiments, the antibody binds to an epitope located in the human CTLA-4 region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-39. In some embodiments, the antibody binds to an epitope located in a region of human CTLA-4 consisting of the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody binds to an epitope located in a region of human CTLA-4 consisting of the sequence amino acids from SEQ ID NO: 36. In some embodiments, the antibody binds to an epitope located in the human CTLA-4 region consisting of the amino acid sequence from SEQ ID NO: 35. In some embodiments, the antibody binds to an epitope located in the region human CTLA-4 consisting of the amino acid sequence of SEQ ID NO: 34. In some embodiments, said antibody binds to an epitope located in a region of human CTLA-4 consisting of the amino acid sequence of SEQ ID NO: 38. In some embodiments, said the antibody binds to an epitope located in the human CTLA-4 region consisting of the amino acid sequence of SEQ ID NO: 39.

According to another aspect of the present invention, there is provided an antibody that, when bound to human CTLA-4 protein or a fragment thereof, for example comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33, reduces the exchange of hydrogen for deuterium in the region consisting of the sequence amino acids set forth in SEQ ID NO: 34, relative to the exchange of hydrogen for deuterium in a region consisting of the amino acid sequence set forth in SEQ ID NO: 34, in the absence of the specified antibody, as assessed using a hydrogen-deuterium exchange assay. According to another aspect of the present invention, there is provided an antibody that, when bound to human CTLA-4 protein or a fragment thereof, for example comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33, reduces the exchange of hydrogen for deuterium in the region consisting of the sequence amino acids set forth in SEQ ID NO: 35, relative to the exchange of hydrogen for deuterium in a region consisting of the amino acid sequence set forth in SEQ ID NO: 35, in the absence of the specified antibody, as assessed using a hydrogen-deuterium exchange assay. According to another aspect of the present invention, there is provided an antibody that, when bound to human CTLA-4 protein or a fragment thereof, for example comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33, reduces the exchange of hydrogen for deuterium in the region consisting of the sequence amino acids set forth in SEQ ID NO: 36, relative to the exchange of hydrogen for deuterium in a region consisting of the amino acid sequence set forth in SEQ ID NO: 36, in the absence of the specified antibody, as assessed using a hydrogen-deuterium exchange assay. According to another aspect of the present invention, there is provided an antibody that, when bound to human CTLA-4 protein or a fragment thereof, for example comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33, reduces the exchange of hydrogen for deuterium in the region consisting of the sequence amino acids set forth in SEQ ID NO: 37, relative to the exchange of hydrogen for deuterium in a region consisting of the amino acid sequence set forth in SEQ ID NO: 37, in the absence of the specified antibody, as assessed using a hydrogen-deuterium exchange assay. According to another aspect of the present invention, there is provided an antibody that, when bound to human CTLA-4 protein or a fragment thereof, for example comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33, reduces the exchange of hydrogen for deuterium in the region consisting of the sequence amino acids set forth in SEQ ID NO: 38, relative to the exchange of hydrogen for deuterium in a region consisting of the amino acid sequence set forth in SEQ ID NO: 38, in the absence of the specified antibody, as assessed using a hydrogen-deuterium exchange assay. According to another aspect of the present invention, there is provided an antibody that, when bound to human CTLA-4 protein or a fragment thereof, for example comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33, reduces the exchange of hydrogen for deuterium in the region consisting of the sequence amino acids set forth in SEQ ID NO: 39, relative to the exchange of hydrogen for deuterium in a region consisting of the amino acid sequence set forth in SEQ ID NO: 39, in the absence of the specified antibody, as assessed using a hydrogen-deuterium exchange assay.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to the same epitope of human CTLA-4 as any antibody according to

- 5 044966 to the present invention. In some embodiments, the antibody, when bound to human CTLA-4 protein or a fragment thereof, for example, comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33, reduces the exchange of hydrogen for deuterium in the region consisting of the amino acid sequence shown in SEQ ID NO: 34, regarding the exchange of hydrogen for deuterium in the region consisting of the amino acid sequence presented in SEQ ID NO: 34, in the absence of the specified antibody, as assessed using hydrogen-deuterium exchange analysis. In some embodiments, the antibody, when bound to human CTLA-4 protein or a fragment thereof, for example, comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33, reduces the exchange of hydrogen for deuterium in the region consisting of the amino acid sequence shown in SEQ ID NO: 35, regarding the exchange of hydrogen for deuterium in the region consisting of the amino acid sequence presented in SEQ ID NO: 35, in the absence of the specified antibody, as assessed using hydrogen-deuterium exchange analysis. In some embodiments, the antibody, when bound to human CTLA-4 protein or a fragment thereof, for example, comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33, reduces the exchange of hydrogen for deuterium in the region consisting of the amino acid sequence shown in SEQ ID NO: 36, regarding the exchange of hydrogen for deuterium in the region consisting of the amino acid sequence presented in SEQ ID NO: 36, in the absence of the specified antibody, as assessed using hydrogen-deuterium exchange analysis. In some embodiments, the antibody, when bound to human CTLA-4 protein or a fragment thereof, e.g., comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33, reduces the exchange of hydrogen for deuterium in the region consisting of the amino acid sequence represented by in SEQ ID NO: 37, regarding the exchange of hydrogen for deuterium in the region consisting of the amino acid sequence presented in SEQ ID NO: 37, in the absence of the specified antibody, as assessed using hydrogen-deuterium exchange analysis. In some embodiments, the antibody, when bound to human CTLA-4 protein or a fragment thereof, e.g., comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33, reduces the exchange of hydrogen for deuterium in the region consisting of the amino acid sequence represented by in SEQ ID NO: 38, regarding the exchange of hydrogen for deuterium in the region consisting of the amino acid sequence presented in SEQ ID NO: 38, in the absence of the specified antibody, as assessed using hydrogen-deuterium exchange analysis. In some embodiments, the antibody, when bound to human CTLA-4 protein or a fragment thereof, e.g., comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33, reduces the exchange of hydrogen for deuterium in the region consisting of the amino acid sequence represented by in SEQ ID NO: 39, regarding the exchange of hydrogen for deuterium in the region consisting of the amino acid sequence presented in SEQ ID NO: 39, in the absence of the specified antibody, as assessed using hydrogen-deuterium exchange analysis.

In some embodiments, the antibody is a human antibody. In a certain embodiment, said antibody is a bispecific antibody.

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

In some embodiments, the antibody is conjugated to a cytotoxic agent, cytostatic agent, toxin, radionuclide, or detectable label.

In some embodiments, the N-terminal amino acid residue of the variable heavy chain and/or variable light chain of the antibody has been converted to pyroglutamate.

In one embodiment, the present invention provides an antibody of the present invention for use as a medicament.

In one embodiment, the present invention relates to the use of an antibody of the present invention for the preparation of pharmaceutical compositions or medicaments for immunotherapy. In some embodiments, the immunotherapy is intended to enhance T cell activation in response to an antigen in a subject, not necessarily to treat a cancer, or to treat or prevent an infectious disease.

In one embodiment, the present invention provides an antibody of the present invention for use as a diagnostic agent.

In one embodiment, the present invention relates to the use of an antibody of the present invention for in vitro detection of human CTLA-4 in a biological sample.

- 6 044966

According to another aspect of the present invention, there is provided a pharmaceutical composition comprising an anti-CTLA-4 antibody described herein and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient.

According to another aspect of the present invention, there is provided an isolated polynucleotide encoding the heavy and/or light chain of an antibody described herein. According to another aspect of the present invention, a vector containing said polynucleotide is provided. According to another aspect of the present invention, a recombinant host cell is provided containing said polynucleotide or said vector. According to another aspect of the present invention, there is provided a method for producing an antibody that binds to human CTLA-4, the method comprising culturing a host cell such that expression of said polynucleotide and production of said antibody occur.

According to another aspect of the present invention, there is provided 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 or pharmaceutical composition described herein. According to another aspect of the present invention, there is provided a method of treating cancer in a subject, the method comprising administering to said subject an effective amount of an anti-CTLA-4 antibody or pharmaceutical composition described herein.

In some embodiments, the subject has cancer. In some embodiments, the subject has a metastatic or locally advanced tumor (eg, a solid tumor). In some embodiments, said cancer is treated in accordance with a method described herein as the first anticancer therapy after diagnosis of said metastatic or locally advanced tumor (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2 , 3, 4, 6, 8 or 12 weeks; or 1, 2, 3, 4, 6, 8 or 12 months after diagnosis). In some embodiments, the cancer is treated in accordance with the method described herein as the first anticancer therapy after tumor progression is diagnosed (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or 1, 2, 3, 4, 6, 8, or 12 months after diagnosis of tumor progression) that occurred despite prior treatment of said tumor with another anticancer therapy, not necessarily the method described herein, is used as a second anticancer therapy used. In some embodiments, the cancer is treated in accordance with the method described herein as the first anticancer therapy after toxicity of another anticancer therapy has been diagnosed (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 another anticancer therapy), optionally the method described herein being used as the second anticancer therapy used therapy. In some embodiments, the cancer treated in accordance with the methods described herein is a metastatic or locally advanced cancer (eg, a solid tumor) for which there is no available standard therapy. In other embodiments, the cancer treated in accordance with the methods described herein is a metastatic or locally advanced cancer (e.g., a solid tumor) that has failed treatment with standard therapy (i.e., the cancer has progressed after receiving standard therapy). therapy). In some embodiments, therapy is unsuccessful if the cancer is refractory to said therapy. In some embodiments, a therapy is unsuccessful if the cancer recurs after a response, complete or partial, to the therapy. In some embodiments, metastatic or locally advanced cancer (eg, a solid tumor) has been confirmed histologically or cytologically.

In some embodiments, the cancer expresses PD-L1. In some embodiments, the percentage of tumor cells in a cancer sample exhibiting detectable membranous expression (e.g., partial or complete membranous expression) of PD-L1 is at least 1% (e.g., at least 2%, 3%, 4%, 5 %, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90%). In some embodiments, the percentage of tumor cells in a cancer sample exhibiting detectable membrane expression (eg, partial or complete membrane expression) of PD-L1 is at least 1%. In some embodiments, the percentage of tumor cells in a cancer sample exhibiting detectable membrane expression (eg, partial or complete membrane expression) of PD-L1 is at least 5%. In some embodiments, the percentage of tumor cells in a cancer sample exhibiting detectable membrane expression (eg, partial or complete membrane expression) of PD-L1 is at least 25%. In some embodiments, the percentage of tumor cells in a cancer sample exhibiting detectable membrane expression (eg, partial or complete membrane expression) of PD-L1 is at least 50%.

- 7 044966

In some embodiments, the metastatic or locally advanced tumor expresses PD-L1. In some embodiments, the percentage of tumor cells in a metastatic or locally advanced tumor sample that exhibit detectable membranous expression (e.g., partial or complete membranous expression) of PD-L1 is at least 1% (e.g., at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90%). In some embodiments, the percentage of tumor cells in a metastatic or locally advanced tumor sample that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 1%. In some embodiments, the percentage of tumor cells in a metastatic or locally advanced tumor sample that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 5%. In some embodiments, the percentage of tumor cells in a metastatic or locally advanced tumor sample that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 25%. In some embodiments, the percentage of tumor cells in a metastatic or locally advanced tumor sample that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 50%.

In some embodiments, said cancer is cervical cancer. In some embodiments, the cancer is a metastatic or locally advanced cancer (eg, a solid tumor). In some embodiments, said metastatic or locally advanced cancer (eg, solid tumor) is a metastatic or locally advanced unresectable squamous cell carcinoma, glandular squamous cell carcinoma, or adenocarcinoma of the cervix. In some embodiments, there is no available standard therapy for the cancer (eg, cervical cancer), or metastatic or locally advanced tumor (eg, solid tumor). In some embodiments, the cancer (eg, cervical cancer), or metastatic or locally advanced tumor (eg, solid tumor) is refractory or refractory to standard therapy. In some embodiments, the cancer (eg, cervical cancer) or metastatic or locally advanced tumor (eg, solid tumor) has recurred after standard therapy. In some embodiments, said standard therapy includes platinum-containing chemotherapy. In some embodiments, said standard therapy is a platinum-containing doublet chemotherapy. In some embodiments, the cancer (e.g., cervical cancer) is a metastatic or locally advanced unresectable squamous cell carcinoma, glandular squamous cell carcinoma, or adenocarcinoma of the cervix that has recurred after platinum-double chemotherapy administered to treat advanced (recurrent, unresectable, or metastatic) disease . In some embodiments, said cancer (eg, cervical cancer), or said metastatic or locally advanced tumor, is HPV positive. In some embodiments, said cancer, or said metastatic or locally advanced solid tumor, is a head and neck cancer, melanoma, renal cell carcinoma, urothelial carcinoma, or endometrial carcinoma. In some embodiments, said cancer (eg, cervical cancer), or said metastatic or locally advanced solid tumor(s), is associated with microsatellite instability.

In some embodiments, the subject has cervical cancer (e.g., metastatic or locally advanced unresectable squamous cell carcinoma, glandular squamous cell carcinoma, or cervical adenocarcinoma), and the method comprises administering to the subject an effective amount of an anti-CTLA-4 antibody described herein, for example, AGEN1884.H3 (IgG1 S239D/A330L/I332E), or a pharmaceutical composition containing such an anti-CTLA-4 antibody, as the first anticancer therapy after diagnosis of cervical cancer (for example, 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 the anti-CTLA-4 antibody described herein document, for example, AGEN1884.H3 (IgG1 S239D/A330L/I332E), or a pharmaceutical composition containing such anti-CTLA-4 antibody is administered at a dosage and frequency selected from the group consisting of 0.3 mg/kg every four weeks, 1 mg/kg every four weeks, 3 mg/kg every four weeks, 0.3 mg/kg every six weeks, 1 mg/kg every six weeks, 3 mg/kg every six weeks, 0.3 mg/kg every 12 weeks, 1 mg/kg every 12 weeks, and 3 mg/kg every 12 weeks. In some embodiments, the subject has cervical cancer (e.g., metastatic or locally advanced unresectable squamous cell carcinoma, glandular squamous cell carcinoma, or cervical adenocarcinoma), and the method includes administering to the subject an effective amount of a therapeutic combination,

- 8 044966 containing an anti-CTLA-4 antibody described herein, for example, AGEN1884.H3 (IgG1 S239D/A330L/I332E), or a pharmaceutical composition containing such an anti-CTLA-4 antibody, and pembrolizumab, as the first anti-cancer therapy after diagnosis of cervical cancer (eg, 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), wherein optionally an anti-CTLA-4 antibody described herein, for example, AGEN1884.H3 (IgG1 S239D/A330L/I332E), or a pharmaceutical composition containing such an anti-CTLA-4 antibody is administered in a dosage and at frequencies 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 12 weeks, 1 mg/kg every 12 weeks, and 3 mg/kg every 12 weeks, and pembrolizumab is administered at a dose of 200 mg every three weeks.

In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, said NSCLC is stage IV NSCLC. In some embodiments, the percentage of tumor cells in an NSCLC sample that exhibit detectable membranous expression (e.g., partial or complete membranous expression) of PD-L1 is at least 1% (e.g., at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90%). In some embodiments, the percentage of tumor cells in an NSCLC sample that exhibit detectable membrane expression (eg, partial or complete membrane expression) of PD-L1 is at least 1%. In some embodiments, the percentage of tumor cells in an NSCLC sample that exhibit detectable membrane expression (eg, partial or complete membrane expression) of PD-L1 is at least 5%. In some embodiments, the percentage of tumor cells in an NSCLC sample that exhibit detectable membrane expression (eg, partial or complete membrane expression) of PD-L1 is at least 25%. In some embodiments, the percentage of tumor cells in an NSCLC sample that exhibit detectable membrane expression (eg, partial or complete membrane expression) of PD-L1 is at least 50%. In some embodiments, said NSCLC is not characterized by genomic tumor aberrations of rEGF or ALK (anaplastic lymphoma kinase). In some embodiments, the metastatic or locally advanced NSCLC is not characterized by a sensitizing eEGF mutation (eg, a mutation that is responsive to treatment with a tyrosine kinase inhibitor, including erlotinib, gefinitib, or afatinib) or an ALK translocation. In some embodiments, the subject has not previously received systemic chemotherapy treatment for NSCLC.

In some embodiments, said metastatic or locally advanced solid tumor is metastatic or locally advanced non-small cell lung cancer (NSCLC). In some embodiments, said metastatic or locally advanced solid tumor is metastatic non-small cell lung cancer (NSCLC). In some embodiments, said metastatic or locally advanced solid tumor is stage IV metastatic or locally advanced NSCLC. In some embodiments, said metastatic or locally advanced solid tumor is stage IV metastatic NSCLC. In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced NSCLC that exhibit detectable membranous expression (e.g., partial or complete membranous expression) of PD-L1 is at least 1% (e.g., at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90%). In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced NSCLC that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 1%. In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced NSCLC that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 5%. In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced NSCLC that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 25%. In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced NSCLC that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 50%. In some embodiments, said metastatic or locally advanced NSCLC is not characterized by genomic tumor aberrations of rEGF or ALK. In some embodiments, the subject has not previously received systemic chemotherapy treatment for metastatic or locally advanced NSCLC.

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In some embodiments, the subject has NSCLC (e.g., metastatic or locally advanced stage IV NSCLC), optionally characterized in that the percentage of tumor cells in the NSCLC sample that exhibit detectable expression (e.g., membranous expression, partial or complete membranous expression) of PD-L1 , is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%, and said method comprises administering to said subject an effective amount of an anti-CTLA-4 antibody described herein, for example, AGEN1884.H3 (IgG1 S239D/A330L/I332E), or a pharmaceutical composition containing such antibody anti-CTLA-4, as first anticancer therapy after diagnosis of cervical cancer (eg, 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 an anti-CTLA-4 antibody described herein, for example, AGEN1884.H3 (IgG1 S239D/A330L/I332E), or a pharmaceutical composition containing such anti-CTLA-4 antibody, administered at a dosage and frequency selected from the group consisting of 0.3 mg/kg every four weeks, 1 mg/kg every four weeks, 3 mg/kg every four weeks, 0.3 mg/kg every six weeks, 1 mg/kg every six weeks, 3 mg/kg every six weeks, 0.3 mg/kg every 12 weeks, 1 mg/kg every 12 weeks, and 3 mg/kg every 12 weeks. In some embodiments, the subject has NSCLC (e.g., metastatic or locally advanced stage IV NSCLC), optionally characterized in that the percentage of tumor cells in the NSCLC sample that exhibit detectable expression (e.g., membranous expression, partial or complete membranous expression) of PD-L1 , is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%, and said method comprises administering to said subject a therapeutic combination containing an anti-CTLA-4 antibody described herein, for example, AGEN1884.H3 (IgG1 S239D/A330L/I332E), or a pharmaceutical composition containing such anti-CTLA-4 antibody, and pembrolizumab, as the first anticancer therapy after diagnosis of cervical cancer (eg, 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 an anti-CTLA-4 antibody described herein, e.g. AGEN1884.H3 (IgG1 S239D/A330L/I332E), or a pharmaceutical composition containing such an anti-CTLA-4 antibody is administered at a dosage and frequency selected from the group consisting of 0.3 mg/kg every four weeks, 1 mg/kg every four weeks, 3 mg/kg every four weeks, 0.3 mg/kg every six weeks, 1 mg/kg every six weeks, 3 mg/kg every six weeks, 0.3 mg/kg every 12 weeks, 1 mg/kg every 12 weeks, and 3 mg/kg every 12 weeks, and pembrolizumab is administered at a dose of 200 mg every three weeks.

In some embodiments, the cancer is cutaneous squamous cell carcinoma (cSCC). In some embodiments, the metastatic or locally advanced solid tumor is a stage IV cutaneous squamous cell carcinoma (cSCC). In some embodiments, the cSCC is diagnosed histologically or cytologically according to the American Joint Committee on Cancer 8th edition cancer staging manual. In some embodiments, the cSCC is not curable with radiation therapy. In some embodiments, the subject has cSCC (e.g., stage IV cSCC), and the method includes administering to the subject an effective amount of an anti-CTLA-4 antibody described herein, for example, AGEN1884.H3 (IgG1 S239D/A330L/I332E), or a pharmaceutical composition containing such an anti-CTLA-4 antibody as the first anticancer 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 an anti-CTLA-4 antibody described herein, e.g. AGEN1884.H3 (IgG1 S239D/A330L/I332E), or a pharmaceutical composition containing such an anti-CTLA-4 antibody is administered at a dosage and frequency selected from the group consisting of 0.3 mg/kg every four weeks, 1 mg/kg every four weeks, 3 mg/kg every four weeks, 0.3 mg/kg every six weeks, 1 mg/kg every six weeks, 3 mg/kg every six weeks, 0.3 mg/kg every 12 weeks, 1 mg/kg every 12 weeks, and 3 mg/kg kg every 12 weeks. In some 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 containing such an anti-CTLA-4 antibody, and pembrolizumab, as the first anticancer 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 an anti-CTLA-4 antibody described herein, e.g., AGEN1884.H3 (IgG1 S239D/A330L/I332E), or a pharmaceutical composition containing such an anti-CTLA-4 antibody, is administered at a dosage and frequency selected from the group consisting of 0.3 mg/kg every four weeks, 1 mg/kg every four weeks , 3 mg/kg every four weeks, 0.3 mg/kg every six weeks, 1 mg/kg every six weeks, 3 mg/kg every six weeks, 0.3 mg/kg every 12 weeks, 1 mg/kg every 12 weeks, and 3 mg/kg every 12 weeks, and pembrolizumab is administered at a dose of 200 mg every three weeks.

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In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intravenously. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intravenously at a dose of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg, optionally at biweekly intervals. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intravenously at a dose of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg or 10 mg/kg, optionally at three-week intervals. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intravenously at a dose of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg, optionally at four-week intervals. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intravenously at a dose of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg, optionally at six-week intervals. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intravenously at a dose of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg, optionally at 12-week intervals. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intratumorally. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intratumorally in an amount of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, or 3 mg/kg, optionally at intervals of once every three weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intratumorally in an amount of 0.03 mg/kg, 0.1 mg/kg, or 0.3 mg/kg, optionally at intervals of once every three weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intratumorally at a dose of up to 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60 -fold, 70-fold, 80-fold, 90-fold, 100-fold, or 200-fold lower than the systemically administered dose. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intratumorally at a dose that is up to 10 times lower than the systemically administered dose. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intratumorally at a dose up to 100-fold lower than the systemically administered dose. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered (eg, intratumorally or systemically) as monotherapy. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intratumorally, and the method further includes administering an additional therapeutic agent to the subject. In some embodiments, said additional therapeutic agent is administered systemically. In some embodiments, the subject has a solid tumor and the additional therapeutic agent is an anti-PD-1 antibody. In some embodiments, the anti-PD1 antibody is pembrolizumab or nivolumab. In some embodiments, pembrolizumab is administered at a dose of 200 mg every three weeks. In some embodiments, the subject has squamous cell carcinoma of the head and neck, and the additional therapeutic agent is an anti-eGF antibody. In some embodiments, the anti-eGF antibody is cetuximab. In some embodiments, the subject has HER2+ breast cancer and the additional therapeutic agent is an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the methods further comprise administering a chemotherapeutic agent to the subject. In some embodiments, the chemotherapeutic agent is administered systemically. In some embodiments, the chemotherapeutic agent is gemcitabine. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intratumorally and the subject has an advanced or metastatic solid tumor. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intratumorally and the subject has head and neck cancer (eg, relapsed/refractory squamous cell carcinoma of the head and neck). In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered intratumorally and the subject has breast cancer (eg, relapsed/refractory HER2+ breast cancer). In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein

- 11 044966 they are delivered to the lymph node draining the tumor. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is delivered via localized administration (eg, subcutaneous administration). In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is delivered via localized administration (e.g., subcutaneous administration) at a dose of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg or 3 mg/kg. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is delivered via localized administration (eg, subcutaneous administration) at a dose of up to 5-fold, 10-fold, 20-fold, 30-fold, 40-fold. 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or 200-fold lower than the systemically administered dose. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is delivered via localized administration (eg, subcutaneous administration) at a dose up to 10-fold lower than the systemically administered dose. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is delivered via localized administration (eg, subcutaneous administration) at a dose up to 100-fold lower than the systemically administered dose. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is delivered via localized administration (eg, subcutaneous administration), and the method further includes administering an additional therapeutic agent to the subject. In some embodiments, said additional therapeutic agent is a vaccine. In some embodiments, the vaccine comprises a heat shock protein-peptide complex (HSPPC) comprising a heat shock protein complexed with an antigenic peptide. In one embodiment, said heat shock protein is gp96 and is complexed with a tumor-associated antigenic peptide, wherein said HSPPC is derived from a tumor obtained from the subject. In some embodiments, the heat shock protein is selected from the group consisting of hsc70, hsp70, hsp90, hsp110, grp170, gp96, calreticulin, mutants, and combinations of two or more of these proteins. In some embodiments, the heat shock protein is hsc70. In some embodiments, the heat shock protein is hsp70. In some embodiments, the antigenic peptide is synthetic. In some embodiments, the subject has cancer. In some embodiments, the subject suffers from an infectious disease. In some embodiments, the methods further comprise administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent or a checkpoint-targeting agent. In some embodiments, the checkpoint-targeting agent is selected from the group consisting of an anti-PD-1 antagonist antibody, an anti-PD-L1 antagonist antibody, an anti-PD-L2 antagonist antibody, an anti-CTLA-4 antagonist antibody, an anti-TIM-3 antagonist antibody, anti-LAG-3 antagonist antibody, anti-CEACAM1 antagonist antibody, anti-GITR agonist antibody, anti-OX40 agonist antibody and anti-CD137 agonist antibody, anti-DR3 agonist antibody, anti-TNFSF14 agonist antibody, and agonist antibody against CD27. In some embodiments, said additional therapeutic agent is radiation therapy. In some 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 some embodiments, said additional therapeutic agent is a vaccine. In some embodiments, the vaccine comprises a heat shock protein-peptide complex (HSPPC) comprising a heat shock protein complexed with an antigenic peptide. In one embodiment, said heat shock protein is gp96 and is complexed with a tumor-associated antigenic peptide, wherein said HSPPC is derived from a tumor obtained from the subject.

According to another aspect of the present invention, there is provided a method of treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of an anti-CTLA-4 antibody or pharmaceutical composition described herein. According to another aspect of the present invention, there is provided a method of 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.

In one embodiment, the present invention provides 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.

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In one embodiment, the present invention provides 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.

In one embodiment, the present invention relates to the use of 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 in vitro detection of human CTLA-4 in a biological sample.

According to one aspect of the present invention, there is provided a pharmaceutical composition comprising an anti-CTLA-4 antibody described herein and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient for use as a medicament.

According to one aspect of the present invention, there is provided a pharmaceutical composition comprising an anti-CTLA-4 antibody described herein and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient for use as a diagnostic agent.

According to one aspect of the present invention, there is provided a pharmaceutical composition comprising an anti-CTLA-4 antibody described herein, a polynucleotide of the present invention, a vector of the present invention and/or a recombinant host cell of the present invention, and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient. In one aspect, said pharmaceutical composition is intended for use as a medicament and/or diagnostic agent.

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

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

In one aspect, the present invention provides an antibody, polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition of the present invention for use in a method of increasing T cell activation in response to an antigen in a subject, comprising administering to said subject an effective amount of the antibody, polynucleotide , vector, recombinant host cell and/or pharmaceutical composition according to the present invention.

In one aspect, the present invention provides an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention for use in a method of treating cancer.

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

In one aspect, the present invention provides an antibody, polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition of the present invention for use in a method of treating cancer in a subject, comprising administering to said subject an effective amount of the antibody, polynucleotide, vector, recombinant host cell and/or a pharmaceutical composition according to the present invention.

In one aspect, the present invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention; and (b) an additional therapeutic agent, preferably an anti-PD-1 antibody, for use as a medicament.

In one aspect, the present invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention; and (b) an additional therapeutic agent, preferably an anti-PD-1 antibody, for use in a method of treating cancer. In a preferred embodiment, said cancer is a solid tumor. In another preferred embodiment, said antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention is administered intratumorally to said subject, preferably intratumorally administered to said subject at a dose of 0.01 mg/kg, 0.03 mg /kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg or 3 mg/kg, optionally at three-week intervals.

According to one aspect, the present invention relates to a pharmaceutical composition, kit or composite kit containing (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention; and (b) an additional therapeutic agent, preferably an anti-PD-1 antibody.

In one aspect, the present invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention; and (b) an anti-rEGF antibody; and, optionally, (c) a chemotherapeutic agent, for use as a medicament.

In one aspect, the present invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention; and (b) an anti-rEGF antibody; and, optionally, (c) a chemotherapeutic agent, for use in a method of treating cancer. In a preferred embodiment, said cancer is squamous cell carcinoma of the head and neck. In another preferred embodiment, said antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention is administered intratumorally to said subject, preferably intratumorally administered to said subject at a dose of 0.01 mg/kg, 0.03 mg /kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg or 3 mg/kg, optionally at three-week intervals.

According to one aspect, the present invention relates to a pharmaceutical composition, kit or composite kit containing (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention; and (b) anti-rEGF antibody; and, optionally, (c) a chemotherapeutic agent.

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

In one aspect, the present invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention; and (b) an anti-HER2 antibody; and, optionally, (c) a chemotherapeutic agent, for use in a method of treating HER2+ breast cancer. In another preferred embodiment, said antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention is administered intratumorally to said subject, preferably intratumorally administered to said subject at a dose of 0.01 mg/kg, 0.03 mg /kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg or 3 mg/kg, optionally at three-week intervals.

According to one aspect, the present invention relates to a pharmaceutical composition, kit or composite kit containing (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention; and (b) anti-HER2 antibody; and, optionally, (c) a chemotherapeutic agent.

In one aspect, the present invention relates to an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention for use in a method of treating cancer, characterized in that said antibody, polynucleotide, vector, recombinant host cell and/or the pharmaceutical composition according to the present invention is administered intratumorally to the specified subject, preferably administered intratumorally to the specified subject at a dose of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg /kg or 3 mg/kg, optionally at three-week intervals.

In one aspect, the present invention relates to an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention for use in a method of treating cancer, characterized in that said antibody, polynucleotide, vector, recombinant host cell and/or the pharmaceutical composition according to the present invention is administered subcutaneously or intravenously to the specified subject, preferably administered intravenously to the specified subject at a dose of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg /kg, 3 mg/kg, 6 mg/kg or 10 mg/kg, optionally at three-week intervals.

In one aspect, the present invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention; and (b) an additional therapeutic agent, for use as a medicament. In a preferred embodiment, said additional therapeutic agent is a chemotherapeutic agent, or a checkpoint-targeting agent, or an indoleamine 2,3-dioxygenase (IDO) inhibitor, or a vaccine.

In one aspect, the present invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention; and (b) an additional therapeutic agent for use in a method of treating cancer. In a preferred embodiment, said additional therapeutic agent is a chemotherapeutic agent, or a checkpoint-targeting agent, or an indoleamine 2,3-dioxygenase (IDO) inhibitor, or a vaccine.

According to one aspect, the present invention relates to a pharmaceutical composition, kit or composite kit containing (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention; and (b) an additional therapeutic agent. In a preferred embodiment, said additional therapeutic agent is a chemotherapeutic agent, or a checkpoint-targeting agent, or an indoleamine 2,3-dioxygenase (IDO) inhibitor, or a vaccine.

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In one aspect, the present invention provides an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention for use in a method of treating cancer and/or for use in a method of increasing T cell activation in response to an antigen, characterized in that said antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention is delivered to a tumor-draining lymph node.

In one aspect, the present invention relates to the use of an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention in a method of treating cancer and/or in a method of increasing T cell activation in response to an antigen in a subject, characterized in that that said antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention is delivered to a tumor-draining lymph node.

In one aspect, the present invention relates to the use of an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention, for the production of medicaments for immunotherapy, for example, for increasing the activation of T cells in response to an antigen in a subject, treating cancer , or the treatment or prevention of infectious diseases.

In one aspect, the present invention relates to the use of an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention for the production of medicaments for immunotherapy, for example, for increasing the activation of T cells in response to an antigen in a subject, treating cancer, or treatment or prevention of infectious diseases, characterized in that said antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention is delivered to a tumor-draining lymph node.

In one aspect, the present invention relates to the use of (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention and (b) an anti-HER2 antibody; and, optionally, (c) a chemotherapeutic agent, to produce a medicament for immunotherapy, for example, to enhance T cell activation in response to an antigen in a subject, treat cancer, or treat or prevent infectious diseases.

In one aspect, the present invention relates to the use of (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention and (b) an anti-HER2 antibody; and, optionally, (c) a chemotherapeutic agent, for the production of a medicament for immunotherapy, for example, for increasing the activation of T cells in response to an antigen in a subject, treating cancer, or treating or preventing infectious diseases, characterized in that said antibody, polynucleotide, the vector, recombinant host cell and/or pharmaceutical composition according to the present invention are delivered to a tumor-draining lymph node.

Brief description of drawings

In fig. 1A-1G are flow cytometry histograms showing the binding of anti-CTLA-4 antibodies or an IgG1 isotype control antibody to Jurkat cells engineered to express human CTLA-4 on the cell surface. The following anti-CTLA-4 antibodies were tested: AGEN1884.H1.1 (IgG1), AGEN1884.H1.2 (IgG1), AGEN1884.H1.3 (IgG1), AGEN1884.H2.1 (IgG1), AGEN1884. H2. 2 (IgG1), AGEN1884.H2.3 (IgG1) HAGEN1884.H3 (IgG1).

In fig. 2 is a graph showing the production of IL-2 by primary human PBMCs after incubation with suboptimal stimulation with staphylococcal enterotoxin A (SEA) superantigen in the absence or presence of anti-CTLA-4 antibody AGEN1884.H3 (IgG1) or isotype control antibody (IgG1). The analysis was performed in 8 replicates for each group; the average values for 8 replicates are marked with a black line.

In fig. 3 is a graph showing the results of an IL-2-luciferase reporter assay demonstrating that CTLA-4 blockade leads to T cell activation. A graph of the fold response in the form of expression of luciferase, a surrogate marker of IL-2 gene activation, is plotted against a range of concentrations of the AGEN1884.H3 antibody (IgG1) or the isotype control antibody (IgG1).

In fig. 4 is a graph showing the results of a reporter assay where simultaneous recruitment of AGEN1884.H3 (IgG1) to target cells (via CTLA-4 binding) and effector cells (via FcyRIIIA binding) triggers luciferase expression by effector lineage cells. Luciferase activity is a surrogate marker of FcγRHA signaling. A graph of the fold response as RLU values versus the concentration range of AGEN1884.H3 antibody (IgG1) and isotype control antibody (IgG1) concentrations is shown.

In fig. 5A-5D are flow cytometry histograms showing CTLA-4 expressing Jurkat cells incubated with anti-CTLA-4 antibody AGEN1884.H3 (IgG1), AGEN1884.H3 (IgG1 S239D/I332E), AGEN1884.H3 (IgG1 S239D/A330L/I332E ) or AGEN1884.H3 (IgG1

- 15 044966

L235V/F243L/R292P/Y300L/P396L), or isotype control antibody. Antibody binding was detected using a fluorochrome-conjugated secondary antibody.

In fig. 6A and 6B are graphs showing the blocking of binding between human CTLA-4 and its ligands, CD80 and CD86, respectively, by the antibody 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), anti-CTLA-4 reference antibody, or isotype control antibody (IgG1) and then stained with the specified fluorescently labeled ligand. Ligand binding was then assessed by flow cytometry.

In fig. 7A-7C are graphs depicting IL-2 production by primary human PBMCs cultured under suboptimal stimulation with SEA superantigen in the absence or presence of isotype control antibody (IgG1) or anti-CTLA-4 antibody. In fig. 7A and 7B are graphs depicting IL-2 production stimulated with either a single dose or titrated doses of isotype control antibody (IgG1) or anti-CTLA-4 antibodies AGEN1884.H3 (IgG1), AGEN1884.H3 (IgG1 S239D/I332E), AGEN1884 .H3 (IgG1 S239D/A330L/I332E) and AGEN1884.H3 (IgG1 L235V/F243L/R292P/Y300L/P396L). In the study presented in FIG. 7B, in addition to isotype control antibody (IgG ₁ ) or anti-CTLA-4 antibody, cells in each sample were also incubated with isotype control antibody IgG ₄ S228P. In fig. 7C is a graph showing IL-2 production stimulated with titrated doses of isotype control antibody (IgG ₁ ) or anti-CTLA-4 antibodies AGEN1884 (IgG ₁ ), AGEN1884 (IgG ₁ S239D/I332E), AGEN1884 (IgG1 S239D/A330L/I332E) and afucosylated AGEN1884 (IgG1).

In fig. 8A shows immunoblot analysis for 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). In fig. 8B is a graph showing normalized densitometric analysis of the data presented in FIG. 8A.

In fig. 9A-9D are graphs depicting the antitumor efficacy and intratumoral depletion of regulatory T cells (Treg) induced by Fc variants of the mouse anti-CTLA-4 antibody 9D9. In fig. 9A shows tumor growth in CT26 mice following treatment with a single dose of mouse anti-CTLA-4 antibody 9D9 (mIgG2a), Fc-silenced 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). The top panel shows the median tumor volume versus time for each treatment group. The remaining panels show tumor volume versus time for individual animals in each treatment group. In fig. 9B shows the effect of anti-CTLA-4 antibody treatment on T cell populations from tumor infiltrates collected from mice treated with single doses of anti-CTLA-4 antibody 9D9 (mIgG2a), anti-CTLA-4 antibody 9D9 (mIgG2a-N297A), anti-CTLA-4 antibody 9D9 (mIgG2a-N297A), CTLA-4 9D9 (mIgG2aS239D/A330L/I332E) or isotype control antibody (mIgG2a). Tumor infiltrates were collected and analyzed by flow cytometry at specified time points after antibody injection. Cell populations analyzed include: FoxP3+ Tregs (upper left panel), CD45+ leukocytes (upper right panel), and CD4+ non-regulatory T cells (lower left panel). The lower right panel shows the ratio of CD8+ T cells to Tregs observed in tumor infiltrates. In fig. 9C shows FoxP3+ Treg populations as a function of time in tumor-draining lymph nodes collected from mice treated as described for FIG. 9B. In fig. 9D shows the fold change in the number of splenic FoxP3+ Tregs 72 hours after treatment as described for FIG. 9B.

In fig. 10 shows a series of graphs reflecting the antitumor efficacy of mouse anti-CTLA-4 antibodies in combination with an antitumor vaccine derived from an HPV+ tumor (E6/E7 viral antigens). Tumor volume versus time is shown for individual untreated mice treated with isotype control antibody (mIgG2a), anti-CTLA-4 antibody 9D9 (mIgG2a), or the Fc variant of anti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E). . The graphs in the top row show the results for animals that received only the indicated antibody during treatment. The graphs in the bottom row show the results for animals that were treated with the indicated antibody in combination with an antitumor vaccine.

In fig. 11A and 11B are graphs depicting gene expression and CpG methylation in human T cell populations. CD4+ CD25 ^+/- FOXP3 ^- non-regulatory T cells (Teff) and CD4+ CD25+ FOXP3+ regulatory T cells (Treg) were isolated from the peripheral blood of healthy donors, expanded and activated. In fig. 11A shows the levels of FOXP3, intracellular CTLA-4 and membrane CTLA-4 in each activated T cell population determined by flow cytometry. In fig. 11B shows the level of CpG methylation at CpG regions within the FOXP3 (top panel) and CTLA4 (bottom panel) loci in naïve T cells, activated effector T cells, and activated regulatory T cells derived from the same donor.

- 16 044966

In fig. 12A and 12B are graphs depicting the time course of antibody-dependent cellular cytotoxicity (ADCC) of human CTLA-4 positive target cells following incubation with anti-CTLA-4 antibody AGEN1884.H3 (IgG1) or Fc variants thereof. NK-92 cells (expressing FcyRIIIA 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). A multiparameter microscopic caspase 3/7 activation assay was then used to quantify ADCC activity. In fig. 12A shows ADCC activity 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 isotype control antibody (IgG1). In fig. 12B shows ADCC activity in primary activated human effector T cells (left panel) or regulatory T cells (right panel) when incubated with the indicated antibodies.

In fig. 13A-13D are graphs depicting the effects of anti-CTLA-4 antibody variants on T cell function when administered alone or in combination with an anti-PD-1 antibody. Human PBMCs were isolated from two donors and incubated under stimulating culture conditions with anti-CTLA-4 antibody AGEN1884.H3 (IgG1), Fc variant of anti-CTLA-4 antibody AGEN1884.H3 (IgG1 S239D/A330L/I332E) or isotype control antibody (IgG1 ) in combination with a reference anti-PD-1 antibody or an isotype control antibody (IgG ₄ ), as indicated. For each of the listed treatment conditions, titrated doses of the antibody from the first antibodies listed and a fixed concentration (5 μg/ml) of the antibody from the second antibodies listed were used. This experiment was performed twice, obtaining a total of two replicates for each donor. The levels of IL-2 production induced by each antibody combination on PBMC collected from the first donor are shown in FIG. 13A (repeat 1) and FIG. 13V (repeat 2). The levels of IL-2 production induced by each antibody combination on PBMCs collected from the second donor are shown in FIG. 13C (replication 1) and FIG. 13D (repeat 2).

In fig. 14A-14C show a series of sequence alignments. In fig. 14A shows a sequence alignment of human CTLA-4 (SEQ ID NO: 33), cynomolgus CTLA-4 (SEQ ID NO: 40), mouse CTLA-4 (SEQ ID NO: 41) and rat CTLA-4 (SEQ ID NO : 42). The dots indicate residues identical to the corresponding human residues. The symbol * (asterisk) indicates positions containing a single completely conserved residue. The symbol: (colon) indicates conservatism in groups with pronounced similarity of properties. Symbol. (dot) indicates conservatism in groups with weak similarity of properties. In fig. 14B and 14C show sequence alignments of human CTLA-4 (residues 1-144 and residues 145-223 SEQ ID NO: 33, respectively), cynomolgus CTLA-4 (residues 1-144 and residues 145-223 SEQ ID NO: 40 , respectively), human CD28 (residues 1-127 and residues 128-220 SEQ ID NO: 43, respectively), human ICOS (residues 1-124 and residues 125-199 SEQ ID NO: 44, respectively), human BTLA (residues 1-125 and residues 126-289 SEQ ID NO: 45, respectively) and human PD-1 (residues 1-143 and residues 144-288 SEQ ID NO: 46, respectively). In fig. 14A-14C highlight two regions that demonstrate a marked reduction in deuterium uptake upon binding of human CTLA-4 to AGEN1884-Fab: 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 of the Invention

The present invention provides antibodies that specifically bind to CTLA-4 (eg, human CTLA-4) and antagonize the function of CTLA-4, eg, CTLA-4-mediated immune suppression. Also provided are pharmaceutical compositions containing said antibodies, nucleic acids encoding said antibodies, expression vectors and host cells for producing said antibodies; and methods of treating a subject using said antibodies. The antibodies described herein are particularly useful for enhancing T cell activation in response to an antigen (eg, a tumor antigen or an infectious disease antigen) and, therefore, for treating cancer in a subject, or treating or preventing an infectious disease in a subject. In all cases, the isolated antibodies described herein further include antibodies that may, but are not necessarily, isolated. In all cases, the isolated polynucleotides described herein further include polynucleotides that may, but are not necessarily, isolated. In all cases, the antibodies described herein further include antibodies that may, but are not necessarily, isolated. In all cases, the polynucleotides described herein further include polynucleotides that may, but are not necessarily, isolated.

One skilled in the art will recognize that the amino acid residue glutamate (E) or glutamine (Q) 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) maybe in

- 17 044966 under certain conditions, spontaneously convert into pyroglutamate due to post-translational cyclization with a free amino group to form a lactam. Accordingly, in certain embodiments of each and every method, application, pharmaceutical composition or kit described herein, the N-terminal amino acid residue of one or more of the antibody variable region heavy chains and/or variable region light chains has been converted to a pyroglutamate (eg , as a result of post-translational cyclization of the free amino group of the N-terminal residue E or Q).

Definitions.

As used herein, the terms about and approximately, when used to modify a numerical value or range of numerical values, indicate that variations in the stated value or range are from 5 to 10% upward (e.g., up to 5 to 10% upward) and from 5 to 10% down (for example, up to 5-10% down) is included in the intended scope of the value or range mentioned.

As used herein, the term CTLA-4 refers to cytotoxic T-lymphocyte-associated protein 4. As used herein, human CTLA-4 refers to the human CTLA-4 protein encoded by the wild-type human CTLA-4 gene, for example, GenBank™ accession number NM_005214. 4 or NM_001037631.2. An example of the immature human CTLA-4 amino acid sequence is shown in SEQ ID NO: 33.

As used herein, the terms antibody and antibodies include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing CDR regions, VH regions, or VL regions of antibodies. 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, tetramer antibodies containing two heavy chain molecules and two light molecules chains, antibody light chain monomer, antibody heavy chain monomer, antibody light chain dimer, antibody heavy chain dimer, pair of antibody light chain and antibody heavy chain, intrabodies, heteroconjugate antibodies, antibody drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), camellized antibodies, affibodies, Fab fragments, F(ab') ₂ fragments linked by disulfide bonds Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, antibodies to anti- I-antibodies), as well as antigen-binding fragments of any of the above antibodies. In some embodiments, the antibodies described herein are polyclonal antibody populations. Antibodies can be of any type (eg IgG, IgE, IgM, IgD, IgA or IgY), any class (eg IgG1, IgG ₂ , IgG ₃ , IgG ₄ , IgA1 or IgA ₂ ) or any subclass (eg IgG _2a or IgG _2b ) immunoglobulin molecules. In some embodiments, the antibodies described herein are IgG antibodies, or belong to a class (eg, human IgG1 or _IgG4 ) or subclass thereof. In a specific embodiment, said antibody is a humanized monoclonal antibody. In another specific embodiment, said antibody is a human monoclonal antibody.

As used herein, the terms VH region and VL region refer to the single variable regions of the heavy and light chains of an antibody, respectively, comprising FRs (framework regions) 1, 2, 3 and 4, and CDRs (complementarity determining regions) 1, 2 and 3 (see Source: 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).

As used herein, the term CDR or complementarity determining region refers to the non-contiguous antigen binding sites found in the variable region of both heavy and light chain polypeptides. These specific areas have been described in: 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), each of which is incorporated herein by reference in its entirety, wherein said definitions include, when compared to each other, overlapping amino acid residues or subsets of amino acid residues. In some embodiments, the term CDR is a CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991). In some embodiments, the term CDR refers to a CDR as defined by Chothia et al., J. Mol. Biol. 196:901-917 (1987). In some embodiments, the term CDR refers to a CDR as defined by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A. Protein Sequence and Structure Analysis of Antibody Variable Domains, in Antibody Engineering, Rontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

As used herein, the term framework (FR) amino acid residues refers to amino acids in the framework region of an immunoglobulin chain. The term frame region or FR region in the present

- 18 044966 document includes amino acid residues that are part of the variable region, but are not part of the CDR regions (for example, when using the Kabat or Chothia definition of CDR).

As used herein, the terms variable region and variable domain are used interchangeably and are well known in the art. A variable region generally refers to a portion of an antibody, typically a portion of the light or heavy chain, typically comprising approximately 110-125 amino-terminal amino acids in the mature heavy chain and approximately 90-115 amino acids in the mature light chain, the sequence of which varies widely between antibodies and is involved in the binding and specificity of a particular antibody for a particular antigen. Sequence variability is concentrated in regions called complementarity determining regions (CDRs), while the more highly conserved regions of the variable domain are called framework regions (FRs). Without being bound by any particular mechanism or any particular theory, it is believed that the CDR regions of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen and the specificity of the antibody for the antigen. In some embodiments, said variable region is a human variable region. In some embodiments, the variable region comprises rodent or mouse CDR regions and human framework regions (FR). In certain embodiments, said variable region is a primate (eg, non-human primate) variable region. In some embodiments, the variable region comprises rodent or mouse CDR regions and primate (eg, non-human primate) framework regions (FR).

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

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

As used herein, the terms constant region and constant domain are used interchangeably and are well known in the art. A constant region is a portion of an antibody, such as the carboxy-terminal portion of the light and/or heavy chain, that is not directly involved in antibody-antigen binding, but may exhibit various effector functions, such as interaction with an Fc receptor (e.g., Fc receptor gamma) . The constant region of an immunoglobulin molecule usually contains a more conserved sequence of amino acids compared to the variable domain of an immunoglobulin.

As used herein, the term heavy chain, when used in reference to an antibody, can refer to any of the distinct types, for example, alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on amino acid sequence constant domain, which give rise to the antibody classes IgA, IgD, IgE, IgG and IgM, respectively, including the IgG subclasses, for example, IgG1, _IgG2 , IgG3 and IgG4.

As used herein, the term light chain, when used in relation to an antibody, can refer to any of the distinct types, such as kappa (k) or lambda (λ), based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art.

As used herein, the term EU numbering system refers to the adopted EU numbering for antibody constant regions as described in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

Binding affinity generally refers to the strength of the combined non-covalent interactions between one binding site of a molecule (eg, antibody) and its binding partner (eg, antigen). Unless otherwise specified, as used herein, binding affinity refers to the true binding affinity, which reflects the 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can typically be represented by a dissociation constant (K _D ). Affinity can be measured and/or expressed in several ways known in the art, including, but not limited to, the equilibrium dissociation constant (KD) and the equilibrium binding constant (K _A ). K _D is calculated as the quotient of koff/kon, and K _A is calculated as the quotient of k _on /ko _ff . k _on refers to the binding rate constant, eg antibody to antigen, ak _off refers to the dissociation rate constant, eg antibody to antigen. k _on and kf can be determined using techniques known to one skilled in the art, such as BIAcore® or KinExA. As used herein, lower affinity refers to higher KD.

As used herein, the terms specifically binds, specifically recognizes, immunospecifically binds, and immunospecifically recognizes are analogous in the context of antibodies and refer to molecules that bind to an antigen (eg, an epitope or immune complex) in the sense that such binding understood by one skilled in the art. For example, a molecule that specifically binds an antigen may bind to other peptides or polypeptides, typically with lower affinity as assessed using, for example, immunoassays, BIAcore®, the KinExA 3000 instrument (Sapidyne Instruments, Boise, Idaho) or other assays , known in the art. In a specific embodiment, molecules that specifically bind an antigen bind to said antigen with a K _A of at least 2 log orders (i.e., 10-fold), 2.5 log orders, 3 log orders, 4 log orders or more than K _A upon nonspecific binding of these molecules to another antigen.

In another specific embodiment, molecules that specifically bind an antigen do not cross-react with other proteins under similar binding conditions. In another specific embodiment, molecules that specifically bind to CTLA-4 do not cross-react with other non-CTLA-4 proteins. According to a specific embodiment of the present invention, there is provided an antibody that binds to CTLA-4 (eg, human CTLA-4) with greater affinity compared to another unrelated antigen. Some embodiments of the present invention provide an antibody that binds to CTLA-4 (eg, human CTLA-4) at 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater affinity compared to another, unrelated antigen as assessed using, for example, a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In a specific embodiment, the degree 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 said antibody to a CTLA-4 protein as assessed using , for example, radioimmunoassay.

As used herein, the term afucosylation or afucosylated in the context of Fc refers to the absence of substantially fucose covalently attached, directly or indirectly, to residue 297 of the human IgG1 Fc region according to the EU numbering system, or to the corresponding residue of non-IgG1 or non-IgG1 immunoglobulins. being human IgG1. Accordingly, in a composition comprising a plurality of afucosylated antibodies, at least 70% of said antibodies are not fucosylated, either directly or indirectly (e.g., through intermediate Sugars), at residue 297 of the Fc region of said antibodies, and, in some embodiments, at least 80%, 85%, 90%, 95% or 99% is not fucosylated, directly or indirectly, at residue 297 of the Fc region.

As used herein, the term epitope corresponds to a term known in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope may be, for example, contiguous amino acids of a polypeptide (linear or continuous epitope), or an epitope may, for example, be composed of two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous or non-contiguous epitope). In some embodiments, the epitope to which the antibody binds can be determined using, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen-deuterium exchange coupled to mass spectrometry (e.g., liquid chromatography/mass spectrometry electrospray), oligopeptide scan-on-chip assays (e.g., peptide constraint using CLIPS (Chemical Linkage of Peptides onto Scaffolds) technology to map discontinuous or conformational epitopes), and/or mutagenesis mapping (e.g., site-specific mapping mutagenesis). In X-ray crystallography, crystallization can be carried out using any methods known in the art (for example, see references: Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350; McPherson A (1990) ) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, each of which is incorporated herein by reference in its entirety). Antibody:antigen crystals can be examined using well-known X-ray diffraction techniques, and their structure can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.,; U.S. 2004/0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1): 37-60; Bricogne G (1997) ) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316-1323), each of which is incorporated herein by reference in its entirety. Mutagenesis mapping studies can be carried out using any method known to one skilled in the art; see, for example, for descriptions of mutagenesis techniques, including alanine scanning, in Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085 , each of which is incorporated herein by reference in its entirety. CLIPS (Chemical Linkage of Peptides onto Scaffolds) is a technology for presenting one or more Peptides

- 20 044966 tides in a structurally limited configuration, behaving as functional mimetics of complex protein domains; see, for example, US Publications US 2008/0139407 A1 and US 2007/099240 A1, and US Patent No. 7,972,993, each of which is incorporated herein by reference in its entirety. In a specific embodiment, the antibody epitope is determined in an alanine scanning mutagenesis study. In a specific embodiment, an antibody epitope is determined using hydrogen-deuterium exchange coupled to mass spectrometry. In a specific embodiment, the antibody epitope is determined using CLIPS epitope mapping technology from Pepscan Therapeutics.

As used herein, the term epitope located in the human CTLA-4 region, consisting of a particular amino acid sequence or group of amino acid residues, refers to an epitope containing one or more of the amino acid residues of a given region, wherein said specified region includes the first specified amino acid residue and the last specified amino acid residue of the human CTLA-4 region. In some embodiments, the epitope comprises each of the amino acid residues located in a given region. In some embodiments, one or more additional human CTLA-4 amino acid residues outside a given region bind to an antibody in association with an epitope located in a given region.

As used herein, the terms T cell receptor and TCR are used interchangeably and refer to full-length heterodimeric αβ or γδ TCRs, antigen-binding fragments of full-length TCRs, and molecules containing CDR regions or TCR variable regions. Examples of TCRs include, but are not limited to, full-length TCRs, antigen-binding fragments of full-length TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single-chain TCRs containing TCR variable regions attached by a flexible linker, engineered disulfide-linked TCR chains, monospecific TCRs, multispecific TCRs TCRs (including bispecific TCRs), fusion TCRs, human TCRs, humanized TCRs, chimeric TCRs, recombinantly derived TCRs and synthetic TCRs. The term includes wild-type TCRs and genetically engineered TCRs (eg, a chimeric TCR containing a chimeric TCR chain that includes a first portion of a first-species TCR and a second portion of a second-species TCR).

As used herein, the terms major histocompatibility complex and MHC are used interchangeably and refer to an MHC class I molecule and/or an MHC class II molecule.

As used herein, the term peptide-MHC complex refers to the MHC molecule (MHC class I or MHC class II) to which the peptide is bound in the art-known binding pocket of said MHC molecule.

As used herein, the terms treat, treatment and treatment refer to the therapeutic or prophylactic measures described herein. Treatment methods involve administering an antibody to a subject with a disease or disorder, or predisposed to such a disease or disorder, to prevent, cure, delay, reduce the severity of, or alleviate one or more symptoms of the disease or disorder, or a recurrent disease or disorder, or to prolong the survival of the subject. beyond the period expected in the absence of such treatment.

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

As used herein, in relation to cancer response to therapy, the terms refractory and resistant have meanings known in the art and are used interchangeably.

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

Determining the percent identity between two sequences (eg, amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A specific non-limiting example of a mathematical algorithm for comparing two sequences is provided by the algorithm from Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified from Karlin S & Altschul SF (1993) PNAS 90: 5873-5877; each of these references is incorporated herein by reference in its entirety. Such an algorithm is included in the NBLAST and XBLAST programs according to Altschul SF et al., (1990) J Mol Biol 215: 403, which is incorporated herein by reference in its entirety. A BLAST nucleotide search can be performed using the NBLAST nucleotide program parameter set, eg, where weight=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search can be performed using a set of XBLAST program parameters, for example, where weight=50, word length=3, yielding amino acid sequences homo

- 21 044966 logical to the protein molecule described in this document. Gapped BLAST can be used to obtain gap alignments for comparison purposes 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 iterative searches that detect distant relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI Blast programs, the default settings of the corresponding programs (for example, XBLAST and NBLAST) may apply (see, for example, the National Center for Biotechnology Information (NCBI) World Wide Web site, ncbi.nlm.nih.gov ). Another specific, non-limiting example of a mathematical algorithm used for sequence comparison is provided by the algorithm from Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is included in the ALIGN program (version 2.0), which is included in the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 residue substitution weight table, a gap extension penalty of 12, and a sequence gap penalty of 4 can be applied.

The percentage identity of two sequences can be determined using techniques similar to those described above, with or without gaps allowed. When calculating percent identity, typically only exact matches are counted.

Antibodies against CTLA-4.

In one aspect, the present invention provides antibodies that specifically bind to CTLA-4 (eg, human CTLA-4) and antagonize the function of CTLA-4.

The amino acid sequences of example antibodies are given in table. 1-4 in this document.

Table 1

Amino acid sequences of examples of antibodies against CTLA-4*

SEQ ID NO: Description Amino acid sequence 1 AGEN1884 VH EVQLVESGGGLVKPGGSLRLSCAASGFFSSYSMNW VRQAPGKGLEWVS SISSSSSYIYYADSVKGRFTISR DNAKNS LYLQMNS LRAE DTAVYYCARVGLMGP FDIW GQGTMVTVSS 2 AGEN1884_M102F VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVS SISSSSSYIYYADSVKGRFTISR DNAKNS LYLQMNS LRAE DTAVYYCARVGL FGP FDIW GQGTMVTVSS 3 AGEN1884_M113L VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVS SISSSSSYIYYADSVKGRFTISR DNAKNS LYLQMNS LRAE DTAVYYCARVGLMGP FDIW GQGTLVTVSS 4 AGEN1884_D62E VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVS SISSSSSYIYYAE SVKGRFTIS R DNAKNS LYLQMNS LRAE DTAVYYCARVGLMGP FDIW GQGTMVTVSS

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5 AGEN18 8 4_M102 F_M11 3L VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVS SISSSSSYIYYADSVKGRFTISR DNAKNS LYLQMNS LRAE DTAVYYCARVGL FGP EDIW GQGTLVTVSS 6 AGEN1884_D62E_M102 F VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVS SISSSSSYIYYAE SVKGRFTIS R DNAKNS LYLQMNS LRAE DTAVYYCARVGL FGP EDIW GQGTMVTVSS 7 AGEN1884_D62E_M113 L VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVS SISSSSSYIYYAE SVKGRFTIS R DNAKNS LYLQMNS LRAE DTAVYYCARVGLMGP FDIW GQGTLVTVSS 8 AGEN1884_D62E_M102 F_M113L VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVS SISSSSSYIYYAE SVKGRFTIS R DNAKNS LYLQMNS LRAE DTAVYYCARVGL FGP FDIW GQGTLVTVSS 9 AGEN1884 VL EIVLTQSPGTLLSLSPGERATLSCRASQSVSRYLGWY QQKPGQAPRLLIYGASTRATGIPDRFSGSGSGTDFT LTITRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK 10 CDRH1 SYSMN eleven CDRH2 SISSSSSYIYYADSVKG 12 CDRH2 SISSSSSYIYYAESVKG 13 CDRH3 VGLMGPFDI 14 CDRH3 VGLFGPFDI 15 CDRL1 RASQSVSRYLG 16 CDRL2 GASTRAT 17 CDRL3 QQYGSSPWT 18 Consensus subsequence CDRH2 SISSSSSYIYYAXSVKG, where: X represents E or D 19 Consensus subsequence CDRH3 VGLXGPFDI, where: X represents F or M

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20 Consensus subsequence VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVSSISSSSSYIYYAXiSVKGRFTISR DNAKNS L YLQMNS LRAE DTAVY YCARVGLX ₂ GP FDIW GQGTX ₃ VTVSS where: Xi represents E or D, X ₂ represents F or M, and X ₃ represents L or M. 23 Heavy chain AGEN1884.H3 (IgGi) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVS SISSSSSYIYYAE SVKGRFTIS R DNAKNS LYLQMNS LRAE DTAVYYCARVGL FGP FDIW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSS SLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG 24 Heavy chain AGEN1884.H3 (IgGi S239D/I332E) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVS SISSSSSYIYYAE SVKGRFTIS R DNAKNS LYLQMNS LRAE DTAVYYCARVGL FGP FDIW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSS SLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG

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25 Heavy chain AGEN1884.H3 (IgGi S239D/A330L/I332E) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVS SISSSSSYIYYAE SVKGRFTIS R DNAKNS LYLQMNS LRAE DTAVYYCARVGL FGP FDIW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSS SLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG 26 Heavy chain AGEN1884.H3 (IgGi L235V/F243L/R292P/ Y300L/P396L) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVS SISSSSSYIYYAE SVKGRFTIS R DNAKNS LYLQMNS LRAE DTAVYYCARVGL FGP FDIW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSS SLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPPEEQYNSTLRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTP LVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG

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47 Heavy chain AGEN1884.H3 (IgGi N297A) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVS SISSSSSYIYYAE SVKGRFTIS R DNAKNS LYLQMNS LRAE DTAVYYCARVGL FGP FDIW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSS SLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG 48 Heavy chain AGEN1884.H3 (IgGi S267E/L328F) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVS SISSSSSYIYYAE SVKGRFTIS R DNAKNS LYLQMNS LRAE DTAVYYCARVGL FGP FDIW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSS SLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVEHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG 27 Light chain AGENT 884.NZ EIVLTQSPGTLSLSPGERATLSCRASQSVSRYLGWY QQKPGQAPRLLIYGASTRATGIPDRFSGSGSGTDFT LTITRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC

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28 IgGi ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTV LHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG 29 IgGi S239D/I332E ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTV LHQDWLNGKEYKCKVSNKALPAPEE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG thirty IgGi S239D/A330L/I332E ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTV LHQDWLNGKEYKCKVSNKALPLPEE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG 31 IgGi L235V/F243L/R292P/ Y300L/P396L ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYN STLRWSVLTV LHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG 32 Light chain constant region RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

* CDR areas are defined according to the Kabat numbering system.

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table 2

Amino acid sequences of CDR heavy chains of example antibodies against CTLA-4*

VH (SEQ ID NO:) CDRH1 (SEQ ID NO: ) CDRH2 (SEQ ID NO:) CDRH3 (SEQ ID NO:) AGEN1884 VH (1) SYSMN (10) SISSSSSYIYYADSV KG (11) VGLMGPFDI (13) AGEN1884_M102F VH (2) SYSMN (10) SISSSSSYIYYADSV KG (11) VGLFGPFDI (14) AGEN1884_M113L VH (3) SYSMN (10) SISSSSSYIYYADSV KG (11) VGLMGPFDI (13) AGEN1884_D62E VH (4) SYSMN (10) SISSSSSYIYYAESV KG (12) VGLMGPFDI (13) AGEN18 8 4_M102 F_M113L VH (5) SYSMN (10) SISSSSSYIYYADSV KG (11) VGLFGPFDI (14) AGEN1884_D62E_M102F VH (6) SYSMN (10) SISSSSSYIYYAESV KG (12) VGLFGPFDI (14) AGEN1884_D62E_M113L VH (7) SYSMN (10) SISSSSSYIYYAESV KG (12) VGLMGPFDI (13) AGEN1884_D62E_M102F_ M113L VH (8) SYSMN (10) SISSSSSYIYYAESV KG (12) VGLFGPFDI (14)

* Defined according to the Kabat numbering system.

Table 3

Amino acid sequences of CDR light chains of example antibodies against CTLA-4*

VL (SEQ ID NO:) CDRL1 (SEQ ID NO:) CDRL2 (SEQ ID NO:) CDRL3 (SEQ ID NO:) AGEN1884 VL (9) RASQSVSRYLG (15) GASTRAT (16) QQYGSSPWT (17)

* Defined according to the Kabat numbering system.

Table 4

Examples of anti-CTLA-4 antibodies

Antibody Heavy chain variable region SEQ ID NO: Light chain variable region SEQ ID NO: AGEN1884 AGEN1884 VH 1 AGEN1884 VL 9 AGEN1884.Hl.1 AGEN1884_M102F VH 2 AGEN1884 VL 9 AGEN1884.Hl.2 AGEN1884_M113L VH 3 AGEN1884 VL 9 AGEN18 84.Hl .3 AGEN1884_D62E VH 4 AGEN1884 VL 9 AGEN1884.H2.1 AGEN18 8 4_M102 F_M113L VH 5 AGEN1884 VL 9 AGEN1884.H2.2 AGEN1884_D62E_M102F VH 6 AGEN1884 VL 9 AGEN1884.H2.3 AGEN1884_D62E_M113L VH 7 AGEN1884 VL 9 AGEN1884.H3 AGEN1884_D62E_M102F_ M113L VH 8 AGEN1884 VL 9

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Table 5

Nearby germline genes

SEQ ID NO: Nearest germline gene Amino acid sequence 21 IGHV3-21*01 EVQLVESGGGLVKPGGSLRLSCAASGFFSSYSM NWVRQAPGKGLEWVS SISSSSSYIYYADSVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCAR 22 IGKV3-20*01 EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYL AWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAVYYCQQYGSSP

Table 6

Example sequences of CTLA-4 and family members

SEQ ID NO: Description Amino acid sequence 33 Immature protein CTLA-4 humans (P16410) MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKA MHVAQPAWLAS SRGI AS FVCE YAS PGKATEVRVTVL RQADSQVTEVCAATYMMGNELTFLDSICTGTSSGNQ VNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQ IYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVS LSKMLKKRSPL TTGVYVKMPPTEPECEKQFQPYFIPI N 34 CTLA-4 epitope YLGI 35 CTLA-4 epitope YPPPYYLGI 36 CTLA-4 epitope YLGIGNGTQI 37 CTLA-4 epitope YPPPYYLGIGNGTQI 38 CTLA-4 epitope MYPPPYY 39 CTLA-4 epitope QVT 40 CTLA-4 macaque- crabeater (G7PL88) MACLGFQRHKARLNLATRTRPYTLLFSLLFIPVFSKA MHVAQ PAWLANS RG IAS FVCE YAS PGKATEVRVTVL RQADSQVTEVCAATYMMGNELTFLDSICTGTSSGNQ VNLTIQGLRAMDTGLYICKVELMYPPPYYMGIGNGTQ IYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVS LSKMLKKRSPLTT GVYVKMPPTEPECEKQFQPYFIPI N 41 CTLA-4 mouse (P09793) MACLGLRRYKAQLQLPSRTWPFVALLTLLFIPVFSEA IQVTQPSWLASSHGVASFPCEYSPSHNTDEVRVTVL RQTNDQMTEVCATTFTEKNTVGFLDYPFCSGTFNESR VNLTIQGLRAVDTGLYLCKVELMYPPPYFVGMGNGTQ IYVIDPEPCPDSDFLLWILVAVSLGLFFYSFLVSAVS LSKMLKKRSPLTTGV YVKMPPTEPECEKQFQPYFIPI N

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42 CTLA-4 rat (Q62859) MACLGLQRYKTHLQLPSRTWPFGVLLSLLFIPIFFSEA IQVTQPSVVLASSHGVASFPCEYASSHNTDEVRVTVL RQTNDQVTEVCATTFTVKNTLGFLDDPFCSGTFNESR VNLTIQGLRAADTGLYFCKVELMYPPPYFVGMGNGTQ IYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLVTAVS LNRTLKKRSPLTTGVY VKMPPTEPECEKQFQPYFIPI N 43 Human CD28 (P10747) MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVN LSCKYSYNLFSREFRASLHKGLDSAVEVCWYGNYSQ QLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYF CKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPG PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRS KRSR LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 44 Human ICOS (Q9Y6W8) MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGG VQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNT VSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNL SIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCA AFVWC ILGCILICWLTKKKYS S SVHDPNGEYMFMRA VNTA KKSRLTDVTL 45 Human BTLA (Q7Z6A9) MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQ LYIKRQSEHSILAGDPFELECPVKYCANRPHVTWCKL NGTTCVKLEDRQTSWKEEKNISFFILHFEPVLPNDNG SYRCSANFQSNLIESHSTTLYVTDVKSASERPSKDEM ASRPWLLYRLLPLGGLPLLITTCFCLFCCLRRHQGKQ NELSDTAGRE INLVDAHLKSEQTEASTRQNSQVLLSE TGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYAS LNHSVIGPNSRLARNVKEAPTEYASICVRS 46 Human PD-1 (Q15116) MQIPQAPWPWWAVLQLGWRPGWFLDSPDRPWNPPTF SPALLWTEGDNATFTCSFSNTSESFVLNWYRMSPSN QTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVV RARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERR AEVPTAHPSPSPRPAGQFQTLWGWGGLLGSLVLLV WVLAVICSRAARGTIGARRTG QPLKEDPSAVPVFSVD YGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGT SSPARRGSADGPRSAQPLRPEDGHCSWPL

In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (eg, human CTLA-4) comprising a VH domain comprising one, two, or all three of the VH domain CDR regions shown in Table 1. 1 in this document. In some embodiments, the antibody comprises CDRH1 of one of the VH domains shown in Table 1. 1. According to some embodiments, the antibody comprises CDRH2 of one of the VH domains presented in table. 1. In some embodiments, the antibody comprises CDRH3 of one of the VH domains shown in Table. 1.

In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (eg, human CTLA-4) comprising a VL domain comprising one, two, or all three of the VL domain CDR regions listed in Table 1. 1 in this document. In some embodiments, the antibody comprises CDRL1 of one of the VL domains shown in Table 1. 1. In some embodiments, the antibody comprises CDRL2 of one of the VL domains presented in Table 1. In some embodiments, the antibody comprises CDRL3 of one of the VL domains presented in Table 1. 1.

In some embodiments, antibody CDR regions can be defined 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.

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In some embodiments, antibody CDR regions may be defined according to the Chothia numbering scheme, which relates to the location of structural loops of immunoglobulins (see, for example, Chothia C & 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): 17 5-82; and US Pat. No. 7,709,226, each of which is incorporated herein by reference in its entirety). Typically, using Kabat numbering, the Chothia CDRH1 loop is located on heavy chain amino acids 26-32, 33 or 34, the Chothia CDRH2 loop is located on heavy chain amino acids 52-56, and the Chothia CDRH3 loop is located on heavy chain amino acids 95. -102, and the CDRL1 Chothia loop is located on light chain amino acids 24-34; the Chothia loop CDRL2 is located on light chain amino acids 50-56 and the Chothia loop CDRL3 is located on light chain amino acids 89-97. The end of the CDRH1 loop according to Chothia, when using the accepted Kabat numbering, varies from H32 to H34 depending on the length of the loop (this is due to the fact that in the Kabat numbering scheme the insertions are located in positions H35A and H35B; in the absence of both 35A and 35B, the loop ends at position 32; if only 35A is present, the loop ends at position 33; if both 35A and 35B are present, the loop ends at position 34).

In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (eg, human CTLA-4) containing the Chothia VH CDR regions of the VHs listed in Table 1. 1 in this document. In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (eg, human CTLA-4) comprising the Chothia VL CDR regions of the VLs listed in Table 1. 1 in this document. In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (eg, human CTLA-4) comprising the Chothia antibody CDR VH regions and the Chothia antibody CDR VL regions set forth in Table 1. 1 in this document. In some embodiments, antibodies that specifically bind to CTLA-4 (eg, human CTLA-4) contain one or more CDR regions, wherein the Chothia and Kabat CDR regions have the same amino acid sequence. In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (eg, human CTLA-4) and contains combinations of Kabat CDR regions and Chothia CDR regions.

In some embodiments, antibody CDR regions may be defined according to the IMGT numbering system as described in Lefranc M-P (1999) The Immunologist 7: 132-136 and Lefranc M-P et al. (1999) Nucleic Acids Res 27: 209-212, each of which is incorporated herein by reference in its entirety.

According to some embodiments of the present invention, antibodies are provided that specifically bind to CTLA-4 (eg, human CTLA-4) and contain the antibody CDR regions shown in table. 1 herein, when applying the IMGT numbering system, for example, as described in Lefranc M-P (1999), supra, and Lefranc M-P et al., (1999), supra.

In some embodiments, antibody CDR regions may be defined according to an AbM numbering scheme that refers to AbM hypervariable regions that are a compromise between Kabat CDR regions and Chothia structural loops, and are used in Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), incorporated herein in its entirety by reference. According to a specific embodiment of the present invention, antibodies are provided that specifically bind to CTLA-4 (eg, human CTLA-4) and contain the antibody CDR regions shown in table. 1 in this document when using the AbM numbering scheme.

In some embodiments, the present invention 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 sequences 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 containing the amino acid sequences of the CDRL1, CDRL2 and CDRL3 regions of the VL domain shown in SEQ ID NO: 9, each CDR defined by MacCallum, Kabat, Chothia , according to a combination of the Kabat and Chothia definitions, according to the IMGT numbering system or the CDR definition according to AbM.

In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), comprising:

(a) CDRH1 containing the amino acid sequence SYSMN (SEQ ID NO: 10); and/or (b) CDRH2 containing the amino acid sequence SISSSSSYIYYAXSVRG (SEQ ID NO: 18), where X is E or D; and/or (c) CDRH3 containing the amino acid sequence VGLXGPFDI (SEQ ID NO: 19), where X represents F or M; and/or (d) CDRL1 containing the amino acid sequence RASQSVSRYLG (SEQ ID NO: 15); and/or

- 31 044966 (e) CDRL2 containing the amino acid sequence GASTRAT (SEQ ID NO: 16); and/or (f) CDRL3 containing the amino acid sequence QQYGSSPWT (SEQ ID NO: 17), and wherein the CDRH1, CDRH2 and CDRH3 sequences of said antibody are not the sequences of SEQ ID NO: 10, 11 and 13, respectively.

In some embodiments, CDRH2 contains the amino acid sequence of SEQ ID NO: 11. In some embodiments, CDRH2 contains the amino acid sequence of SEQ ID NO: 12. In some embodiments, CDRH3 contains the amino acid sequence of SEQ ID NO: 13. In some embodiments, CDRH3 contains the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) comprising a VH domain comprising the amino acid sequences CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NOs: 10, 11, and 14; 10, 12 and 13; or 10, 12 and 14, respectively. Some embodiments of the present invention provide an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) comprising a VH domain comprising the amino acid sequences CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NOs: 10, 12, and 14 , respectively.

In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), comprising a heavy chain variable region comprising the CDRH1, CDRH2, and CDRH3 regions, and a light chain variable region comprising the CDRL1 regions, CDRL2 and CDRL3, wherein said regions CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 contain 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 some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), comprising a heavy chain variable region comprising the CDRH1, CDRH2, and CDRH3 regions, and a light chain variable region comprising the CDRL1 regions, CDRL2 and CDRL3, wherein said regions CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 contain the amino acid sequences set forth in SEQ ID NOs: 10, 12, 14, 15, 16 and 17, respectively.

Some embodiments of the present invention provide 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. Some embodiments of the present invention provide an isolated an antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) comprising a heavy chain variable region comprising an amino acid sequence of 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. According to some embodiments of the present invention, there is provided an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) comprising a heavy chain variable region comprising an amino acid sequence of at least 75 %, 80%, 85%, 90%, 95%, 99% or 100% (for example, 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 some 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 some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3. implementation, said antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, said antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, said antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: b. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 8.

Some embodiments of the present invention provide 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 of at least 75%, 80%, 85%, 90 %, 95%, 99% or 100% (for example, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical amino acid sequence, set forth in SEQ ID NO: 9. In some embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 9.

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In some embodiments, the present invention 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 sequence amino acids from SEQ ID NO: 9. According to some embodiments of the present invention, there is provided an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) comprising a heavy chain variable region comprising an amino acid sequence of at least 75% , 80%, 85%, 90%, 95%, 99% or 100% (for example, 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 containing an amino acid sequence of 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 represented by in SEQ ID NO: 9. According to some embodiments of the present invention, there is provided an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) comprising a heavy chain variable region comprising an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, 99% or 100% (for example, 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 at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid sequence ( for example, 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 some embodiments said antibody contains a heavy chain variable region and a light chain variable region with the amino acid sequences set forth in SEQ ID NOs: 2 and 9; 4i9; 5i9; 6 and 9; 7 and 9; or 8 and 9, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 2 and 9, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 3 and 9, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 4 and 9, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 5 and 9, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region with the amino acid sequences set forth in SEQ ID NOs: 6 and 9, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region with the amino acid sequences set forth in SEQ ID NOs: 7 and 9, respectively.

In some embodiments, the antibody comprises 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.

Some embodiments of the present invention provide 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 the human germline IGHV3-21 sequence (e.g., IGHV3- 21*01, for example, with the amino acid sequence from 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) may be derived from the human germline IGHV3-21 sequence (e.g., IGHV3-21* 01, for example, with the amino acid sequence from SEQ ID NO: 21). In one embodiment, all of the regions, framework 1, framework 2, framework 3, CDRH1 and CDRH2, are derived from the human germline IGHV3-21 sequence (e.g., IGHV3-21*01, e.g., with the amino acid sequence of SEQ ID NO: 21 ).

Some embodiments of the present invention provide 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 (eg, IGKV3-20*01, eg, with the amino acid sequence of 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) may be derived from a human germline sequence selected from the group consisting of IGKV3-20 (eg, IGKV3-20*01, eg, with the amino acid sequence of SEQ ID NO: 22). In one embodiment, all of the regions, scaffold 1, scaffold 2, scaffold 3, CDRL1 and CDRL2, are derived from a human germline sequence selected from the group consisting of IGKV3-20 (e.g., IGKV3-20*01, e.g., with the sequence amino acids from SEQ ID NO: 22).

- 33 044966

Some embodiments of the present invention provide 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 the human germline IGHV3-21 sequence (e.g., IGHV3- 21*01, for example, with 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 IGKV3-2 0 (for example, IGKV3-20 *01, for example, with the amino acid sequence of SEQ ID NO: 22).

In some embodiments, the present invention provides an isolated antibody that cross-competes for binding to CTLA-4 (eg, 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 some embodiments, the present invention provides an isolated antibody that cross-competes for binding to CTLA-4 (e.g., human CTLA-4) with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 3 and 9. respectively.

In some embodiments, the present invention provides an isolated antibody that binds to the same or overlapping CTLA-4 epitope (e.g., human CTLA-4 epitope) as an antibody described herein, e.g., an antibody comprising amino acid sequences of the severe and light chains presented in SEQ ID NO: 2 and 9; 4 and 9; 5 and 9; 6 and 9; 7 and 9; or 8 and 9, respectively. In some embodiments, the present invention provides an isolated antibody that binds to the same or overlapping CTLA-4 epitope (e.g., human CTLA-4 epitope) as an antibody described herein, e.g., an antibody comprising amino acid sequences of the severe and light chains shown in SEQ ID NO: 3 and 9, respectively. In some embodiments, said antibody epitope can be determined using, for example, NMR spectroscopy, surface plasmon resonance (BIAcore®), X-ray diffraction crystallography studies, ELISA assays, hydrogen-deuterium exchange coupled to mass spectrometry (e.g. liquid chromatography/electrospray mass spectrometry), oligopeptide scanning chip assays, and/or mutagenesis mapping (eg, site-directed mutagenesis mapping). In X-ray crystallography, crystallization can be carried out using any methods known in the art (for example, see references: Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350; McPherson A (1990) ) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, each of which is incorporated herein by reference in its entirety). Antibody:antigen crystals can be examined using well-known X-ray diffraction techniques, and their structure can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., sources: Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.; US patent application no. 2004/0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1): 37-60 ; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316-1323, each of which is incorporated herein in its entirety via link). Mutagenesis mapping studies can be carried out using any method known to one skilled in the art; see, for example, for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, in Champe M et al., (1995), supra, and Cunningham BC & Wells JA (1989), supra. In a specific embodiment, the antibody epitope is determined using alanine scanning mutagenesis studies. In addition, antibodies that recognize and bind the same or overlapping epitopes of CTLA-4 (e.g., human CTLA-4) can be identified using routine techniques such as immunoassays, for example, by demonstrating the ability of one antibody to block the binding of another antibody with the target antigen, i.e. competitive binding assay. Competitive binding assays can also be used to determine whether two antibodies have similar epitope binding specificity. Competitive binding can be determined in an assay where the test immunoglobulin inhibits the specific binding of a reference antibody to a common antigen such as CTLA-4 (eg, 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 (ELISA), competitive sandwich assay (see Stahli C et al., (1983) Methods Enzymol 9: 242-253); direct phase biotin-avidin ELISA (see Kirkland TN et al., (1986) J Immunol 137: 3614-9); solid phase direct labeling assay, solid phase direct labeling sandwich assay (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phase direct RIA labeled 1-125 (see Morel GA et al., (1988) Mol

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Immunol 25(1): 7-15); direct biotin-avidin ELISA (see Cheung RC et al., (1990) Virology 176: 546-52); and direct labeled RIA (see Moldenhauer G et al., (1990) Scand J Immunol 32: 7782), each of which is incorporated herein by reference in its entirety. Typically, such an assay involves the use of purified antigen (eg, CTLA-4, such as human CTLA-4) bound to a solid surface, or cells bearing one of these Igs, an unlabeled test immunoglobulin, and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to a solid surface or cells in the presence of a test immunoglobulin. Usually the immunoglobulin being tested is present in excess. Typically, when the competing antibody is present in excess, it inhibits the 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 performed in a variety of different formats using either a labeled antigen or a labeled antibody. In the general version of this assay, the antigen is immobilized on a 96-well plate. The ability of unlabeled antibodies to block the binding of labeled antibodies to antigen is then measured using radioactive or enzyme labels. Additional detailed information can be found, for example, in the sources: Wagener C et al., (1983) J Immunol 130: 2308-2315; Wagener C et al., (1984) J Immunol Methods 68: 269-274; Kuroki M et al., (1990) Cancer Res 50: 4872-4879; Kuroki M et al., (1992) Immunol Invest 21: 523-538; Kuroki M et al., (1992) Hybridoma 11: 391-407; and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D editors, supra, pp. 386-389, each of which is incorporated herein by reference in its entirety.

In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 26. In some embodiments, In some embodiments, said antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, said antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, said antibody comprises a heavy chain a chain comprising the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 26.

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

In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) 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: 23 NO: 27. According to some embodiments of the present invention, there is provided an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4), comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 24, and a light chain containing the sequence amino acids from SEQ ID NO: 27.

In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) 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: 25 : 27. Some embodiments of the present invention provide an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 26 and a light chain comprising the amino acid sequence from SEQ ID NO: 27.

Any Ig constant region can be used in the antibodies described herein. In some embodiments, the Ig region is a human immunoglobulin molecule IgG, IgE, IgM, IgD, IgA, or IgY, an immunoglobulin molecule of any class (e.g., IgG1, _IgG2 , _IgG3 , _IgG4 , IgA1, and _IgA2 ) or any subclass (eg IgG _2a and IgG _2b ).

In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 28, 29, 30, or 31. In some embodiments, Embodiments of the present invention provide an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) comprising a light chain constant region containing the amino acid sequence of SEQ ID NO: 32.

Some embodiments of the present invention provide an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) comprising a heavy chain constant region, e.g., an IgG1 constant region or fragment thereof, containing a mutation selected from the group consisting of S239D, I332E and their combinations according to the EU numbering system. In some embodiments, the antibody comprises a constant region

- 35 044966 IgG1 heavy chain containing mutations S239D and I332E in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 29.

Some embodiments of the present invention provide an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) 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 from S239D, A330L, I332E and their combinations according to the EU numbering system. In some embodiments, the antibody comprises an IgG1 heavy chain constant region containing mutations S239D, A330L, and I332E in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 30.

Some embodiments of the present invention provide an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) comprising a heavy chain constant region, e.g., an IgG1 constant region or fragment thereof, containing a mutation selected from the group consisting of L235V, F243L, R292P, Y300L, P396L and their combinations in accordance with the EU numbering system. In some embodiments, the antibody comprises an IgG1 heavy chain constant region containing mutations L235V, F243L, R292P, Y300L, and P396L in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 31.

In some embodiments, the IgG regions of the antibodies described herein have increased affinity for FcyRIIIA, for example, compared to an antibody containing a wild-type Fc region, such as an IgG1 Fc. Sequence changes that result in increased affinity for FcyRIIIA are known in the art, see, for example, Kellner et al., Methods 65: 105-113 (2014), Lazar et al., Proc Natl Acad Sci 103 : 40054010 (2006), Shields et al., J Biol Chem. 276 (9): 6591-6604 (2001), each of which is incorporated herein by reference in its entirety. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region or fragment thereof, containing a mutation selected from the group consisting of G236A, S239D, F243L, T256A, K29OA, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T and P396L, as well as their combinations, in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, containing S239D in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region or fragment thereof, containing T256A in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region or fragment thereof, containing K290A in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region or fragment thereof, containing S298A in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, containing I332E in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, containing E333A in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region or fragment thereof, containing K334A in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region or fragment thereof, containing A339T in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region or fragment thereof, comprising S239D and I332E in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, comprising S239D, A330L, and I332E in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region or fragment thereof, comprising S298A, E333A, and K334A in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, comprising G236A, S239D, and I332E in accordance with the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region or fragment thereof, comprising F243L, R292P, Y300L, V305I, and P396L in accordance with the EU numbering system.

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In some embodiments, the antibodies described herein exhibit antibody-dependent cellular cytotoxicity (ADCC activity). In some embodiments, the antibodies described herein initiate natural killer cell-mediated cell depletion. In some embodiments, the antibodies described herein are used to treat a tumor infiltrated by natural killer cells. In some embodiments, the antibodies described herein exhibit antibody-dependent cellular phagocytosis (ADCP activity). In some embodiments, the antibodies described herein initiate macrophage-mediated cell depletion. In some embodiments, the antibodies described herein are used to treat a tumor infiltrated by macrophages. In some embodiments, the antibodies described herein selectively deplete intratumoral regulatory T cells.

In some embodiments, an antibody described herein is an activated antibody that binds human CTLA-4 protein when activated. In some embodiments, said activated antibody comprises a masking moiety that inhibits binding of said antibody in an uncleaved state to human CTLA-4 protein, and at least one cleavable moiety conjugated to said antibody, wherein, for example, said cleavable moiety is a polypeptide , which functions as a protease substrate that is enriched in the tumor microenvironment. Examples of activated antibodies are described, for example, in US Patent No. 8513390 and No. 8518404, and in published US patent applications US 2014/0255313, US 2014/0010810, US 2014/0023664, which are incorporated herein by reference. In some embodiments, the activated antibody comprises a human IgG heavy chain constant region that is a wild-type human IgG heavy chain constant region variant, wherein the human IgG heavy chain constant region variant binds to human FcyRIIIA with greater affinity than the binding affinity of the human IgG constant region. wild-type human IgG heavy chain region with human FcyRIIIA.

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

In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) so as to change the number of cysteine residues in the hinge region (e.g., increase or decrease) as described, e.g. in US 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 changed, for example, to facilitate the assembly of light and heavy chains, or to change (eg, increase or decrease) the stability of the antibody.

In a specific embodiment, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant domain or an FcRn binding fragment thereof (preferably an Fc domain or hinge-Fc fragment) to alter (e.g., decrease or increase) the timing half-life of the antibody in vivo; see, for example, international publications No. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Patent No. 5,869,046, No. 6,121,022, No. 6,277,375, and No. 6,165,745, each of which is incorporated herein by reference in its entirety, with examples of mutations that alter (eg, decrease or increase) the half-life of an antibody in vivo. In some embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant domain or an FcRn binding fragment thereof (preferably an Fc domain or hinge-Fc fragment) to reduce the half-life of the antibody in vivo. In other embodiments, one, two, or more amino acid mutations (eg, substitutions, insertions, or deletions) are introduced into an IgG constant domain or an FcRn binding fragment thereof (preferably an Fc domain or hinge-Fc fragment) to increase the in vivo half-life of the antibody. In a specific embodiment, said antibodies may contain one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and/or third constant (CH3) domain (residues 341-447 of human IgG1) according to the EU numbering system. In a specific embodiment, the constant region of an IgG1 antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic substitution acid (E) at position 256 according to the EU numbering system; see US Pat. No. 7,658,921, which is incorporated herein by reference in its entirety. The indicated type of mutant IgG, nase

- 37 044966 mutant YTE has been shown to exhibit a 4-fold increased half-life compared to wild-type variants of the same antibody (see source: Dall'Acqua WF et al., (2006) J Biol Chem 281: 23514- 24, which is incorporated herein by reference in its entirety). In some embodiments, the antibody comprises an IgG constant domain containing one, two, three, or more amino acid residue substitutions at positions 251-257, 285-290, 308-314, 385-389, and 428-436 according to the EU numbering system.

In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of an antibody described herein (e.g., the CH2 domain (residues 231-340 of human IgG1), and/or the CH3 domain (residues 341 -447 human IgG1), and/or a hinge region in accordance with the EU numbering system, to increase or decrease the affinity of the specified antibody for an Fc receptor (for example, an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody, which decrease or increase the affinity of an antibody for the Fc receptor, and techniques for introducing such mutations into the Fc region or fragment thereof are known to one skilled in the art.Examples of mutations in the Fc region of an antibody that can be made to change the affinity of the antibody for the Fc receptor , are described, for example, in Smith P et al., (2012) PNAS 109: 6181-6186, in US patent No. 6737056 and international publications WO 02/060919; WO 98/23289; and WO 97/34631, each of which are incorporated herein by reference in their entirety.

In a further embodiment, one, two, or more amino acid substitutions are introduced into the Fc region of the IgG constant domains to alter the effector function or 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 according to the EU numbering system may be replaced by another amino acid residue such that the affinity of the antibody for the effector ligand changes, but it retains the antigen-binding ability of the original antibody. The effector ligand for which the affinity is changed may be, for example, an Fc receptor or a complement component C1. This approach is described in more detail in US Pat. No. 5,624,821 and US Pat. No. 5,648,260, each of which is incorporated herein by reference in its entirety. In some embodiments, deletion or inactivation (by point mutations or other methods) of the constant region domain can reduce Fc receptor binding by circulating antibody, thereby increasing tumor localization; see, for example, US Pat. No. 5,585,097 and US Pat. No. 8,591,886, each of which is incorporated herein by reference in its entirety, for mutations that delete or inactivate the constant domain and thereby increase tumor localization. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region that may reduce Fc receptor binding (see, for example, 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 may be made in the constant region of an antibody described herein: N297A substitution; replacement N297Q; replacement of L235A and replacement of L237A; replacement of L234A and replacement of L235A; replacement of E233P; replacement L234V; replacement L235A; deletion C236; replacing P238A; replacement D265A; A327Q replacement; or replacing P329A in accordance with the EU numbering system. In some embodiments, a mutation selected from the group consisting of D265A, P329A, and combinations thereof according to the EU numbering system may be introduced into the constant region of an antibody described herein.

In a specific embodiment, the antibody described herein contains an IgG1 constant domain with an amino acid substitution N297Q or N297A in accordance with the EU numbering system. In one embodiment, an antibody described herein comprises an IgG1 constant domain with a mutation selected from the group consisting of D265A, P329A, and combinations thereof according to the EU numbering system. In another embodiment, an antibody described herein comprises an IgG1 constant domain with a mutation selected from the group consisting of L234A, L235A, and combinations thereof according to the EU numbering system. In some embodiments, the amino acid residues in the constant region of an antibody described herein at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to the EU numbering system are not L, L, and D, respectively. This approach is described in detail in international publication 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 the human IgG1 heavy chain are F, E and A; or A, A and A, respectively, according to the EU numbering system.

In some embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 in the constant region of an antibody described herein, in accordance with the EU numbering system, may be replaced by another amino acid residue so as to alter the binding of the antibody. with C1q, and/or decrease or elimination

- 38 044966 prevention of complement-dependent cytotoxicity (CDC) mediated by it. This approach is described in more detail in US 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 are altered, thereby altering the complement fixation ability of the antibody, within amino acid positions 231-238 of the N-terminal region of the CH2 domain of the antibody described herein, in accordance with the EU numbering system. This approach is described in more detail in international publication WO 94/29351, which is incorporated herein by reference in its entirety. In some embodiments, the Fc region of an antibody described herein is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for the Fcγ receptor by mutation of one or more amino acids (e.g., introduction of amino acid substitutions) in the following positions: 238, 239, 248, 249, 2 52, 254, 255, 256, 258, 265, 267, 268, 269, 270,

272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315,

320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389,

398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 according to the EU numbering system. This approach is described in more detail in international publication WO 00/42072, which is incorporated herein by reference in its entirety.

In some embodiments, an antibody described herein comprises an IgG ₄ antibody constant region and a serine in place of a proline at amino acid residue 228 of the heavy chain in accordance with the EU numbering system.

In some 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 comprising two heavy chain constant regions.

In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (eg, human CTLA-4) and functions as an antagonist.

Some embodiments of the present invention provide an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) and reduces the activity of CTLA-4 (e.g., human CTLA-4) by at least 5%, 10%, 15 %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% as assessed using methods described herein and/or known to one skilled in the art, compared to CTLA-4 activity (e.g., human CTLA-4) without any antibody or in the presence of an unrelated antibody (eg, antibodies that do not specifically bind to CTLA-4 (eg, human CTLA-4)). In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) and reduces the activity of CTLA-4 (e.g., human CTLA-4) by at least about 1.2-fold, 1 ,3x, 1.4x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x , 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 -fold, 90-fold, or 100-fold, as assessed using methods described herein and/or known to one skilled in the art, compared to the activity of CTLA-4 (e.g., human CTLA-4) without any antibody or in the presence of an unrelated antibody (eg, an antibody that does not specifically bind to CTLA-4 (eg, human CTLA-4)). Non-limiting examples of CTLA-4 (e.g., human CTLA-4) activity may include CTLA-4 signaling (e.g., human CTLA-4), binding of CTLA-4 (e.g., human CTLA-4) to a CTLA-4 ligand (e.g., human CTLA-4) (eg CD80 or CD86) and inhibition of cytokine production (eg IL-2, IFN-γ or TNF-α). In some embodiments, the present invention provides an isolated antibody that specifically binds to CTLA-4 (eg, human CTLA-4) and inactivates, reduces, or inhibits the activity of CTLA-4 (eg, human CTLA-4). In specific embodiments, the reduction in CTLA-4 (eg, human CTLA-4) activity is assessed as described in the examples below.

According to specific embodiments of the present invention, an isolated antibody is provided that specifically binds to CTLA-4 (eg, human CTLA-4) and reduces the binding of CTLA-4 (eg, human CTLA-4) to its ligand (eg, CD80 or CD86) by by at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 95%, 98%, or 99%, as assessed using methods known to one skilled in the art, compared 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 unrelated antibody (eg, an antibody that does not specifically bind CTLA-4 (eg, human CTLA-4)). According to specific embodiments of the present invention, an isolated antibody is provided,

- 39 044966 which specifically binds to CTLA-4 (eg, human CTLA-4) and reduces the binding of CTLA-4 (eg, human CTLA-4) to its ligand (eg, CD80 or CD86) by at least about 1,2- multiple, 1.3x, 1.4x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x, 70x fold, 80-fold, 90-fold, or 100-fold, as assessed using methods known to one skilled in the art, compared to the binding of CTLA-4 (e.g., human CTLA-4) to its ligand (e.g., CD80 or CD86) without any antibody or in the presence of an unrelated antibody (eg, an antibody that does not specifically bind to CTLA-4 (eg, human CTLA-4)).

Specific embodiments of the present invention provide an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) and increases the production of cytokines (e.g., IL-2, IFN-γ, or TNF-α) by at least about 5 %, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, as assessed using the methods described herein (see examples below) or known to one skilled in the art, compared to cytokine production without or in the presence of any antibody an unrelated antibody (eg, an antibody that does not specifically bind to CTLA-4 (eg, human CTLA-4)). Specific embodiments of the present invention provide an isolated antibody that specifically binds to CTLA-4 (e.g., human CTLA-4) and increases the production of cytokines (e.g., IL-2, IFN-γ, or TNF-α) by at least about 1. 2x, 1.3x, 1.4x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x, 70x, 80-fold, 90-fold, or 100-fold, as assessed using the methods described herein (see examples below) or known to one skilled in the art, compared to cytokine production without any antibody or in the presence of an unrelated antibody (eg, antibodies that do not specifically bind to CTLA-4 (eg, human CTLA-4)).

In some embodiments, IL-2 production by 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) is increased at least about 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times multiple, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold compared to PBMCs stimulated with SEA alone without any antibody or in the presence of an unrelated antibody (eg, an antibody that does not specifically bind CTLA-4 (eg, human CTLA-4)), as assessed using methods described herein (see examples below) or known to one skilled in the art.

Pharmaceutical compositions.

The present invention provides compositions containing the anti-CTLA4 antibody described herein, having the required degree of purity, in a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; 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; complexes with metals (for example, complexes of Zn with protein); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In a specific embodiment, the pharmaceutical compositions comprise an anti-CTLA-4 antibody described herein and, optionally, one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In a specific embodiment, the pharmaceutical compositions contain an effective amount of an antibody described herein or an antigen binding fragment thereof and, optionally, one or more additional prophylactic or therapeutic agents in a pharmaceutically

- 40 044966 acceptable media. In some embodiments, the antibody is the only active ingredient included in the pharmaceutical composition. The pharmaceutical compositions described herein may be suitable for use in inhibiting CTLA-4 activity and treating a condition such as cancer or an infectious disease.

According to one aspect of the present invention, there is provided a pharmaceutical composition comprising an anti-CTLA-4 antibody of the present invention and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient for use as a medicament.

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

Pharmaceutically acceptable carriers used in parenteral formulations include aqueous bases, non-aqueous bases, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable agents. Examples of aqueous bases include sodium chloride injection, Ringer's solution for injection, isotonic dextrose injection, sterile water for injection, and dextrose lactated Ringer's solution for injection. Non-aqueous parenteral bases include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations may be added to parenteral formulations packaged in multi-dose containers, which include phenols or cresols, mercury-containing substances, benzyl alcohol, chlorobutanol, methyl and propyl phydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone.

Emulsifying agents include Polysorbate 80 (TWEEN® 80). The metal ion sequestering or chelating agent includes EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water-miscible bases; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid to adjust the pH.

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, characterized by subcutaneous, intramuscular or intravenous injection, is also contemplated by the present invention. Injectable compositions may be prepared in standard form, either as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in liquid prior to injection, or as emulsions. Injectable formulations, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions for administration may also contain minor amounts of non-toxic co-materials such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers and other such agents such as, for example, sodium acetate, sorbitan monolaurate, triethanol aminoleate and cyclodextrins.

Formulations for parenteral administration of the antibody include prepared sterile injectable solutions, sterile dry soluble products, such as lyophilized powders, ready to be combined with a vehicle immediately before use, including subcutaneous tablets, prepared sterile injectable suspensions, sterile dry insoluble products, ready to be combined with the base immediately before use, and sterile emulsions. These solutions can be either aqueous or non-aqueous.

For intravenous administration, suitable carriers include physiological saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

Topical mixtures containing the antibody are prepared as described for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, or the like, and may be formulated into creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, drenches, sprays, suppositories, dressings, dermal patches or any other formulations suitable for topical administration.

The anti-CTLA-4 antibody described herein can be formulated into a topical aerosol, for example, by inhalation (see, for example, US Pat. No. 4,044,126,

- 41 044966 No. 4414209 and No. 4364923, which describe aerosols for the delivery of a steroid suitable for the treatment of inflammatory diseases, in particular asthma, and are incorporated herein in their entirety by reference). Said compositions for administration to the respiratory tract may take the form of an aerosol or nebulizer solution, or a micronized powder for insufflation, alone or in combination with an inert carrier such as lactose. In this case, the particles of the specified composition have, according to one embodiment, diameters of less than 50 μm, according to one embodiment, less than 10 μm.

The anti-CTLA-4 antibody described herein can be formulated for topical or topical use, for example, for topical application to the skin and mucous membranes, such as the eyes, in the form of gels, creams and lotions, and for application to eyes; or for intracisternal or intraspinal use. Topical administration includes transdermal delivery, as well as ocular or mucosal administration or inhalation therapy. Nasal antibody solutions may also be administered alone or in combination with other pharmaceutically acceptable excipients.

Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those skilled in the art and can be used to administer the antibody. For example, such patches are described in US Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, each of which is incorporated herein by reference in its entirety. .

In some embodiments, a pharmaceutical composition comprising an antibody described herein, or an antigen-binding fragment thereof, is a lyophilized powder that can be reconstituted for administration as solutions, emulsions, and other mixtures. It can also be reconstituted and incorporated into solid formulations or gels. A lyophilized powder is prepared by dissolving an antibody described herein, or an antigen binding fragment thereof, or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain an excipient that enhances stability or other pharmacological component of the powder or reconstituted solution obtained from said powder. Suitable excipients include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate, or other similar buffer known to those skilled in the art, in one embodiment having an approximately neutral pH value. Subsequent sterilizing filtration of the solution, followed by lyophilization under standard conditions known to those skilled in the art, provides the desired composition. In one embodiment, the resulting solution is distributed into lyophilization vessels. Each vial contains a single dose or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions, for example, from about 4°C to room temperature. Reconstitution of said lyophilized powder with water for injection provides a composition for parenteral use. For 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.

The anti-CTLA-4 antibodies described herein and other compositions provided by the present invention may also be formulated to target a specific tissue, receptor, or other area of the body of the subject being treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated by the present invention for use in the present compositions. Non-limiting examples of targeting methods are described, for example, in US patents No. 6316652, No. 6274552, No. 6271359, No. 6253872, No. 6139865, No. 6131570, No. 6120751, No. 6071495, No. 6060082, No. 6048736, No. 6039975, No. 6004534, No. 5985307, No. 5972366, No. 5900252, No. 5840674, No. 5759542 and No. 5709874, each of which is incorporated herein by reference in its entirety. In a specific embodiment, an antibody described herein, or an antigen-binding fragment thereof, is targeted to a tumor.

Compositions for in vivo administration may be sterile. Sterility can be easily achieved by filtration through, for example, sterilizing filtration membranes.

Methods and applications

According to another aspect of the present invention, there is provided a method of treating a subject using an anti-CTLA-4 antibody described herein. Treatment of any disease or disorder in a subject that would benefit from inhibition of CTLA-4 function can be achieved using the anti-CTLA-4 antibody described herein. Antibodies against CTLA-4 described herein are particularly suitable for inhibiting immune system tolerance to tumors and accordingly can be used as immunomodulators.

- 42 044966 notherapy for subjects suffering from cancer. For example, some embodiments of the present invention provide 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 or a pharmaceutical composition containing the same as described herein. In some embodiments, the present invention 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.

Cancers that may be treated with antibodies, therapeutic combinations, or pharmaceutical compositions described herein include, but are not limited to, solid cancer (eg, relapsed or refractory solid cancer, and advanced or metastatic solid cancer), carcinoma, sarcoma , melanoma (eg, stage III or stage IV melanoma), small cell lung cancer, non-small cell lung cancer, urothelial cancer, ovarian cancer, prostate cancer (eg, metastatic hormone-refractory prostate cancer and advanced metastatic prostate cancer), pancreatic cancer glands, breast cancer (eg, HER2+ breast cancer (eg, relapsed/refractory HER2+ breast cancer)), head and neck cancer (eg, relapsed/refractory head and neck squamous cell carcinoma (HNSCC)), glioma, malignant glioma, glioblastoma multiforme, brain metastases, Merkel cell cancer, gastric cancer, gastroesophageal cancer, renal cell cancer, uveal melanoma, colon cancer, cervical cancer, lymphoma (eg, relapsed or refractory lymphoma), non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia and multiple myeloma.

In some embodiments, the cancer is treated by intratumoral administration of an antibody, therapeutic combination, or pharmaceutical composition described herein. Cancers that may be treated by intratumoral administration of antibodies, therapeutic combinations, or pharmaceutical compositions described herein include, but are not limited to, solid tumors (eg, advanced or metastatic solid tumors), head and neck cancers (eg, recurrent/ refractory head and neck squamous cell carcinoma (HNSCC)) and breast cancer (eg, HER2+ breast cancer (eg, relapsed/refractory HER2+ breast cancer)).

In some embodiments, the cancer being treated in accordance with the methods described herein is a solid tumor. In some embodiments, the cancer treated in accordance with the methods described herein is a metastatic or locally advanced cancer (eg, a metastatic or locally advanced solid tumor). In some embodiments, said cancer is treated in accordance with a method described herein as the first anticancer therapy after diagnosis of said metastatic or locally advanced tumor (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2 , 3, 4, 6, 8 or 12 weeks; or 1, 2, 3, 4, 6, 8 or 12 months after diagnosis). In some embodiments, the cancer is treated in accordance with the method described herein as the first anticancer therapy after tumor progression is diagnosed (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or 1, 2, 3, 4, 6, 8, or 12 months after diagnosis of tumor progression) that occurred despite prior treatment of said tumor with another anticancer therapy, not necessarily the method described herein, is used as a second anticancer therapy used. In some embodiments, the cancer is treated in accordance with the method described herein as the first anticancer therapy after toxicity of another anticancer therapy has been diagnosed (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 another anticancer therapy), optionally the method described herein being used as the second anticancer therapy used therapy. In some embodiments, the cancer treated in accordance with the methods described herein is a metastatic or locally advanced cancer (eg, a solid tumor) for which there is no available standard therapy. In other embodiments, the cancer treated in accordance with the methods described herein is a metastatic or locally advanced cancer (e.g., a solid tumor) that has failed treatment with standard therapy (i.e., the cancer has progressed after receiving standard therapy). therapy). In some embodiments, therapy is unsuccessful if the cancer is refractory to said therapy. In some embodiments, a therapy is unsuccessful if the cancer recurs after a response, complete or partial, to the therapy. In some embodiments, metastatic or locally advanced cancer (eg, a solid tumor) has been confirmed histologically or cytologically.

In some embodiments, the cancer is a solid tumor.

- 43 044966

In some embodiments, the cancer (eg, a solid tumor) expresses PD-L1. In some embodiments, the percentage of tumor cells in a cancer sample (e.g., solid tumor) that exhibit detectable expression (e.g., partial or complete expression) of PD-L1 is at least 1% (e.g., at least 2%, 3% , 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90%). In some embodiments, the percentage of tumor cells in a cancer sample (e.g., a solid tumor) that exhibit detectable membranous expression (e.g., partial or complete membranous expression) of PD-L1 is at least 1% (e.g., at least 2% 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90%). In some embodiments, the percentage of tumor cells in a cancer sample (eg, solid tumor) that exhibit detectable membrane expression (eg, partial or complete membrane expression) of PD-L1 is at least 1%. In some embodiments, the percentage of tumor cells in a cancer sample (eg, solid tumor) that exhibit detectable membrane expression (eg, partial or complete membrane expression) of PD-L1 is at least 5%. In some embodiments, the percentage of tumor cells in a cancer sample (eg, solid tumor) that exhibit detectable membrane expression (eg, partial or complete membrane expression) of PD-L1 is at least 25%. In some embodiments, the percentage of tumor cells in a cancer sample (eg, solid tumor) that exhibit detectable membrane expression (eg, partial or complete membrane expression) of PD-L1 is at least 50%.

In some embodiments, said metastatic or locally advanced cancer (eg, solid tumor) expresses PD-L1. In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced cancer (e.g., a solid tumor) that exhibit detectable expression (e.g., partial or complete expression) of PD-L1 is at least 1% (e.g., at least 2% , 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90%). In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced cancer (e.g., a solid tumor) that exhibit detectable membranous expression (e.g., partial or complete membranous expression) of PD-L1 is at least 1% (e.g., at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90% ). In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced cancer (eg, solid tumor) that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 1%. In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced cancer (eg, solid tumor) that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 5%. In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced cancer (eg, solid tumor) that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 25%. In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced cancer (eg, solid tumor) that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 50%.

For any and all of the methods described herein that require a certain percentage of cells in a sample to exhibit detectable expression (e.g., membrane expression, partial or complete membrane expression) of PD-L1, PD-L1 expression may be detected by any method well known in the art, including, but not limited to, by immunohistochemical examination.

Examples of immunohistochemical assays for measuring PD-L1 expression in tumor cells are suggested in: Hirsch et al. (2017, J. Thoracic Oncol. 12(2): 208-222), Rimm et al. (2017, JAMA Oncol. 3(8): 1051–1058), and Diggs and Hsueh (2017, Biomarker Res. 5:12), which are incorporated by reference herein in their entirety.

In some embodiments, the cancer being treated in accordance with the method described herein is metastatic or locally advanced non-small cell lung cancer (NSCLC). In some embodiments, the cancer treated in accordance with the method described herein is metastatic non-small cell lung cancer (NSCLC). In some embodiments, the cancer being treated in accordance with the method described herein is stage IV metastatic or locally advanced NSCLC. In some embodiments, the cancer being treated in accordance with the method described herein is stage IV metastatic NSCLC. In some embodiments, the percentage of tumor cells in the sample

- 44 044966 metastatic or locally advanced NSCLC that exhibit detectable expression (eg, partial or complete expression) of PD-L1 is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20 %, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90%. In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced NSCLC that exhibit detectable membranous expression (e.g., partial or complete membranous expression) of PD-L1 is at least 1%, 2%, 3%, 4%, 5% , 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90%. In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced NSCLC that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 1%. In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced NSCLC that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 5%. In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced NSCLC that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 25%. In some embodiments, the percentage of tumor cells in a sample of metastatic or locally advanced NSCLC that exhibit detectable membranous expression (eg, partial or complete membranous expression) of PD-L1 is at least 50%. In some embodiments, said metastatic or locally advanced NSCLC is not characterized by genomic tumor aberrations of rEGF or ALK. In some embodiments, the metastatic or locally advanced NSCLC is not characterized by a sensitizing eEGF mutation (eg, a mutation that is responsive to treatment with a tyrosine kinase inhibitor, including erlotinib, gefinitib, or afatinib) or an ALK translocation. In some embodiments, the subject having metastatic or locally advanced NSCLC has not previously received systemic chemotherapy treatment for metastatic or locally advanced NSCLC. In some embodiments, said metastatic or locally advanced NSCLC is treated in accordance with the method described herein as the first anticancer therapy after diagnosis (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3 4, 6, 8, or 12 weeks; or 1, 2, 3, 4, 6, 8, or 12 months after diagnosis) of metastatic or locally advanced NSCLC. In some embodiments, the method comprises treating a subject suffering from NSCLC (e.g., stage IV metastatic or locally advanced NSCLC) using an anti-CTLA-4 antibody described herein, e.g., AGEN1884.H3 (IgG1 S239D/A330L/I332E), or a pharmaceutical composition containing such an anti-CTLA-4 antibody, wherein the percentage of tumor cells in the NSCLC sample that exhibit detectable membranous expression (e.g., partial or complete membranous expression) of PD-L1 is at least 1%, 2%, 3 %, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90%, and wherein this method is proposed as the first anticancer therapy after diagnosis of cervical cancer (for example, within 1, 2, 3, 4, 5 or 6 days; 1, 2, 3, 4, 6, 8 or 12 weeks; or 1, 2 , 3, 4, 6, 8 or 12 months after diagnosis).

In some embodiments, the cancer being treated in accordance with the methods described herein is cervical cancer. In some embodiments, the cancer treated in accordance with the methods described herein is metastatic or locally advanced unresectable squamous cell carcinoma, glandular squamous cell carcinoma, or adenocarcinoma of the cervix. In some embodiments, the cancer being treated in accordance with the methods described herein is unresectable or metastatic cervical cancer. In some embodiments, said cervical cancer has progressed after standard therapy (eg, recurred after said standard therapy or is refractory to said standard therapy). In some embodiments, said standard therapy includes platinum-containing chemotherapy. In some embodiments, the platinum-containing chemotherapy is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, picoplatin, triplatin, phenanthriplatin, iproplatin, lobaplatin, heptaplatin, lipoplatin, and combinations thereof. In some embodiments, said standard therapy further includes a second chemotherapy. In some embodiments, said second chemotherapy is selected from the group consisting of a nucleotide analog (eg, gemcitabine), a folate antimetabolite (eg, pemetrexed), and a taxane (eg, paclitaxel). In some embodiments, said standard therapy is platinum-based doublet chemotherapy (PT-DC) (also known as platinum doublet chemotherapy) known in the art. In some embodiments, said RT-DC includes cisplatin and gemcitabine, cisplatin and pemetrexed, cisplatin and paclitaxel, carboplatin and paclitaxel, or cisplatin and topotecan. Standard therapy (eg, including

- 45 044966

RT-DC) may optionally further include one or more additional therapies, such as bevacizumab. In some embodiments, the standard therapy includes paclitaxel and topotecan. In some embodiments, said cervical cancer is HPV positive. In some embodiments, said cervical cancer is associated with microsatellite instability. In some embodiments, the cancer treated in accordance with the methods described herein is metastatic or locally advanced unresectable squamous cell carcinoma, glandular squamous cell carcinoma, or adenocarcinoma of the cervix that has relapsed after platinum-containing doublet chemotherapy administered to treat advanced (recurrent, unresectable or metastatic) disease. In some embodiments, the cervical cancer is treated in accordance with the method described herein as the first anticancer 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). In some embodiments, the cervical cancer is treated in accordance with the method described herein as the first anticancer therapy after tumor progression is diagnosed (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or 1, 2, 3, 4, 6, 8, or 12 months after diagnosis of tumor progression) that occurred despite prior treatment for cervical cancer with other anticancer therapy, optionally the method described herein is used as the second anticancer therapy used. In some embodiments, the cervical cancer is treated in accordance with the method described herein as the first anticancer therapy after toxicity of another anticancer therapy has been diagnosed (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 another anticancer therapy), optionally the method described herein being used as a second anticancer therapy used. In some embodiments, the method comprises treating a subject suffering from cervical cancer (e.g., metastatic or locally advanced unresectable squamous cell carcinoma, glandular squamous cell carcinoma, or adenocarcinoma of the cervix) using an anti-CTLA-4 antibody described herein, e.g., AGEN1884 .H3 (IgG1 S239D/A330L/I332E), or a pharmaceutical composition containing such an anti-CTLA-4 antibody, which method is proposed as the first anti-cancer therapy after diagnosis of cervical cancer (for example, within 1, 2, 3, 4 , 5 or 6 days; 1, 2, 3, 4, 6, 8 or 12 weeks; or 1, 2, 3, 4, 6, 8 or 12 months after diagnosis). In some embodiments, the method comprises treating a subject suffering from cervical cancer (e.g., metastatic or locally advanced unresectable squamous cell carcinoma, glandular squamous cell carcinoma, or cervical adenocarcinoma) using an anti-CTLA-4 antibody described herein, e.g., AGEN1884 .H3 (IgG1 S239D/A330L/I332E), or a pharmaceutical composition containing such an anti-CTLA-4 antibody, wherein the method is applied after diagnosis of tumor progression (for example, within 1, 2, 3, 4, 5 or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or 1, 2, 3, 4, 6, 8, or 12 months after diagnosis of tumor progression) that occurred despite prior treatment for cervical cancer with other anticancer therapy , or used after toxicity of another anticancer therapy has been diagnosed (eg, 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 another anticancer therapy), and the method described herein is proposed as the second anticancer therapy used.

In some embodiments, the cancer being treated in accordance with the methods described herein is cutaneous squamous cell carcinoma (cSCC). In some embodiments, the cancer being treated in accordance with the methods described herein is stage IV cutaneous squamous cell carcinoma (cSCC). In some embodiments, the cSCC (eg, stage IV cSCC) is not curable with radiation therapy. In some embodiments, said stage IV cSCC is diagnosed histologically or cytologically according to the American Joint Committee on Cancer Staging Manual, 8th edition (AJCC-8). In some embodiments, said cSCC (e.g., stage IV cSCC) is treated in accordance with the method described herein as the first anticancer therapy after diagnosis of said cSCC (e.g., stage IV cSCC) (e.g., within 1, 2, 3 , 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or 1, 2, 3, 4, 6, 8, or 12 months after diagnosis). In some embodiments, the specified cSCC (e.g., stage IV cSCC) is treated in accordance with the method described herein as the first anticancer therapy after diagnosis of tumor progression (e.g., within 1, 2, 3, 4, 5, or 6 days ; 1, 2, 3, 4, 6, 8, or 12 weeks; or 1, 2, 3, 4, 6, 8, or 12 months after diagnosis of tumor progression) that did not occur

- 46 044966 based on prior treatment of said cSCC (eg, stage IV cSCC) with another anticancer therapy, optionally the method described herein being used as the second anticancer therapy used. In some embodiments, the specified cSCC (e.g., stage IV cSCC) is treated according to the method described herein as the first anticancer therapy after toxicity of another anticancer therapy has been diagnosed (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 another anticancer therapy), not necessarily the method described herein , is used as a second-line anticancer therapy.

In some embodiments, the cancer treated in accordance with the methods described herein is a B-cell lymphoma (e.g., B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin's lymphoma, cutaneous B-cell lymphoma, diffuse large B-cell lymphoma, cellular lymphoma), basal cell carcinoma, bladder cancer, blastoma, brain metastases, breast cancer, Burkitt's lymphoma, carcinoma (eg, adenocarcinoma (eg, gastroesophageal junction)), cervical cancer, colon cancer, colon cancer and rectal cancer (colon and rectal cancer), endometrial carcinoma, esophageal cancer, Ewing's sarcoma, follicular lymphoma, gastric cancer, gastroesophageal junction carcinoma, gastrointestinal cancer, glioblastoma (eg, glioblastoma multiforme, such as newly diagnosed or recurrent), glioma, head and neck cancer (eg, squamous cell carcinoma of the head and neck), liver metastases, Hodgkin lymphoma and non-Hodgkin lymphoma, kidney cancer (eg, renal cell carcinoma and Wilms tumors), laryngeal cancer, leukemia (eg, chronic myelocytic leukemia, hairy cell leukemia), liver cancer (eg, liver carcinoma and hepatoma), lung cancer (eg, 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., pancreatic ductal adenocarcinoma), prostate cancer (e.g., hormone-refractory (e.g., castration-resistant), metastatic, metastatic hormone-refractory (eg, castration-resistant androgen-independent)), renal cell carcinoma (eg, metastatic), salivary gland carcinoma, sarcoma (eg, rhabdomyosarcoma), skin cancer (eg, melanoma (eg, metastatic melanoma)), sarcoma soft tissue, solid tumor, squamous cell carcinoma, synovial sarcoma, testicular cancer, thyroid cancer, transitional cell carcinoma (urothelial cell cancer), uveal melanoma (eg, metastatic), verrucous carcinoma, vulvar cancer, and Waldenström's macroglobulinemia.

In some embodiments, the cancer treated in accordance with the methods described herein is a human sarcoma or carcinoma, for example, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma (eg, metastatic), hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma or retinoblastoma.

In some embodiments, the cancer treated in accordance with the methods described herein is an angiosarcoma.

In some embodiments, the cancer treated in accordance with the methods described herein is acute lymphocytic leukemia or acute myelocytic leukemia (eg, 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; lymphoma of the small intestine; or marginal zone lymphoma of the spleen.

In some embodiments, the cancer treated in accordance with the methods described herein is multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, gastrointestinal stromal tumors, head and/or neck cancer (e.g., hypopharyngeal squamous cell carcinoma, laryngeal squamous cell carcinoma , cell carcinoma

- 47 044966 mu 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 metastases, uveal melanoma, uveal melanoma with metastases to the liver, non-small cell lung cancer, colorectal cancer, or myelodysplastic syndrome. In some embodiments, the cancer being treated according to the methods is metastatic.

In some embodiments, the cancer treated in accordance with the methods described herein is prostate cancer, breast cancer, lung cancer, colorectal cancer, melanoma, bronchial cancer, bladder cancer, brain cancer, or central nervous system, peripheral nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, non-Hodgkin's lymphoma, thyroid cancer, kidney cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer , squamous cell carcinoma, mesothelioma, osteocarcinoma, thymoma/thymus carcinoma, glioblastoma, myelodysplastic syndrome, soft tissue sarcoma, diffuse brain stem cell tumor DIPG, adenocarcinoma, osteosarcoma, chondrosarcoma, leukemia or pancreatic cancer. In some embodiments, the cancer treated in accordance with the methods described herein includes carcinoma (eg, adenocarcinoma), lymphoma, blastoma, melanoma, sarcoma, or leukemia.

In some embodiments, the cancer treated in accordance with 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 (such as liver carcinoma and hepatoma), bladder cancer, breast cancer, inflammatory breast cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, stomach cancer, bladder cancer, endometrial carcinoma, myeloma (eg multiple myeloma), salivary gland carcinoma, kidney cancer (eg renal cell carcinoma and Wilms tumors), 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 some embodiments, cancers treated in accordance with the methods described herein include desmoplastic melanoma, inflammatory breast cancer, thymoma, rectal cancer, anal cancer; or surgically treatable or nonsurgically treatable brainstem glioma. In a specific embodiment, said cancer is a solid tumor. In another specific embodiment, said cancer is glioblastoma multiforme. In some embodiments, said glioblastoma multiforme is recurrent. In some embodiments, said glioblastoma multiforme is first diagnosed. In some embodiments, said glioblastoma multiforme is observed in a subject with unmethylated MGMT promoters. In some embodiments, the glioblastoma multiforme is refractory to bevacizumab therapy. In some embodiments, said glioblastoma multiforme is observed in a subject who has not received bevacizumab therapy.

In some embodiments, the cancer treated in accordance with the methods described herein is metastatic melanoma (eg, resistant metastatic melanoma), metastatic ovarian cancer, or metastatic renal cell cancer. In some embodiments, the cancer being treated in accordance with the methods described herein is ipilimumab-resistant melanoma. In some embodiments, the cancer being treated in accordance with the methods described herein is melanoma that is resistant to nivolumab or pembrolizumab. In some embodiments, the cancer being treated in accordance with the methods described herein is melanoma that is resistant to ipilimumab and nivolumab or pembrolizumab.

In some embodiments, the cancer treated in accordance with the methods described herein is breast cancer (e.g., Herceptin-resistant breast cancer and trastuzumab-DM1-resistant (T-DM1-resistant) breast cancer), prostate cancer glands, glioblastoma multiforme, colorectal cancer, sarcoma, bladder cancer, cervical cancer, HPV-associated cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer, rectal cancer, oropharyngeal cancer, multiple myeloma, renal cell cancer, ovarian cancer, hepatocellular cancer, endometrial cancer, pancreatic cancer, lymphoma and leukemia (eg, advanced leukemia, acute myeloid leukemia (AML) and advanced AML).

In some embodiments, the cancer being treated in accordance with the methods described herein is metastatic malignant melanoma (on

- 48 044966 example, cutaneous or intraocular malignant melanoma), kidney cancer (for example, clear cell carcinoma), prostate cancer (for example, hormonally refractory prostate adenocarcinoma), breast cancer, colon cancer, lung cancer (for example, non-small cell lung cancer), bone cancer, pancreatic cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, carcinoma cervix, vaginal carcinoma, vulvar carcinoma, esophageal cancer, small intestinal cancer, endocrine cancer, 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, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvic carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, angiogenesis tumors, spinal tumor, brainstem glioma, glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, T-cell lymphoma, cancer induced by environmental factors, including asbestos-induced, esophageal cancer, liver cancer, refractory or recurrent malignancies, metastatic cancers, PD-L1 expressing cancers, and combinations of these cancers.

In some embodiments, the subject has previously received immunotherapy. In some embodiments, the subject has not previously received any immunotherapy. In some embodiments, the cancer is an advanced or metastatic cancer.

In some embodiments, the present invention provides a method of preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of an anti-CTLA-4 antibody or a pharmaceutical composition containing the same as described herein. According to one embodiment of the present invention, methods are provided for preventing and/or treating an infection (eg, a viral infection, a bacterial infection, a fungal infection, a protozoal infection, or a parasitic infection). The infection prevented and/or treated in accordance with these methods may be caused by an infectious agent identified herein. In a specific embodiment, the anti-CTLA-4 antibody described herein, or a composition containing it, is the only active agent that is administered to the subject. In some embodiments, an anti-CTLA-4 antibody described herein, or a composition comprising it, is used in combination with an anti-infective intervention (eg, antiviral, antibacterial, antifungal, or anthelmintic agents) to treat infectious diseases.

Infectious diseases for which anti-CTLA-4 antibodies or pharmaceutical compositions described herein may be used to treat and/or prevent are caused by infectious agents, including, but not limited to, bacteria, parasites, fungi, protozoa, and viruses. In a specific embodiment, the infectious disease to be 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 in accordance with 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 zoster virus, adenovirus, herpes simplex virus type I (HSV-I), herpes simplex virus type II (HSV-II), rinderpest virus, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus , papovavirus, cytomegalovirus, echinovirus, arbovirus, hantavirus, Coxsackie virus, mumps virus, measles virus, rubella virus, poliovirus, variola virus, Epstein-Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus II type (HIV-II) and agents of viral diseases such as viral meningitis, encephalitis, dengue fever or smallpox.

Bacterial infections that can be prevented and/or treated include those caused by Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris, Staphylococcus viridans and Pseudomonas aeruginosa. Bacterial diseases caused by bacteria (eg, 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 not limited to those caused by Mycobacteria rickettsia, Mycoplasma, Neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease), Bacillus anthracis (anthrax), tetanus; caused by streptococcus, staphylococcus, mycobacteria; whooping cough, cholera, plague, diphtheria, chlamydia; caused by S. aureus and Legionella.

Protozoal diseases or protozoal infections caused by protozoa that can be prevented and/or treated in accordance with the methods described herein include, but are not limited to, leishmaniasis, coccidiosis, trypanosomiasis, schistosomiasis, or malaria. Parasitic diseases or parasitic infections caused by parasites that can be prevented and/or treated in accordance with the methods described herein include, but are not limited to, those caused by chlamydia and rickettsia.

Fungal diseases or fungal infections that can be prevented and/or treated in accordance with the methods described herein include, but are not limited to, Candida infections, zygomycosis, Candida mastitis, progressive disseminated trichosporonosis with latent trichosporonemia, disseminated candidiasis , pulmonary paracoccidioidomycosis, pulmonary aspergillosis, pneumonia caused by Pneumocystis carinii, cryptococcal meningitis, coccidioidal meningoencephalitis and cerebrospinal vasculitis, Aspergillus niger infection, Fusarium keratitis, mycoses of the paranasal sinuses, endocarditis caused by Aspergillus fumigatus, dyschemia ondroplasia of the tibia, vaginitis caused by Candida glabrata, oropharyngeal candidiasis, chronic granulomatous disease with X-linked inheritance, tinea pedis, cutaneous candidiasis, mycotic placentitis, disseminated trichosporonosis, allergic bronchopulmonary aspergillosis, mycotic keratitis, Cryptococcus neoformans infection, mycotic peritonitis, Curvularia geniculata infection, staphylococcal endophthalmitis, spore orichosis and dermatophytosis.

In some embodiments, said infectious disease is acute. In some embodiments, said infectious disease is chronic. In some embodiments, the infectious disease is caused by a flavivirus, such as West Nile virus, St. Louis encephalitis virus, Powassan virus, tick-borne encephalitis virus, dengue virus, Zika virus, Kyasanur Forest disease virus, yellow fever virus, and chikungunya virus. In some embodiments, the infectious disease is caused by the Ebola virus. In some embodiments, the infectious disease is caused by an influenza virus. In some embodiments, the infectious disease is caused by human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV). In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition comprising it as described herein promotes viral control. In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition comprising it as described herein eliminates viral reservoirs.

The present invention relates, in one aspect, to an anti-CTLA-4 antibody according to the present invention and/or a pharmaceutical composition according to the present invention comprising an anti-CTLA-4 antibody according to the present invention and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient, for use as a medicament .

The present invention relates, in one aspect, to the anti-CTLA-4 antibody of the present invention and/or its use in combination with pharmaceutically acceptable carriers or excipients for the preparation of pharmaceutical compositions or medicaments for immunotherapy (for example, immunotherapy to enhance T cell activation in response to an antigen in a subject, treatment of cancer, or treatment or prevention of infectious diseases).

The present invention relates, in one aspect, to an anti-CTLA-4 antibody according to the present invention and/or a pharmaceutical composition according to the present invention comprising an anti-CTLA-4 antibody according to the present invention and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient, for use in a method of treatment cancer.

The present invention relates, in one aspect, to an anti-CTLA-4 antibody according to the present invention and/or a pharmaceutical composition according to the present invention containing an anti-CTLA-4 antibody according to the present invention and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient for use in a method of inhibition tolerance of the immune system to tumors and/or for immunotherapy in subjects suffering from cancer.

The present invention relates, in one aspect, to an anti-CTLA-4 antibody according to the present invention and/or a pharmaceutical composition according to the present invention comprising an anti-CTLA-4 antibody according to the present invention and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient, for use in a method of treatment infectious disease.

In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered as monotherapy.

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

In some embodiments, the present invention provides (a) 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 pharmaceutically acceptable excipient; and (b) an additional therapeutic agent for use as a medicament. In a preferred embodiment, said additional therapeutic agent is a chemotherapeutic agent or a checkpoint-targeting agent.

In some embodiments, the present invention provides (a) 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 pharmaceutically acceptable excipient; and (b) an additional therapeutic agent for use in a method of treating cancer.

In some embodiments, the present invention provides (a) 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 pharmaceutically acceptable excipient; and (b) an additional therapeutic agent for use in a method of treating an infectious disease.

In some embodiments, an anti-CTLA-4 antibody described herein is administered to a subject in combination with a compound that targets an immunomodulatory enzyme or enzymes, such as IDO (indoleamine (2,3) dioxygenase) and/or TDO (tryptophan 2, 3-dioxygenase). In some embodiments, such a compound is selected from the group consisting of epacadostat (Incyte Corp; see, for example, WO 2010/005958, which is incorporated herein by reference in its entirety), F001287 (Flexus Biosciences), indoximod (NewLink Genetics), and NLG919 (NewLink Genetics). In one embodiment, the compound is epacadostat. According to another embodiment, said compound is F001287. In another embodiment, the compound is indoximod. In another embodiment, the compound is NLG919.

In some embodiments, the present invention provides (a) 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 pharmaceutically acceptable 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.

In some embodiments, the present invention provides (a) 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 pharmaceutically acceptable excipient; and (b) a compound that targets an immunomodulatory enzyme for use in a method of treating cancer. In a preferred embodiment, the compound targets IDO and/or TDO.

In some embodiments, an anti-CTLA-4 antibody described herein is administered to a subject in combination with a vaccine. In some embodiments, the vaccine is a heat shock protein tumor vaccine or a heat shock protein pathogen vaccine. In a specific embodiment, an anti-CTLA-4 antibody described herein is administered to a subject in combination with a heat shock protein tumor vaccine. Heat shock proteins (HSPs) are a family of highly conserved proteins widely distributed across species. Significantly higher levels of their expression can be dramatically induced by heat shock or

- 51 044966 other forms of stress, including exposure to toxins, oxidative stress or glucose deprivation. Five families have been classified according to molecular weight: HSP-110, -90, -70, -60 and -28. HSP delivers immunogenic peptides through a cross-presentation pathway to antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs), leading to T cell activation. HSPs function as chaperones that carry tumor-associated antigenic peptides, forming complexes capable of inducing tumor-specific immunity. When released from dying tumor cells, HSP-antigen complexes are engulfed by antigen-presenting cells (APCs), and these antigens are converted into peptides that bind MHC class I and class II molecules, resulting in the activation of antitumor CD8+ and CD4+ T cells. Immunity induced by tumor-derived HSP complexes is directed specifically at the unique repertoire of antigenic peptides expressed by each subject's cancer.

Heat shock protein-peptide complex (HSPPC) is a protein-peptide complex consisting of heat shock protein non-covalently complexed with antigenic peptides. HSPPCs trigger both innate and adaptive immune responses. In a specific embodiment, said antigenic peptide or said antigenic peptides exhibit antigenicity to the cancer being treated. APCs efficiently capture HSPPCs through membrane receptors (mainly CD91) or through binding to Toll-like receptors.

Internalization of HSPPC leads to functional maturation of APCs with the production of chemokines and cytokines, leading to the activation of natural killer (NK) cells, monocytes, and Th1 and Th-2 mediated immune responses. In some embodiments, the HSPPCs used in the methods described herein comprise one or more heat shock proteins from the hsp60, hsp70, or hsp90 family of stress proteins in complex with antigenic peptides. In some embodiments, the HSPPCs comprise hsc70, hsp70, hsp90, hsp110, grp170, gp96, calreticulin, or combinations of two or more of these proteins.

In a specific embodiment, an anti-CTLA-4 antibody described herein is administered to a subject in combination with a heat shock protein-peptide complex (HSPPC), such as heat shock protein-peptide complex-96 (HSPPC-96), for the treatment of cancer. HSPPC-96 contains the 96-kDa heat shock protein (Hsp), gp96, complexed with antigenic peptides. HSPPC-96 is a cancer immunotherapy agent derived from a tumor of a subject and contains the antigenic fingerprint of the cancer. In some embodiments, the fingerprint contains unique antigens that are present only on specific cancer cells of the specific subject, and the vaccine injection is designed to stimulate the subject's immune system to recognize and attack any cells bearing the specific cancer fingerprint.

In some embodiments, said HSPPC, for example, HSPPC-96, is obtained from tumor tissue of a subject. In a specific embodiment, said HSPPC (eg, HSPPC-96) is derived from a tumor of the type of cancer or metastasis thereof being treated. In another specific embodiment, said HSPPC (eg, HSPPC-96) is autologous to the subject being treated. In some embodiments, said tumor tissue is non-necrotic tumor tissue. In some 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, etc.) is used to prepare a vaccine for a treatment regimen. at least 7, at least 8, at least 9, or at least 10 grams) of non-necrotic tumor tissue. In some embodiments, after surgical resection, non-necrotic tumor tissue is frozen until used in vaccine preparation. In some embodiments, the HSPPC, eg, HSPPC-96, is isolated from tumor tissue using purification techniques, filtered, and prepared for an injectable vaccine. In some embodiments, the subject is administered 6-12 doses of said HSPPC, for example, HSPCC-96. In such embodiments, doses of said HSPPC, eg, HSPPC-96, may be administered weekly for the first 4 doses and then biweekly for 2-8 additional doses.

Additional examples of HSPPC that may be used in accordance with the methods described herein are described in the following patents and patent applications, which are incorporated herein by reference in their entirety: US Pat. No. 6,391,306, US Pat. No. 6,383,492, US Pat. No. 6,403,095, US Pat. No. 6,410,026, US Pat. No. 6,436,404 , No. 644778θ, No. 6447781 and No. 6610659, each of which is incorporated herein by reference in its entirety.

In some embodiments, the present invention provides (a) 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 pharmaceutically acceptable excipient; and (b) a vaccine,

- 52 044966 for use as a medicine. In a preferred embodiment, the vaccine is a heat shock protein anti-tumor vaccine or a heat shock protein anti-pathogen vaccine. In a preferred embodiment, the vaccine is a heat shock protein based antiviral vaccine.

In some embodiments, the present invention provides (a) 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 pharmaceutically acceptable excipient; and (b) a vaccine, for use in a method of treating cancer. In a preferred embodiment, said vaccine is a heat shock protein based antitumor vaccine.

The anti-CTLA-4 antibody and the additional therapeutic agent (eg, a chemotherapeutic agent, a checkpoint-targeted agent, an IDO inhibitor, and/or a vaccine) may 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.

In some embodiments, an anti-CTLA-4 antibody described herein is administered to a subject intratumorally. In some embodiments, an anti-CTLA-4 antibody described herein is administered intratumorally to a subject in combination with an additional therapeutic agent. In some embodiments, said additional therapeutic agent is administered systemically. In some embodiments, the subject has solid tumors. In some embodiments, the subject has head and neck squamous cell carcinoma (HNSCC). In some embodiments, the subject has HER2+ breast cancer. In some embodiments, the additional therapeutic agent that is administered systemically is an anti-PD-1 antibody (eg, pembrolizumab or nivolumab). In some embodiments, the additional therapeutic agent that is administered systemically is an anti-eGF antibody (eg, cetuximab). In some embodiments, the additional therapeutic agent that is administered systemically is an anti-HER2 antibody (eg, trastuzumab). In some embodiments, said additional therapeutic agent that is administered systemically is a chemotherapeutic agent (eg, gemcitabine). In some embodiments, the subject has solid tumors and the additional therapeutic agent that is administered systemically is an anti-PD-1 antibody (eg, pembrolizumab or nivolumab). In some embodiments, the anti-PD-1 antibody is pembrolizumab, which is administered in an amount of 200 mg every three weeks. In some embodiments, the subject has head and neck squamous cell carcinoma (HNSCC) and the additional therapeutic agent that is administered systemically is an anti-eGF antibody (eg, cetuximab). In some embodiments, the subject has HER2+ breast cancer and the additional therapeutic agent that is administered systemically is an anti-HER2 antibody (eg, trastuzumab). In some embodiments, the subject additionally received a chemotherapeutic agent (eg, gemcitabine). In one aspect, the present invention provides an anti-CTLA-4 antibody and/or a pharmaceutical composition according to the present invention and, optionally, an additional therapeutic agent, for use in a method of treating cancer, wherein said anti-CTLA-4 antibody and/or pharmaceutical composition according to the present invention administered intratumorally to a specified subject. In one preferred embodiment, the additional therapeutic agent is administered to the subject, more preferably, the additional therapeutic agent is administered to the subject systemically.

In some embodiments, an anti-PD-1 antibody is used in the methods described herein. In some embodiments, the anti-PD1 antibody is nivolumab, also known as BMS-936558 or MDX1106, developed by Bristol-Myers Squibb. In some embodiments, the anti-PD-1 antibody is pembrolizumab, also known as lambrolizumab or MK-3475, developed by Merck & Co. In some embodiments, the anti-PD-1 antibody is pidilizumab, also known as CT-011, developed by CureTech. In some embodiments, the anti-PD-1 antibody is MEDI0680, also known as AMP-514, developed by Medimmune. In some embodiments, the anti-PD-1 antibody is PDR001 developed by Novartis Pharmaceuticals. In some embodiments, the anti-PD-1 antibody is REGN2810, developed by Regeneron Pharmaceuticals. In some embodiments, the anti-PD-1 antibody is PF-06801591 developed by Pfizer. In some embodiments, the anti-PD-1 antibody is BGB-A317, developed by BeiGene. In some embodiments, the anti-PD-1 antibody is TSR-042, developed by AnaptysBio and Tesaro. In some embodiments, the anti-PD-1 antibody is SHR-1210 developed by Hengrui.

Additional non-limiting examples of anti-PD-1 antibodies that may be used in the treatments described herein are described in the following patents and patent applications, which are incorporated herein by reference in their entirety for all purposes: Patent

US No. 6808710; US Patent No. 7332582; US Patent No. 7488802; US Patent No. 8008449; patent

US No. 8114845; US Patent No. 8168757; US Patent No. 8354509; US Patent No. 8686119; patent

US No. 8735553; US Patent No. 8747847; US Patent No. 8779105; US Patent No. 8927697; patent

US No. 8993731; US Patent No. 9102727; US Patent No. 9205148; US publication US 2013/0202623 A1; US publication US 2013/0291136 A1; US publication US 2014/0044738 A1; US publication US 2014/0356363 A1; US publication US 2016/0075783 A1; and PCT publication WO 2013/033091 A1; PCT publication WO 2015/036394 A1; PCT publication WO 2014/179664 A2; PCT publication WO 2014/209804 A1; PCT publication WO 2014/206107 A1; PCT publication WO 2015/058573 A1; PCT publication WO 2015/085847 A1; PCT publication WO 2015/200119 A1; PCT publication WO 2016/015685 A1; and PCT publication WO 2016/020856 A1.

In some embodiments, an anti-PD-L1 antibody is used in the methods described herein. In some embodiments, the anti-PDL1 antibody is atezolizumab developed by Genentech. In some embodiments, the anti-PD-L1 antibody is durvalumab, developed by AstraZeneca, Celgene and Medimmune. In some embodiments, the anti-PD-L1 antibody is avelumab, also known as MSB0010718C, developed by Merck Serono and Pfizer. In some embodiments, the anti-PD-L1 antibody is MDX1105 developed by Bristol-Myers Squibb. In some embodiments, the anti-PD-L1 antibody is AMP-224, developed by Amplimmune and GSK.

Non-limiting examples of anti-PD-L1 antibodies that may be used in the treatments described herein are described in the following patents and patent applications, which are incorporated herein by reference in their entirety for all purposes: US Patent No. 7,943,743; US Patent No. 8168179; US Patent No. 8217149; US Patent No. 8552154; US Patent No. 8779108; US Patent No. 8981063; US Patent No. 9175082; US publication US 2010/0203056 A1; US publication US 2003/0232323 A1; US publication US 2013/0323249 A1; US publication US 2014/0341917 A1; US publication US 2014/0044738 A1; US publication US 2015/0203580 A1; US publication US 2015/0225483 A1; US publication US 2015/0346208 A1; US publication US 2015/0355184 A1; and PCT publication WO 2014/100079 A1; PCT publication WO 2014/022758 A1; PCT publication WO 2014/055897 A2; PCT publication WO 2015/061668 A1; PCT publication WO 2015/109124 A1; PCT publication WO 2015/195163 A1; PCT publication WO 2016/000619 A1; and PCT publication WO 2016/030350 A1.

In some embodiments, an anti-LAG-3 antibody is used in the methods described herein. In some embodiments, the anti-LAG-3 antibody is BMS-986016, developed by Bristol-Myers Squibb. In some embodiments, the anti-LAG-3 antibody is LAG525 developed by Novartis. In some embodiments, the anti-LAG-3 antibody is GSK2831781, developed by GSK.

Non-limiting examples of anti-LAG-3 antibodies that may be used in the treatments described herein are described in the following patents and patent applications, which are incorporated herein by reference in their entirety for all purposes: US Patent No. 9244059; US publication US 2011/0150892 A1; US publication US 2014/0093511 A1; US publication US 2014/0286935 A1; US publication US 2015/0259420 A1; and PCT publication WO 2015/042246 A1; PCT publication WO 2015/116539 A1; PCT publication WO 2015/200119 A1; and PCT publication WO 2016/028672 A1.

In some embodiments, an anti-eGF antibody is used in the methods described herein. In some embodiments, the anti-eGF antibody is cetuximab developed by Bristol-Myers Squibb and ImClone, panitumumab developed by Abgenix and Amgen, nimotuzumab developed by CMI Cuba and YM 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 595 developed by Amgen and ImmunoGen, CetuGEX developed by Glycotope, laprituximab emtansine developed by ImmunoGen, JNJ-61186372 developed by Genmab and Janssen Biotech, SCT200 developed by Sinocelltech, LY3164530 developed by Lilly, HLX07 developed by Shanghai Henlius; or SYN004, developed by Synermore.

- 54 044966

In some embodiments, an anti-HER2 antibody is used in the methods described herein. In some embodiments, the anti-HER2 antibody is trastuzumab developed by Genentech and Roche, trastuzumab emtansine 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 & Co, CMAB302, developed by Shanghai CP Guojian, TrasGEX, developed by Glycotope, ARX788, developed by Ambrx and Zhejiang Medicine, SYD985, developed by Synthon, FS102 developed by Bristol-Myers Squibb and F-star, BCD-022 developed by Biocad, ABP 980 developed by Amgen, DS-8201a developed by Daiichi Sankyo, HLX02 developed by Shanghai Henlius; or CANMAb, developed by Biocon and Mylan.

The antibody or pharmaceutical composition described herein can be delivered to a subject through various routes. These routes include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, percutaneous, intravenous, intratumoral, conjunctival, and subcutaneous. Pulmonary administration, for example using an inhaler or nebulizer, and formulation with an aerosol agent for use as a spray may also be used. In some embodiments, said antibody or said pharmaceutical composition described herein is delivered subcutaneously or intravenously. In some embodiments, said antibody or said pharmaceutical composition described herein is delivered intratumorally. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is delivered to a tumor-draining lymph node. In some embodiments, said antibody or said pharmaceutical composition as described herein is delivered via localized administration (eg, subcutaneous administration). In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is delivered systemically. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is delivered locally.

In one aspect, the present invention provides an anti-CTLA-4 antibody and/or a pharmaceutical composition according to the present invention, and optionally an additional therapeutic agent, for use in a method of treating cancer, characterized in that said anti-CTLA-4 antibody and/or pharmaceutical the composition of the present invention is delivered intratumorally to the subject, delivered to a tumor-draining lymph node of the subject, or delivered via localized administration (eg, subcutaneous administration) to the subject.

The amount of antibody or composition that will be effective in treating and/or preventing the condition depends on the nature of the disease, and can be determined using standard clinical techniques.

The exact dosage to be used in the composition also depends on the route of administration and the severity of the infection or disease caused by it, and should be determined in accordance with the judgment of the practitioner and the circumstances surrounding each subject. For example, effective doses may also vary depending on the route of administration, the target site, the physiological condition of the patient (including age, body weight and health status), whether the patient is human or animal, other drugs administered, or whether whether the treatment is preventive or therapeutic. Typically the patient is a human, but non-human mammals, including transgenic mammals, may also be treated. Treatment dosages are optimally titrated to optimize safety and effectiveness.

In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (e.g., by intravenous injection) at a dose of 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, approximately 0.1 mg/kg, approximately 0.3 mg/kg, approximately 1 mg/kg, approximately 3 mg/kg, approximately 6 mg/kg, or approximately 10 mg/kg. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (e.g., by intravenous injection) every week, every two weeks, every three weeks, every four weeks, every six weeks, every eight weeks, every 12 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 some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (eg, by intravenous injection) every three weeks at the dosages described above.

In one aspect, the present invention provides an anti-CTLA-4 antibody and/or a pharmaceutical composition according to the present invention, and optionally an additional therapeutic agent, for use in a method of treating cancer, characterized in that said anti-CTLA-4 antibody and/or a pharmaceutical composition according to the present invention is administered to a subject at a dose of 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, approximately 0.1 mg/kg, approximately 0.3 mg/kg, approximately 1 mg/kg, approximately 3 mg/kg, approximately 6 mg/kg, or approximately 10 mg/kg , more preferably every two weeks, every three weeks, every four weeks, every six weeks or every 12 weeks.

In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (e.g., by intravenous injection) at a dose of 0.1 mg/kg every two weeks, every three weeks, every four weeks, every six weeks, or every 12 weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (e.g., by intravenous injection) at a dose of 0.3 mg/kg every two weeks, every three weeks, every four weeks, every six weeks, or every 12 weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (e.g., by intravenous injection) at a dose of 1 mg/kg every two weeks, every three weeks, every four weeks, every six weeks, or every 12 weeks In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (e.g., by intravenous injection) at a dose of 3 mg/kg every two weeks, every three weeks, every four weeks, every six weeks, or every 12 weeks In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (e.g., by intravenous injection) at a dose of 6 mg/kg every two weeks, every three weeks, every four weeks, every six weeks, or every 12 weeks In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (e.g., by intravenous injection) at a dose of 10 mg/kg every two weeks, every three weeks, every four weeks, every six weeks, or every 12 weeks

In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (eg, by intravenous injection) at a dose of 0.1 mg/kg, or approximately 0.1 mg/kg, every three weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (eg, by intravenous injection) at a dose of 0.3 mg/kg, or approximately 0.3 mg/kg, every three weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (eg, by intravenous injection) at a dose of 1 mg/kg, or approximately 1 mg/kg, every three weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (eg, by intravenous injection) at a dose of 3 mg/kg, or approximately 3 mg/kg, every three weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (eg, by intravenous injection) at a dose of 6 mg/kg, or approximately 6 mg/kg, every three weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject (eg, by intravenous injection) at a dose of 10 mg/kg, or approximately 10 mg/kg, every three weeks.

In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject by intratumoral injection at a dose of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/ kg, 1 mg/kg, 3 mg/kg, approximately 0.01 mg/kg, approximately 0.03 mg/kg, approximately 0.1 mg/kg, approximately 0.3 mg/kg, approximately 1 mg/kg or approximately 3 mg/kg. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject by intratumoral injection every week, every two weeks, every three weeks, every four weeks, every six weeks, every eight weeks, every 12 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 some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject by intratumoral injection every three weeks at the dosages described above.

In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject by intratumoral injection at a dose of 0.01 mg/kg, or approximately 0.01 mg/kg, every three weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject by intratumoral injection at a dose of 0.03 mg/kg, or approximately 0.03 mg/kg, every three weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject by intravenous

- 56 044966 tumor injection at a dose of 0.1 mg/kg or approximately 0.1 mg/kg every three weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject by intratumoral injection at a dose of 0.3 mg/kg, or approximately 0.3 mg/kg, every three weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject by intratumoral injection at a dose of 1 mg/kg, or approximately 1 mg/kg, every three weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject by intratumoral injection at a dose of 3 mg/kg, or approximately 3 mg/kg, every three weeks.

In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject via localized administration (eg, subcutaneous administration) at a dose of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg , 0.3 mg/kg, 1 mg/kg, 3 mg/kg, approximately 0.01 mg/kg, approximately 0.03 mg/kg, approximately 0.1 mg/kg, approximately 0.3 mg/kg, approximately 1 mg/kg or approximately 3 mg/kg. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition as described herein is administered to a subject via localized administration (eg, subcutaneous administration) every week, every two weeks, every three weeks, every four weeks, every six weeks, every eight weeks , every 12 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 some embodiments, the therapeutic combination is administered to a subject for at least 3, 6, 9, 12, 18, or 24 months. In some embodiments, the therapeutic combination is administered to a subject over a period of up to 3, 6, 9, 12, 18, or 24 months.

In some embodiments, the present invention provides a method of treating a subject suffering from angiosarcoma, comprising administering to the subject (eg, intravenously, intratumorally, or subcutaneously) an effective amount of an anti-CTLA-4 antibody or a pharmaceutical composition as described herein. According to some embodiments of the present invention, there is provided a method of treating a subject suffering from angiosarcoma, the method comprising intravenously administering to the subject an antibody that specifically binds to human CTLA-4 at a dose of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, approximately 0.01 mg/kg, approximately 0.03 mg/kg, approximately 0.1 mg/kg, approximately 0 .3 mg/kg, approximately 1 mg/kg, or approximately 3 mg/kg, optionally weekly, every two or every three weeks. In some embodiments, the present invention provides a method of treating a subject suffering from angiosarcoma, the method comprising intravenously administering to the subject an antibody that specifically binds to human CTLA-4 at a dose of 0.1 mg/kg once every three weeks. In some 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 some embodiments, the antibody comprises a heavy chain comprising the sequence amino acids from SEQ ID NO: 23; and a light chain comprising the amino acid sequence of SEQ ID NO: 27. In some 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 some 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 some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 26; and a light chain containing the amino acid sequence of SEQ ID NO: 27.

The anti-CTLA-4 antibody described herein can also be used to analyze CTLA-4 protein levels in a biological sample using classical immunohistological techniques known to those skilled in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA) ), immunoprecipitation or Western blotting. Suitable labels for antibody assays are known in the art and include enzyme labels such as glucose oxidase; radioisotopes such as iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹²¹ In) and technetium ( ⁹⁹ Tc); luminescent tags such as luminol; and fluorescent tags such as fluorescein and rhodamine, as well as biotin. Such labels can be used to label an antibody described herein or an antigen binding fragment thereof. Alternatively, a second antibody that recognizes an anti-CTLA-4 antibody described herein or an antigen binding fragment thereof can be labeled and used in combination with an anti-CTLA-4 antibody or an antigen binding fragment thereof to detect protein levels CTLA-4. According to one embodiment, the present invention relates to the use of an anti-CTLA-4 body according to the present invention for the analysis and/or detection of protein levels

CTLA-4 in a biological sample in vitro.

The CTLA-4 protein expression level analysis is intended to include a qualitative or quantitative measurement or calculation of the CTLA-4 protein level in the first biological sample, either direct (e.g., by determining or calculating the absolute protein level) or comparative (e.g., by comparison with the level of disease-associated protein in the second biological sample). The level of expression of the CTLA-4 polypeptide in a first biological sample may be measured or calculated and compared with a standard level of CTLA-4 protein determined in a second biological sample obtained from an individual not suffering from the disorder or determined based on averaging levels in a population of individuals. who do not suffer from this disorder. As will be appreciated by those skilled in the art, once a standard level of CTLA-4 polypeptide is known, it can be used repeatedly as a reference standard.

As used herein, the term biological sample refers to any biological sample obtained from a subject, cell line, tissue, or other cell source potentially expressing CTLA-4. Methods for obtaining biopsies of tissues and body fluids from animals (eg, humans) are well known in the art. Biological samples include peripheral blood mononuclear cells.

The anti-CTLA-4 antibody described herein, or an antigen-binding fragment thereof, can be used for prognosis, diagnosis, monitoring and screening, including in vitro and in vivo applications well known and standard in the art, and based on the present description . Prognostic, diagnostic, monitoring and screening assays and kits for assessing and determining in vitro status of the immune system and/or immune response may be used for prognosis, diagnosis and monitoring in the evaluation of patient specimens, including those from patients, known or suspected suffering from immune system dysfunction, or in relation to an expected or desired immune system response, response to an antigen, or response to a vaccine. Assessing and determining the status of the immune system and/or immune response is also useful in determining a patient's suitability for participation in a clinical drug trial or for administration of a particular chemotherapeutic agent or antibody or antigen-binding fragment thereof, including combinations thereof, versus another agent, or another antibody or antigen-binding fragment thereof. This type of prognostic and diagnostic monitoring and evaluation using antibodies against the HER2 protein in breast cancer is already in practice (HercepTest™, Dako), and the test is also used to identify patients for antibody therapy with Herceptin®. In vivo applications include targeted cell therapy and modulation of the immune system, as well as radioimaging of the immune response.

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

In one aspect, the present invention provides an anti-CTLA-4 antibody and/or a pharmaceutical composition according to the present invention for use in a method for predicting, diagnosing and/or monitoring immune system dysfunction and/or cancer.

In one embodiment, the present invention relates to the use of an anti-CTLA-4 antibody of the present invention for predicting, diagnosing and/or monitoring immune system dysfunction and/or cancer in a subject by analyzing and/or detecting levels of CTLA-4 protein in a biological sample from the specified subject in vitro.

In one embodiment, an anti-CTLA-4 antibody or antigen-binding fragment thereof can be used in the immunohistochemical examination of biopsy specimens. In another embodiment, an anti-CTLA-4 antibody or antigen-binding fragment thereof can be used to detect levels of CTLA-4 or levels of cells that contain CTLA-4 on the surface of membranes; wherein said levels can then be associated with specific disease symptoms. The anti-CTLA-4 antibodies described herein, or antigen binding fragments thereof, may carry a detectable or functional label. When using fluorescent tags, currently available microscopic and fluorescence-activated cell sorting (FACS) assays, or a combination of both techniques known in the art, can be used to identify and quantify specific binding participants. The anti-CTLA-4 antibodies described herein, or antigen binding fragments thereof, may carry a fluorescent label. Examples of fluorescent labels include, for example, reactive and conjugated probes such as aminocoumarin, fluorescein and Texas Red, Alexa Fluor dyes, cyanine dyes and DyLight dyes. The anti-CTLA-4 antibody or antigen-binding fragment thereof may carry a radiolabel such as the isotopes ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁶⁷ Cu , ⁹⁰ Y, ⁹⁹ Tc, 1 ⁿ In, ¹¹⁷ Lu, ¹²¹ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ¹⁹⁸ Au, ²¹¹ At, ²¹³ Bi, ²²⁵ Ac and ¹⁸⁶ Re. When radioactive labels are used, currently available counting procedures known in the art can be used to identify and quantify the specific binding of an anti-CTLA-4 antibody or antigen binding fragment thereof to CTLA-4 (eg, human CTLA-4). In the case where the specified label is an enzyme, detection can be carried out using any of the currently used colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art. This can be achieved by contacting the sample or control with an anti-CTLA-4 antibody or antigen-binding fragment thereof under conditions that allow formation of a complex between the antibody or antigen-binding fragment thereof and CTLA-4. Any complexes formed between the antibody or antigen-binding fragment thereof and CTLA-4 are detected and compared in the sample and control. Given the specific binding of the antibodies described herein to CTLA-4, these antibodies or antigen binding fragments thereof can be used to detect, in particular, the expression of CTLA-4 on the surface of cells. The antibodies described herein, or antigen binding fragments thereof, can also be used to purify CTLA-4 by immunoaffinity. The present invention also provides an analytical system that can be implemented in the form of a test kit for quantitative analysis of the presence of, for example, CTLA-4 complexes or CTLA-4/CTLA-4 ligand. The test system or test kit may contain a labeled component, such as a labeled antibody, and one or more additional immunochemical reagents.

In one embodiment, the present invention provides an in vitro method for analyzing and/or detecting levels of CTLA-4 protein in a biological sample, comprising (1) contacting the sample and optionally a control sample with an anti-CTLA-4 antibody of the present invention, or its antigen-binding fragment under conditions allowing for the formation of a complex between the antibody or its antigen-binding fragment and CTLA-4; and (2) detection and comparison of complexes formed in the sample and, optionally, in the control.

Polynucleotides, vectors and methods for producing antibodies against CTLA-4.

According to another aspect of the present invention, there are provided polynucleotides comprising a nucleotide sequence encoding an antibody or fragment thereof described herein (e.g., a light chain variable region and/or a heavy chain variable region) that specifically binds to a CTLA-4 antigen (e.g. , human CTLA-4), as well as vectors, for example, vectors containing such polynucleotides for recombinant expression in host cells (eg, E. coli and mammalian cells). The present invention provides polynucleotides containing nucleotide sequences encoding any of the antibodies provided by the present invention, as well as vectors containing such polynucleotide sequences, for example, expression vectors for their efficient expression in host cells, for example, mammalian cells.

As used herein, an isolated polynucleotide or isolated nucleic acid molecule refers to a polynucleotide or nucleic acid molecule separated from other nucleic acid molecules that are present in the natural source (eg, mouse or human) of said nucleic acid molecule. In addition, an isolated nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when produced using recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. For example, the expression substantially free includes compositions with a polynucleotide or nucleic acid molecule containing less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular, less than approximately 10%) other material, such as cellular material, culture medium, other nucleic acid molecules, chemical precursors and/or other chemicals. In a specific embodiment, the nucleic acid molecule or molecules encoding the antibody described herein are isolated or purified.

According to specific aspects of the present invention, there are provided polynucleotides comprising nucleotide sequences encoding antibodies or antigen binding fragments thereof that specifically bind to a CTLA-4 polypeptide (e.g., human CTLA-4) and containing an amino acid sequence as described herein, as well as antibodies that compete with such antibodies for binding to the CTLA-4 polypeptide (eg, in a dose-dependent manner) or bind to the same epitope as such antibodies.

In accordance with certain aspects of the present invention, there are provided polynucleotides comprising a nucleotide sequence encoding the light chain or heavy chain of an antibody described herein. Said polynucleotides may comprise nucleotide sequences encoding a light chain containing FR regions and CDR regions from the VL antibodies described in

- 59 044966 this document (see, for example, Table 1).

The present invention also provides polynucleotides encoding an anti-CTLA-4 antibody, optimized, for example, by optimizing codons/RNA, replacing signal sequences with heterologous ones, and eliminating elements of mRNA instability. Methods for producing nucleic acids encoding an anti-CTLA-4 antibody or fragment thereof (e.g., light chain, heavy chain, VH domain, or VL domain) optimized for recombinant expression by introducing codon changes and/or eliminating inhibitory regions in mRNA can be accomplished by adaptations of optimization methods described, for example, in US patents No. 5965726; No. 6174666; No. 6291664; No. 6414132; and No. 6794498, respectively, each of which is incorporated herein by reference in its entirety. For example, potential splice sites and instability elements (eg, A/T-rich or A/U elements) in RNA can be mutated without changing the amino acids encoded by the nucleic acid sequences to increase the stability of the RNA for recombinant expression. With these changes, the degeneracy of the genetic code is used, for example, an alternative codon for an identical amino acid is used. In some embodiments, one or more codons may need to be changed to encode a conservative mutation, eg, encoding a similar amino acid with a similar chemical structure and properties, and/or functioning like the original amino acid. Such methods can increase the 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. fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to the expression of anti-CTLA-4 antibody encoded by polynucleotides that have not been optimized.

In some embodiments, an optimized polynucleotide sequence encoding an anti-CTLA-4 antibody described herein, or a fragment thereof (e.g., a VL domain and/or a VH domain), can hybridize to an antisense (e.g., complementary) polynucleotide of a non-optimized polynucleotide sequence encoding an anti-CTLA-4 antibody described herein, or a fragment thereof (eg, VL domain and/or VH domain). In specific embodiments, an optimized nucleotide sequence encoding an anti-CTLA-4 antibody described herein, or a fragment thereof, hybridizes under very high stringency conditions to an antisense polynucleotide of a non-optimized polynucleotide sequence encoding an anti-CTLA-4 antibody described herein, or its fragment. In a specific embodiment, an optimized nucleotide sequence encoding an anti-CTLA-4 antibody described herein, or a fragment thereof, is hybridized under high, intermediate, or lower stringency hybridization conditions with an antisense polynucleotide of a non-optimized nucleotide sequence encoding an anti-CTLA-4 antibody described in this document, or a fragment thereof. Information regarding hybridization conditions has been provided, for example, in published US patent application US 2005/0048549 (eg, paragraphs 72-73), which is incorporated herein by reference.

Said polynucleotides may be prepared and the nucleotide sequence of said polynucleotides determined by any method known in the art. Nucleotide sequences encoding the antibodies described herein, for example, the antibodies described in table. 1, and modified versions of these antibodies can be determined using methods well known in the art, i.e. the assembly of nucleotide codons that are known to encode specific amino acids occurs, such that a nucleic acid is formed that encodes the specified antibody. Such an antibody-encoding polynucleotide can be assembled from chemically synthesized oligonucleotides (for example, as described in Kutmeier G et al., (1994) BioTechniques 17: 242-6, incorporated by reference in its entirety), which, in brief, involves the synthesis overlapping oligonucleotides containing parts of the sequence encoding the specified antibody, annulation and ligation of the specified oligonucleotides, followed by amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody described herein can be obtained from nucleic acid from a suitable source (eg, a hybridoma) using methods well known in the art (eg, PCR and other molecular cloning methods).

For example, PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to produce nucleic acids containing the sequence encoding the light chain and/or heavy chain of an antibody. Such PCR amplification methods can be used to produce nucleic acids containing a sequence encoding a light chain variable region and/or a heavy chain variable region of an antibody. Amplified nucleic acids can be cloned into vectors for expression in host cells and

- 60 044966 further cloning, for example, to obtain chimeric and humanized antibodies.

If a clone containing the nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the nucleic acid encoding the specified immunoglobulin can be chemically synthesized or obtained from a suitable source (for example, cDNA libraries of antibodies or cDNA libraries obtained from any tissue or cells, or nucleic acid, preferably poly-A+ RNA, isolated from any tissue or cells expressing the specified antibody, such as hybridoma cells selected for the ability to express the antibody described herein) by PCR amplification using synthetic primers that hybridize with the 3' and 5' ends of the sequence, or by cloning using an oligonucleotide probe specific for a particular gene sequence to identify, for example, a cDNA clone from a cDNA library that encodes the specified antibody. The amplified nucleic acids obtained by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

DNA encoding the anti-CTLA-4 antibodies described herein can be readily isolated and sequenced using standard procedures (eg, using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the said anti-CTLA-4 antibody 4). Hybridoma cells may serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors which are then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells (e.g. CHO cells of the CHO GS System™ expression system (Lonza) ), or myeloma cells that do not otherwise produce immunoglobulin protein, to promote the synthesis of anti-CTLA-4 antibodies in recombinant host cells.

To generate complete antibodies, PCR primers comprising VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify VH or VL sequences in scFv clones. Using cloning techniques known to those skilled in the art, PCR-amplified VH domains can be cloned into vectors expressing a heavy chain constant region, such as the human gamma-4 constant region, and PCR-amplified VL domains can be cloned into vectors expressing a heavy chain constant region, such as the human gamma-4 constant region. expressing a light chain constant region, for example, human kappa or lambda constant regions. In some embodiments, the VH or VL domain expression vectors comprise an EF-1a promoter, a secretion signal, a cloning site for the variable region, constant domains, and a selectable marker such as neomycin. The VH and VL domains can also be cloned into a single vector expressing the necessary constant regions. The heavy chain conversion vectors and the light chain conversion vectors are then co-transfected into cell lines to produce stable or transient cell lines that express full-length antibodies, eg IgG, using techniques known to those skilled in the art.

The DNA may also be modified, for example, by replacing mouse sequences with human heavy and light chain constant domain coding sequences, or by covalently attaching all or part of a non-immunoglobulin polypeptide coding sequence to the immunoglobulin coding sequence.

Also provided are polynucleotides that hybridize under high, intermediate, or lower stringency hybridization conditions to polynucleotides that encode an antibody described herein. In specific embodiments, the polynucleotides described herein hybridize under high, intermediate, or lower stringency hybridization conditions to VH domain-encoding and/or VL domain-encoding polynucleotides of the present invention.

Hybridization conditions have been described in the art and are known to one skilled in the art. For example, hybridization under stringent conditions may involve hybridization with filter-bound DNA in sodium chloride/sodium citrate (SSC) at approximately 45°C followed by one or more washes in 0.2xSSC/0.1% SDS at approximately 50-65 °C; hybridization under very stringent conditions may involve hybridization with filter-bound nucleic acid in 6xSSC at approximately 45°C followed by one or more washes in 0.1xSSC/0.2% SDS at approximately 68°C. Hybridization under other stringent hybridization conditions is known to those skilled in the art and has been described, see, for example, Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, at pp. 6.3.1 through 6.3.6 and 2.10.3, incorporated by reference in their entirety.

In accordance with certain aspects of the present invention, there are provided cells (e.g., host cells) expressing (e.g., recombinantly) antibodies described herein (or an antigen binding fragment thereof) that specifically bind to CTLA-4 (e.g., human CTLA-4), and related polynucleotides and expression vectors. The present invention provides vectors (eg, expression vectors) containing polynucleotides containing nucleotide sequences encoding anti-CTLA-4 antibodies or a fragment thereof for recombinant expression in host cells, preferably mammalian cells. The present invention also provides host cells containing such vectors for recombinant expression of the anti-CTLA-4 antibodies described herein (eg, a human antibody or a humanized antibody). According to a specific aspect of the present invention, methods for producing an antibody described herein are provided, comprising expressing such an antibody by a host cell.

Recombinant expression of an antibody described herein (e.g., a full-length antibody, a heavy and/or light chain antibody, or a single chain antibody described herein) that specifically binds to CTLA-4 (e.g., human CTLA-4) involves constructing an expression vector containing a polynucleotide that encodes said antibody. After obtaining a polynucleotide encoding an antibody molecule, an antibody heavy and/or light chain, or a fragment thereof (e.g., heavy and/or light chain variable regions) described herein, a vector for producing said antibody molecule can be produced using recombinant DNA technology , using techniques well known in the art. Accordingly, methods for producing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody or antibody fragment (eg, a light chain or a heavy chain) are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing coding sequences for an antibody or antibody fragment (eg, light chain or heavy chain) and suitable transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. Also provided are replicable vectors containing a nucleotide sequence encoding an antibody molecule described herein, an antibody heavy or light chain, an antibody heavy or light chain variable region or fragment thereof, or a heavy or light chain CDR operably linked to a promoter. Such vectors may, for example, include a nucleotide sequence encoding the constant region of an antibody molecule (see, for example, WO 86/05807 and WO 89/01036; and US Pat. No. 5,122,464, which are incorporated herein by reference in their entirety), and the antibody variable regions can be cloned into such a vector to express the complete heavy chain, the complete light chain; or both full heavy and full light chains.

The expression vector can be transferred into a cell (eg, a host cell) using standard techniques, and the resulting cells can then be cultured using standard techniques to produce the antibody described herein, or a fragment thereof. Accordingly, the present invention provides host cells containing a polynucleotide encoding an antibody described herein, or fragments thereof; or a heavy or light chain thereof, or a fragment thereof; or a single chain antibody as described herein operably linked to a promoter for expression of such sequences in a host cell. In some embodiments for the expression of double-chain antibodies, vectors encoding both the heavy and light chains individually can be co-expressed in a host cell to express a complete immunoglobulin molecule, as detailed below. In some embodiments, the host cell contains a vector containing a polynucleotide encoding both a heavy chain and a light chain of an antibody described herein, or a fragment thereof. In certain embodiments, the host cell contains two different vectors, the first of which contains a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody described herein or a fragment thereof, and the second of which contains a polynucleotide encoding the light chain or light chain variable region an antibody described herein, or a fragment thereof. In other embodiments, the first host cell contains a first vector containing a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody described herein or a fragment thereof, and the second host cell contains a second vector containing a polynucleotide encoding the light chain or the light chain variable region of an antibody described herein. In certain embodiments, the heavy chain/heavy chain variable region expressed by the first cell binds to the light chain/light chain variable region of the second cell to form an anti-CTLA-4 antibody described herein or an antigen binding fragment thereof. According to some embodiments of the present invention, a population of host cells is provided, comprising such a first host cell and such a second host cell.

According to a specific embodiment of the present invention, a population of vectors is provided, comprising a first vector containing a polynucleotide encoding the light chain/light chain variable region of an anti-CTLA-4 antibody described herein, and a second vector containing a polynucleotide encoding the heavy chain/heavy chain variable region. antibody chains

- 62 044966 against CTLA-4 described herein.

Various host/expression vector systems can be used to express the antibody molecules described herein (see, for example, US Pat. No. 5,807,715). Such host expression systems are represented by frameworks by which coding sequences of interest can be produced and then purified, but are also represented by cells that are capable, after transformation or transfection with suitable nucleotide coding sequences, of expressing an antibody molecule described herein in situ. These cells include, but are not limited to, microorganisms such as bacteria (eg, E. coli and B. subtilis) transformed with recombinant expression vectors with bacteriophage DNA, plasmid DNA or cosmid DNA containing antibody coding sequences; yeast (eg, Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (eg, baculovirus) containing antibody coding sequences; plant cell systems (eg green algae such as Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (eg cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg Ti plasmid), containing antibody coding sequences; or mammalian cell systems (e.g. COS cells (e.g. COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa and NIH 3T3, HEK-293T, HepG2, SP210 , R1.1, B-W, L-M, BSC1, BSC40, YB/20 and BMT10), carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (for example, the metallothionein promoter) or mammalian viruses (for example, the late adenoviral promoter; promoter vaccinia virus size 7.5 kDa). In a specific embodiment, the cells for expressing the antibodies described herein, or an antigen binding fragment thereof, are CHO cells, for example, CHO cells from the CHO GS System™ (Lonza). In a specific embodiment, the cells for expressing the antibodies described herein are human cells, such as human cell lines. In a particular embodiment, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In a particular embodiment, bacterial cells such as Escherichia coli or eukaryotic cells (eg, mammalian cells) are used to express the recombinant antibody molecule, particularly to express the entire recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells in combination with a vector such as the human cytomegalovirus major intermediate early gene promoter element provide an efficient expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45 : 101-5; and Cockett MI et al., (1990) Biotechnology 8(7): 662-7, incorporated herein by reference in its entirety). In some embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In a specific embodiment, the expression of nucleotide sequences encoding antibodies described herein that specifically bind CTLA-4 (eg, human CTLA-4) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

Bacterial systems may provide the advantage of allowing multiple expression vectors to be selected depending on the intended use of the antibody molecule being expressed. For example, if it is necessary to produce significant amounts of such an antibody, the preparation of pharmaceutical compositions with the antibody molecule may require vectors that direct the expression of high levels of fusion protein products that are easy to purify. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (source: Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794, incorporated herein by reference in its entirety), characterized in that the coding sequence the antibody can be individually ligated into the vector in frame with the lac Z coding region such that a fusion protein is produced; pIN vectors (sources: Inouye S & Inouye M (1985) Nuc Acids Res 13: 31Ο1-31θ9; Van Heeke G & Schuster Sm (1989) J Biol Chem 24: 5503-5509, which are incorporated herein by reference in their entirety); and so on. For example, pGEX vectors can also be used to express foreign polypeptides as glutathione 5-transferase (GST) fusion proteins. In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to glutathione agarose matrix 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 gene product can be freed from the GST moiety.

In an insect cell system, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector for the expression of foreign genes. This virus grows in

- 63 044966 cells of Spodoptera frugiperda. The antibody coding sequence can be cloned individually into non-essential regions (eg, the polyhedrin gene) of the virus and placed under the control of the AcNPV promoter (eg, the polyhedrin promoter).

A number of virus-based expression systems can be used in mammalian host cells. In cases where an adenovirus is used as the expression vector, the coding sequence of the antibody of interest can be ligated into an adenoviral transcription/translation control complex, eg, a late promoter and a tripartite leader sequence. Said chimeric gene can then be inserted into the adenoviral genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg the E1 or E3 region) results in a recombinant virus that is viable and capable of expressing the antibody molecule in the infected host (eg Logan J & Shenk T (1984) PNAS 81( 12): 3655-9, incorporated herein by reference in its entirety). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences.

These signals include the ATG start codon and adjacent sequences. In addition, the specified start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insertion. These exogenous translation control signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be increased by including suitable elements - transcription enhancers, transcription terminators, etc. (See, for example, Bitter G et al., (1987) Methods Enzymol. 153: 516-544, incorporated herein by reference in its entirety).

In addition, a host cell strain may be selected that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific desired manner. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for protein function. Different host cells have characteristic, specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells can be used, which have cellular mechanisms for proper processing of the primary transcript, glycosylation and phosphorylation of the gene product. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (for example, COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 and HsS78Bst. In some embodiments, the anti-CTLA-4 antibodies described herein are produced in mammalian cells, such as CHO cells.

In a specific embodiment, the antibodies described herein, or antigen binding fragments thereof, are reduced in fucose or do not contain fucose. Such antibodies can be prepared using techniques known to one skilled in the art. For example, antibodies can be expressed in cells that are deficient in the ability to fucosylate or lack this ability. According to a specific example, cell lines with knockout of both alleles of α-1,6-fucosyltransferase can be used to obtain antibodies or antigen-binding fragments thereof with a reduced fucose content. The Potelligent® system (Lonza) is an example of such a system that can be used to produce antibodies or antigen-binding fragments thereof with reduced fucose content.

For long-term, high-yield production of recombinant proteins, cells can be obtained for stable expression. For example, cell lines can be constructed that stably express the anti-CTLA-4 antibody described herein or an antigen binding fragment thereof. In specific embodiments, a cell of the present invention stably expresses a light chain/light chain variable region and a heavy chain/heavy chain variable region that bind to form an antibody described herein or an antigen binding fragment thereof.

In certain aspects, instead of using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by suitable expression control elements (e.g., promoter sequences, enhancer sequences, transcription terminators, polyadenylation sites, etc.) and selectable marker. After introduction of foreign DNA/polynucleotide, the engineered cells can be allowed to grow for 1-2 days in enriched medium and then switched to selective medium. A selectable marker in a recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines. The use of this method can

- 64 044966 provide the advantage of constructing cell lines that express the anti-CTLA-4 antibody described herein, or a fragment thereof. Such engineered cell lines may be particularly suitable for use in screening and evaluating compositions that interact, directly or indirectly, with an antibody molecule.

A number of selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase genes (Wigler M et al., (1977) Cell 11(1): 223-32), hypoxanthine guanine phosphoribosyltransferase (source: Szybalska EH & Szybalski W (1962) PNAS 48(12): 2026-2034, incorporated herein in its entirety by reference) and adenine phosphoribosyltransferase (source: Lowy I et al., (1980) Cell 22(3): 817-23, incorporated herein in its entirety by reference) in tk-, hgprt-, or aprT- cells, respectively. Antimetabolite resistance can also be used as a 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: 152731); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance to aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217;Nabel GJ & Feigner 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), each of which is incorporated herein by reference in its entirety. To select the desired recombinant clone, methods generally known in the art of recombinant DNA technology can be routinely used; such methods are described, for example, in: Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); Dracopoli N.C. et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994) (chapters 12 and 13); Colbere-Garapin F et al., (1981) J Mol Biol 150: 1-14, which are incorporated by reference herein in their entirety.

Expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 ( Academic Press, New York, 1987), incorporated herein by reference in its entirety). If a marker in a vector system expressing an antibody is amplifiable, increasing the level of inhibitor present in the host cell culture increases the copy number of said marker gene. Since the amplified region is associated with the antibody gene, the level of antibody production will also be increased (source: Crouse GF et al., (1983) Mol Cell Biol 3: 257-66, incorporated herein by reference in its entirety).

A host cell can be cotransfected with two or more expression vectors described herein, the first of which encodes a heavy chain-derived polypeptide and the second of which encodes a light chain-derived polypeptide. The two vectors may contain identical selectable markers to ensure equal expression of the heavy and light chain polypeptides. Host cells can be cotransfected with varying amounts of two or more expression vectors. For example, 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.

Alternatively, a single vector that encodes and is capable of expressing both heavy and light chain polypeptides may be used. In such situations, the light chain must be placed before the heavy chain to avoid excess toxic free heavy chain (sources: Proudfoot NJ (1986) Nature 322: 562-565; and Kohler G (1980) PNAS 77: 2197-2199, incorporated herein entirely by reference). The heavy and light chain coding sequences may comprise cDNA or genomic DNA. Said expression vector may be monocistronic or multicistronic. A multicistronic nucleic acid construct may encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes/nucleotide sequences, or 2-5, 5-10 or 10-20 genes/nucleotide sequences. For example, a bicistronic nucleic acid construct may comprise, in that order, a promoter, a first gene (eg, the heavy chain of an antibody described herein), and a second gene (eg, a light chain of an antibody described herein). In such an expression vector, the transcription of both genes can be driven by a promoter, while the translation of mRNA from the first gene can be represented by a cap-dependent scanning mechanism, and the translation of mRNA from the second gene can be represented by a cap-independent mechanism, for example, involving an IRES.

Once the antibody molecule described herein has been produced by recombinant expression, it can be purified by any method known in the art for purifying an immunoglobulin molecule, for example, using chromatography (e.g., ion exchange, affinity, in particular, affinity for a specific antigen after Protein A chromatography, and size distribution column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. In addition, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or other sequences known in the art to facilitate purification.

In certain embodiments, an antibody or antigen binding fragment thereof described herein is isolated or purified. Typically, the isolated antibody is an antibody substantially free of other antibodies with an antigenic specificity different from that of the isolated antibody. For example, in a particular embodiment, the antibody composition described herein is substantially free of cellular material and/or chemical precursors. The expression essentially free of cellular material includes antibody formulations where the antibody is separated from the cellular components of the cells from which it is isolated or in which it is recombinantly produced. Accordingly, an antibody that is substantially free of cellular material includes antibody formulations containing less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (as appropriate). dry weight) of a heterologous protein (also referred to herein as a contaminant protein), and/or antibody variants, such as otherwise post-translationally modified forms of the antibody or other different antibody variants (eg, antibody fragments). If the antibody is produced recombinantly, it is also usually substantially free of culture medium, i.e. the culture medium constitutes less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% by volume of the protein composition. When an antibody is produced by chemical synthesis, it is usually substantially free of chemical precursors or other chemicals, i.e. separated from chemical precursors or other chemicals that are involved in the synthesis of the specified protein. Accordingly, such antibody compositions contain 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 isolated or purified.

Antibodies or fragments thereof that specifically bind to CTLA-4 (eg, human CTLA-4) can be produced by any method known in the art for synthesizing antibodies, for example, by chemical synthesis or using recombinant expression techniques. The methods described herein employ, unless otherwise indicated, standard techniques of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related techniques known in the art. in this field of technology. These techniques are described, for example, in the sources cited herein and are fully explained in the literature; see, for example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel F. M. et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987, updated annually); Current Protocols in Immunology, John Wiley & Sons (1987, with annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, each of which is incorporated herein by reference in its entirety.

In a specific embodiment, an antibody described herein is an antibody (eg, a recombinant antibody) produced, expressed, generated, or isolated by any method that includes generating, for example, by synthesizing, genetically engineering, DNA sequences. In some embodiments, such an antibody contains sequences (eg, DNA sequences or amino acid sequences) that are not naturally present in the animal or mammalian (eg, human) germline antibody repertoire in vivo.

According to one aspect of the present invention, there is provided a method of producing an antibody or antigen binding fragment thereof that specifically binds to CTLA-4 (eg, human CTLA-4), comprising culturing a cell or host cell as described herein. According to a certain aspect of the present invention, there is provided a method of producing an antibody or antigen binding fragment thereof that specifically binds to CTLA-4 (e.g., human CTLA-4), comprising expressing (e.g., recombinant expression) said antibody or antigen binding fragment thereof using a cell or host cell described herein (eg, a cell or host cell containing polynucleotides encoding an antibody described herein). In a specific embodiment, said cell is an isolated cell. In a specific embodiment, exogenous polynucleotides are introduced into the cell. In a specific embodiment, the method further includes the step of purifying an antibody or antigen binding fragment thereof obtained from said cell or host cell. Preferably, said method is carried out in vitro.

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Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 of the source: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York, included this document in its entirety by reference).

Monoclonal antibodies can be produced using a wide range of techniques known in the art, including hybridoma, recombinant and phage display technology, or combinations thereof.

For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art and described, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), which are incorporated herein by reference in their entirety. The term monoclonal antibody as used herein is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be produced recombinantly from host cells exogenously expressing an antibody described herein, or a fragment thereof, such as the light chain and/or heavy chain of such an antibody.

In specific embodiments, a monoclonal antibody as used herein is an antibody produced by a single cell (e.g., a hybridoma cell or a host cell producing a recombinant antibody), wherein said antibody specifically binds to CTLA-4 (e.g., human CTLA-4), according to the results of, for example, an ELISA or other antigen binding or competitive binding assay known in the art or described in the examples provided herein. In specific embodiments, the monoclonal antibody may be a chimeric antibody or a humanized antibody. In some embodiments, the monoclonal antibody is a monovalent antibody or a multivalent (eg, bivalent) antibody. In certain embodiments, the monoclonal antibody is a monospecific or multispecific antibody (eg, a bispecific antibody). The monoclonal antibodies described herein may, for example, be produced using a hybridoma method as described in Kohler G & Milstein C (1975) Nature 256: 495, incorporated herein by reference in its entirety, or may, for example, be isolated from phage libraries, including using methods as described herein. Other methods for producing clonal cell lines and the monoclonal antibodies they express are well known in the art (see, for example, Chapter 11 of the source: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

Methods for producing and screening specific antibodies using hybridoma technology are common and well known in the art. For example, in the hybridoma method, a mouse or other suitable host animal, such as a sheep, goat, rabbit, rat, hamster, or monkey, is immunized to activate lymphocytes that produce or are capable of producing antibodies that specifically bind to a protein (e.g., CTLA-4 ( e.g. human CTLA-4) used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to produce a hybridoma cell (source: Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986), included this document in its entirety by reference). In addition, the RIMMS (repeated immunization at multiple sites) technique can be used to immunize an animal (source: Kilpatrick KE et al., (1997) Hybridoma 16:381-9, incorporated herein by reference in its entirety).

In some embodiments, mice (or other animals such as rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) can be immunized with an antigen (e.g., CTLA-4 (e.g., human CTLA-4)) and upon detection of an immune response eg, antibodies specific for said antigen in mouse serum, mouse spleens are collected and splenocytes are isolated. The splenocytes are then fused using well known techniques with any suitable myeloma cells, such as the SP20 cell line available from the American Type Culture Collection (ATCC®) (Manassas, VA), to produce hybridomas. Hybridomas are selected and cloned using the limited dilution method. In some embodiments, lymph nodes from immunized mice are harvested and fused with NS0 myeloma cells.

The hybridoma cells thus obtained are seeded and cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the original myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT, or HPRT), hybridoma culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells.

Specific embodiments involve myeloma cells that fuse efficiently, support stable production of high levels of antibody by selected antibody-producing cells, and are sensitive to a medium, such as a HAT medium. These myeloma cell lines include murine myeloma lines, such as the NS0 cell line, or those derived from mouse MOPC-21 and MPC-11 tumors, available from the Salk Institute Cell Distribution Center, San Diego, California, USA. , and SP-2 or X63-Ag8.653 cells, available from the American Type Culture Collection, Rockville, MD, USA. Human myeloma cell lines and mouse/human heteromyeloma cell lines have also been described to produce human monoclonal antibodies (sources: Kozbor D (1984) J Immunol 133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51 -63 (Marcel Dekker, Inc., New York, 1987), incorporated herein by reference in its entirety).

The production of monoclonal antibodies directed against CTLA-4 (eg, human CTLA-4) is analyzed in a culture medium in which hybridoma cells are cultured. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined using methods known in the art, for example, immunoprecipitation, or using an in vitro binding assay, such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA).

Once hybridoma cells are identified that produce antibodies with the desired specificity, affinity and/or activity, these clones can be subcloned using limited dilution procedures and cultured by standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640. In addition, these hybridoma cells can be cultured in vivo as ascites tumors in an animal.

Monoclonal antibodies secreted by these subclones are suitably separated from the culture medium, ascites fluid or serum using standard immunoglobulin purification procedures, such as, for example, protein A/Sepharose purification, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography .

The antibodies described herein include antibody fragments that recognize specific CTLA-4 (eg, human CTLA-4) and can be prepared using any technique known to those skilled in the art. For example, the Fab and F(ab') ₂ fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') ) ₂ -fragments). The Fab fragment corresponds to one of two identical arms of the antibody molecule and contains the complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab') ₂ fragment contains two antigen-binding arms of the antibody molecule connected by disulfide bonds at the hinge region.

In addition, the antibodies described herein, or antigen binding fragments thereof, can also be produced using various phage display methods known in the art. In phage display methods, the functional domains of an antibody are displayed on the surface of phage particles, which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding the VH and VL domains are amplified from animal cDNA libraries (eg, human or mouse diseased tissue cDNA libraries). DNAs encoding the VH and VL domains are recombined with the scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli and E. coli is infected with helper phage. The phage used in these methods is typically a filamentous phage, including fd and M13, and the VH and VL domains are usually recombinantly fused to phage gene III or gene VIII. A phage expressing an antigen-binding domain that binds to a particular antigen can be selected or identified by the antigen, for example, using a labeled antigen or an antigen bound or captured on a solid surface or bead. Examples of phage display methods that can be used to produce the antibodies described herein include those described in: Brinkman U et al., (1995) J Immunol Methods 182: 41-50; Ames RS et al., (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; Burton DR & Barbas CF (1994) Advan Immunol 57: 191-280; PCT application PCT/GB91/001134; international publications WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401 and WO 97/13844; and US patents No. 5698426, No. 5223409, No. 5403484, No. 5580717, No. 5427908, No. 5750753, No. 5821047, No. 5571698, No. 5427908, No. 5516637, No. 5780225, No. 565872 7, No. 5733743 and No. 5969108, each of which is included in this document is incorporated in its entirety by reference.

As described in the above references, once a phage is selected, antibody coding regions from the phage can be isolated and used to produce whole antibodies, including human antibodies, or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells , insect cells, plant cells, yeast and bacteria, for example, as described below. Techniques for recombinant production of antibody fragments such as Fab-, Fab'- and (ab') ₂ - 68 044966 fragments can also be used using methods known in the art, such as those described in the references: PCT publication WO 92 /22324; Mullinax RL et al., (1992) BioTechniques 12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; and Better M et al., (1988) Science 240: 1041-1043, each of which is incorporated herein by reference in its entirety.

In some embodiments, PCR primers comprising VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to produce whole antibodies to amplify VH or VL sequences from a template, such as scFv clones. Using cloning techniques known to those skilled in the art, PCR-amplified VH domains can be cloned into vectors expressing the VH constant region, and PCR-amplified VL domains can be cloned into vectors expressing the VL constant region, such as kappa constant regions or lambda man.

The VH and VL domains can also be cloned into a single vector expressing the necessary constant regions. The heavy chain conversion vectors and the light chain conversion vectors are then cotransfected into cell lines to produce stable or transient cell lines that express full length antibodies, eg IgG, using techniques known to those skilled in the art.

A chimeric antibody is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may comprise the variable region of a mouse or rat monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art; see, for example, Morrison SL (1985) Science 229: 1202-7; Oi VT & Morrison SL (1986) BioTechniques 4: 214-221; Gillies SD et al., (1989) J Immunol Methods 125: 191-202; and US Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415, each of which is incorporated herein by reference in its entirety.

The humanized antibody is capable of binding to a predetermined antigen and contains a framework region substantially containing the amino acid sequence of a human immunoglobulin and CDR regions substantially having the amino acid sequence of a non-human Ig (eg, mouse immunoglobulin). In certain embodiments, the humanized antibody also contains at least a portion of an immunoglobulin constant region (Fc), typically human immunoglobulin. The antibody may also include the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. The humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, _IgG2 , _IgG3 and IgG4. Humanized antibodies can be produced using various techniques known in the art, including, but not limited to, CDR grafting (sources: European patent EP 239400; international publication WO 91/09967; and US patents No. 5225539, No. 5530101 and No. 5585089), masking or altering surface residues (European patents EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4/5): 489-498; Studnicka GM et al., (1994) Prot Engineering 7( 6): 805-814; and Roguska MA et al., (1994) PNAS 91: 969-973), strand permutation (US Pat. No. 5,565,332) and techniques described, for example, in US Pat. No. 6,407,213, US Pat. No. 5,766,886 , international publication no. WO 93/17105; and references: Tan P et al., (2002) J Immunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5): 353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al., (1997) J Biol Chem 272(16): 10678-84; Roguska MA et al., (1996) Protein Eng 9(10): 895 904; Couto JR et al., (1995) Cancer Res. 55 (23 Supp): 5973s-5977s; Couto JR et al., (1995) Cancer Res 55(8): 1717-22; Sandhu JS (1994) Gene 150(2): 409-10 and Pedersen JT et al., (1994) J Mol Biol 235(3): 95973, each of which is incorporated herein by reference in its entirety; See also published US patent application US 2005/0042664 A1 (dated February 24, 2005), which is incorporated herein by reference in its entirety.

Methods for producing multispecific (eg, bispecific) antibodies have been described, see, for example, US Pat. No. 7,951,917; No. 7183076; No. 8227577; No. 5837242; No. 5989830; No. 5869620; No. 6132992 and No. 8586713, each of which is incorporated herein by reference in its entirety.

Single domain antibodies, for example, antibodies lacking light chains, can be produced using methods well known in the art; see sources: Riechmann L & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall SD et al., (2000) Curr Pharm Biotechnol 1(3): 253263; Muyldermans S, (2001) J Biotechnol 74(4): 277-302; US Patent No. 6,005,079; and international publications WO 94/04678, WO 94/25591 and WO 01/44301, each of which is incorporated herein by reference in its entirety.

In addition, antibodies that specifically bind to the CTLA-4 antigen can in turn be used to produce anti-idiotypic antibodies that mimic the antigen using techniques well known to those skilled in the art. (See, for example, Greenspan NS & Bona CA (1989) FASEB J 7(5): 437-444; and Nissinoff A (1991) J Immunol 147(8): 2429-2438, which are incorporated herein in their entirety via links).

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In specific embodiments, an antibody described herein that binds to the same CTLA-4 epitope (e.g., human CTLA-4) as an anti-CTLA-4 antibody described herein is a human antibody or an antigen binding fragment thereof . In specific embodiments, an antibody described herein that competitively blocks (e.g., in a dose-dependent manner) the binding of any of the antibodies described herein to CTLA-4 (e.g., human CTLA-4) is a human antibody or antigen-binding antibody thereof. fragment. Human antibodies can be obtained using any method known in the art. For example, transgenic mice that are unable to express functional endogenous immunoglobulins but are capable of expressing human immunoglobulin genes can be used. In particular, gene complexes of human immunoglobulin heavy and light chains can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region and D region can be introduced into mouse embryonic stem cells along with human heavy and light chain genes. The mouse immunoglobulin heavy and light chain genes can be rendered functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.

Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. These chimeric mice are then crossed to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized in the usual manner with a selected antigen, eg, a complete antigen or a portion of an antigen (eg, CTLA-4). Monoclonal antibodies directed against the specified antigen can be obtained from immunized transgenic mice using standard hybridoma technology. Human immunoglobulin transgenes carried by transgenic mice undergo rearrangement during B cell differentiation and then undergo class switching and somatic mutations. Accordingly, using this technique it is possible to obtain therapeutically useful IgG, IgA, IgM and IgE antibodies; For a review of this human antibody technology, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93, which is incorporated herein by reference in its entirety.

For a detailed description of this technology for the production of human antibodies and human monoclonal antibodies, as well as protocols for the production of such antibodies, see, for example, international publications WO 98/24893, WO 96/34096 and WO 96/33735; and US Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598. Examples of mice capable of producing human antibodies, each of which is incorporated herein by reference in their entirety, include Xenomouse™ ( Abgenix, Inc.; US Patents No. 6075181 and No. 6150184), HuAb-Mouse™ (Mederex, Inc./Gen Pharm; US Patents No. 5545806 and No. 5569825), Trans Chromo Mouse™ (Kirin) and KM Mouse™ (Medarex/ Kirin).

Human antibodies that specifically bind to CTLA-4 (e.g., human CTLA-4) can be produced by various methods known in the art, including the phage display methods described above, using antibody libraries derived from immunoglobulin sequences person; see also sources: US patents No. 4444887, No. 4716111 and No. 5885793; and international publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741, each of which is incorporated herein in its entirety by reference.

In some embodiments, human antibodies can be produced using mouse/human hybridomas. For example, Epstein-Barr virus (EBV)-transformed human peripheral blood lymphocytes can be fused with mouse myeloma cells to produce mouse/human hybridomas that secrete human monoclonal antibodies, and these mouse/human hybridomas can be screened to identify hybridomas that secrete human monoclonal antibodies that specifically bind to the target antigen (eg, CTLA-4 (eg, human CTLA-4)). Such methods are known and described in the art, see, for example, sources: Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31, which are incorporated herein in their entirety by reference.

Sets.

Also provided are kits containing one or more of the antibodies described herein, or a pharmaceutical composition therewith, or conjugates thereof. According to a specific embodiment of the present invention, there is provided a pharmaceutical package or kit containing one or more containers filled with one or more ingredients of the pharmaceutical compositions described herein, such as one or more antibodies provided according to the present invention, or an antigen binding fragment of such an antibody. In some embodiments, the kits contain a pharmaceutical composition described herein

- 70 044966 those and any prophylactic or therapeutic agent such as those described herein. In some embodiments, the kits may contain a T cell mitogen, such as, for example, phytohemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or a TCR complex stimulating antibody, such as an anti-CD3 antibody and an anti-CD28 antibody. Such container or containers may optionally be accompanied by a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products reflecting approval by such agency for manufacture, use, or sale for administration to humans.

Kits are also provided that can be used in the methods described above. In one embodiment, the kit contains an antibody described herein, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits described herein contain substantially isolated CTLA-4 antigen (eg, human CTLA-4) as a control. In another specific embodiment, the kits described herein further comprise a control antibody that does not react with the CTLA-4 antigen. In another specific embodiment, the kits described herein contain one or more elements for detecting the binding of an antibody to a CTLA-4 antigen (for example, the antibody may be conjugated to a detectable substrate, such as a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound, or second antibody, that recognizes the first antibody may be conjugated to the detectable substrate). In specific embodiments, the kit provided by the present invention may include a recombinantly produced or chemically synthesized CTLA-4 antigen. The CTLA-4 antigen included in the kit can also be attached to a solid support. In a more specific embodiment, the detection means of the kit described above include a solid support to which a CTLA-4 antigen is attached. Such a kit may also include an unattached reporter-labeled anti-human or anti-mouse/rat antibody. In this embodiment, binding of an antibody to a CTLA-4 antigen can be detected by binding of said reporter-labeled antibody.

In one embodiment, the present invention relates to the use of a kit according to the present invention for the analysis and/or in vitro detection of human CTLA-4 in a biological sample.

Examples

The examples in this section (i.e., Section 6) are provided for illustration and not limitation.

Example 1 Characterization of novel anti-CTLA-4 antibodies.

This example describes the characterization of antibodies that bind to human CTLA-4. In particular, this example describes the characterization of antibodies that specifically bind to human CTLA-4 and inhibit the function of CTLA-4. Information on the sequences of the variable regions of these antibodies is presented in table. 4. All of these antibodies were expressed as IgG1 antibodies and analyzed in assays as described below.

Binding of anti-CTLA-4 antibodies to CTLA-4-expressing cells.

Jurkat cells engineered to constitutively express human CTLA-4 (Promega) were used for anti-CTLA-4 antibody binding assays. Briefly, the indicated cells at a density of 5x105 cells/well were stained using 2 μg/ml antibody in a 96-well plate for 30 min at 4°C. Cells were washed twice and incubated for 20 min at 4°C with anti-human IgG secondary antibody (Thermo Scientific, cat. no. 31529). Cells were washed and suspended in 50 μl of 2% paraformaldehyde (Alfa Aesar, cat. no. 43368) in PBS. Data were collected using BD FACS Canto II.

As shown in FIG. 1A-1G, all anti-CTLA-4 antibodies tested bound to CTLA-4 expressing cells.

Effect of anti-CTLA-4 antibody on human PBMC following stimulation with staphylococcal enterotoxin A (SEA).

Functional activity of anti-CTLA-4 antibody.

AGEN1884.H3 (IgG1) against primary human PBMCs was assessed after stimulation with staphylococcal enterotoxin A (SEA). Briefly, cryopreserved PBMCs were stimulated with 100 ng/ml SEA superantigen (Toxin Technologies, Cat. No. at101red) in the absence or presence of 10 μg/ml anti-CTLA-4 antibody or isotype control antibody (IgG1) for 5 days at 37°C and 5% CO ₂ . IL-2 concentrations in the culture supernatant were analyzed using AlphaLISA (Perkin Elmer, Cat. No. AL221F).

Anti-CTLA-4 antibody AGEN1884.H3 (IgG1) increased IL-2 production in human PBMCs stimulated with superantigen SEA (Fig. 2).

Effect of anti-CTLA-4 antibody on an IL-2-luciferase reporter cell line.

Further, the functional activity of the anti-CTLA-4 antibody AGEN1884.H3 (IgG1) is additionally

- 71 044966 was analyzed using an IL-2-luciferase reporter assay. Briefly, a human T cell line (Jurkat) endogenously expressing CD3 and CD28 was engineered to constitutively express cell surface CTLA-4 and a luciferase reporter gene driven by the IL-2 promoter. The Jurkat reporter cell line was cocultured with an antigen presenting cell line (Raji) endogenously expressing CD80 and CD86 and engineered to express a proprietary T cell activator (Promega). T cell receptor (TCR) triggering (signal 1) was achieved by a T cell activator; and costimulatory signaling (signal 2) was provided in a transactive manner by CD80 and CD86 expressed on Raji cells. TCR signaling in the Jurkat T cell line triggered IL-2 expression, leading to the production of luciferase, a surrogate marker of T cell activation. Co-culture of the two cell lines resulted in recruitment of the inhibitory co-receptor CTLA-4 (expressed on Jurkat cells), with its natural ligands CD80 and CD86 (expressed on Raji cells) inhibiting T cell activation, as indicated by the absence of luciferase expression. The addition of increasing concentrations of blocking antibodies against CTLA-4 attenuated this inhibition. Luciferase expression was quantified using Bio-Glo™ reagent, and the resulting data was used to determine fold response values (fold increase with AGEN1884.H3 (IgG ₁ ) versus isotype control antibody (IgG ₁ )).

As shown in FIG. 3, anti-CTLA-4 antibody AGEN1884.H3 (IgG ₁ ) dose-dependently abolished CTLA-4-mediated T cell inhibition in the indicated IL-2-luciferase reporter assay.

Effect of anti-CTLA-4 antibody on an Fc receptor gamma IIIA reporter cell line.

The ability of anti-CTLA-4 antibody to simultaneously recruit CTLA-4 and signal by activating Fc receptor gamma was assessed using a reporter cell line expressing Fc receptor gamma IIIA (FcyRIIIA) (Promega). Briefly, Jurkat cells were engineered to constitutively express human CTLA-4 on the cell surface. These target cells were co-cultured with an effector cell line (Jurkat) engineered to express FcyRIIIA. (variant V158), located upstream of the NFAT response element (RE), which controls the expression of firefly luciferase. A titrated dose of AGEN1884.H3 (IgG ₁ ) or isotype control antibody (IgG ₁ ) was added to the co-culture and incubated at 37°C overnight. Simultaneous recruitment of AGEN1884.H3 by a target cell line (binding of the CTLA-4 Fab region) and an effector cell line (binding of the Fc region to FcyRIIIA) triggers activation of the RE NFAT reporter gene and luciferase expression. The next day, Bio-Glo reagent (Promega) was added to the coculture, and luminescence was measured using an EnVison plate reader. Multimode (Perkin Elmer), and relative light units (RLU) were recorded to calculate fold response values (fold increase with AGEN1884.H3 (IgG ₁ ) compared to isotype control antibody (IgG ₁ )).

When bound to target cells expressing human CTLA-4 on the cell surface, the IgG1 antibody AGEN1884.H3 activated FcyRIIIA signaling in effector cells (Fig. 4).

Example 2. Characterization of anti-CTLA-4 antibodies with different Fc regions.

In this example, the effect of Fc/Fc receptor interaction on the functional activity of antibodies against CTLA-4 was analyzed. AGEN1884.H3 was expressed as antibodies containing the Fc region of IgG ₁ with mutations S239D/I332E, S239D/A330L/I332E or L235V/F243L/R292P/Y300L/P396L according to the EU numbering system, and tested in the functional assays described below . Antibody AGEN1884.H3 (IgG ₁ S239D/I332E) contains a heavy chain containing the amino acid sequence from SEQ ID NO: 24, and a light chain containing the amino acid sequence from SEQ ID NO: 27. Antibody AGEN1884.H3 (IgG ₁ S239D/A330l/ I332E) contains a heavy chain containing the amino acid sequence from SEQ ID NO: 25, and a light chain containing the amino acid sequence from SEQ ID NO: 27. Antibody AGEN1884.H3 (IgG ₁ L235V/F243L/R292P/Y300L/P396L) contains a heavy chain , containing the amino acid sequence of SEQ ID NO: 26, and a light chain containing the amino acid sequence of SEQ ID NO: 27. For comparison, AGEN1884 was also expressed as a wild-type IgG1 antibody containing the S239D/I332E or S239D/A330L/I332E mutations according to with the EU numbering system, or afucosylated IgG1 antibody, and tested in several functional assays.

Binding of anti-CTLA-4 antibodies to CTLA-4-expressing cells.

Binding of antibodies against CTLA-4 AGEN1884.H3 (IgG ₁ S239D/I332E), AGEN1884.H3 (IgG ₁ S239D/A330L/I332E) and AGEN1884.H3 (IgG ₁ L235V/F243L/R292P/Y300L/P396L) with expressing and CTLA- 4 cells were characterized in a similar manner as described above. Briefly, Jurkat cells engineered to express human CTLA-4 (Promega) were stained first with 5 μg/ml anti-CTLA-4 antibody or isotype control antibody, followed by a secondary anti-human IgG antibody (Thermo Scientific, Cat. No. 31529). Cells were analyzed using BD FACS Canto II.

As shown in FIG. 5A-5D, all AGEN1884.H3 antibodies with different Fc regions bound to cells expressing human CTLA-4.

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Effect of anti-CTLA-4 antibody on ligand binding to human CTLA-4.

In this example, the ability of the Fc variant of the anti-CTLA-4 antibody was tested

AGEN1884.H3 (IgG1-S239D/A330E/I332E) to block the binding between human CTLA-4 and its ligands, CD80 and CD86.

Briefly, recombinant proteins CD80-FC and CD86-FC were conjugated to Alexa Fluor 647 fluorochrome (Invitrogen, A20186). Jurkat cells were transduced with trCTLA4 (truncated intracellular domain) under the control of the EF1a promoter as described in Nakaseko et al. (J Exp Med. 1999 Sep 20; 190(6): 765774), thereby obtaining a cell line constitutively expressing human CTLA-4 on the cell surface. CTLA-4-expressing cells were incubated with titrated doses of anti-CTLA-4 antibody AGEN1884.H3 (IgG1-S239D/A330E/I332E), anti-CTLA-4 reference antibody, or isotype control antibody (IgG1). The cells were then stained with fluorescently labeled CD80-FC or CD86-FC protein. After staining, fluorescence was analyzed using an LSRFortessa flow cytometer (BD Biosciences). FACS plots were analyzed using a combination of FACS DIVA and WEHI Weasel software. Value graphs were generated using Graphpad Prism software.

As shown in FIG. 6A, both AGEN1884.H3 (IgG1-S239D/A330E/I332E) and the reference anti-CTLA-4 antibody blocked binding between human CTLA-4 and CD80 in a dose-dependent manner, whereas the isotype control antibody (IgG1) had no effect. As shown in FIG. 6B, both AGEN1884.H3 (IgG1-S239D/A330E/I332E) and the reference anti-CTLA-4 antibody also blocked binding between human CTLA-4 and CD86 in a dose-dependent manner, whereas the isotype control antibody (IgG1) had no effect. These data indicate that AGEN1884.H3 (IgG1-S239D/A330E/I332E) functions as a CTLA-4 ligand blocking antibody.

Effect of anti-CTLA-4 antibodies on human PBMCs after stimulation with staphylococcal enterotoxin A (SEA).

In this example, the effect of Fc regions on the functional activity of anti-CTLA-4 antibodies was analyzed using the SEA stimulation assay described above. Briefly, human PBMCs were cultured in vitro with 100 ng/ml SEA peptide (Toxin Technologies, Cat. No. at101red) in the absence or presence of anti-CTLA-4 antibodies with different Fc regions or an isotype control antibody. Five days later, concentrations of IL-2 in the culture supernatant, a marker of T cell activation, were measured using AlphaLISA (Perkin Elmer, Cat. No. AL221F).

As shown in FIG. 7A, three AGEN1884.H3 antibodies containing mutations in the IgG1 Fc regions, all of which increased binding to FcyRIIIA, stimulated greater IL-2 secretion compared with AGEN1884.H3 containing the wild-type IgG1 Fc region.

In similar studies, AGEN1884.H3 or AGEN1884 antibodies with different Fc regions were tested in the SEA stimulation assay. Introduction of the S239D/I332E, S239D/A330L/I332E, or L235V/F243L/R292P/Y300L/P396L substitutions into the Fc region of IgG1 significantly increased the functional activity of AGEN1884.H3 (Fig. 7B). Likewise, AGEN1884 (IgG1 S239D/I332E), AGEN1884 (IgG1 S239D/A330L/I332E), and afucosylated AGEN1884 (IgG1) increased IL-2 production at substantially lower concentrations compared to AGEN1884 with the wild-type IgG1 Fc region (Fig. .7C).

Effect of anti-CTLA-4 antibodies on ZAP70 phosphorylation.

In this example, the effect of Fc regions on the functional activity of anti-CTLA-4 antibodies at the T cell/antigen presenting cell (APC) synapse was analyzed using an assay to measure the level of phosphorylation of the protein tyrosine kinase ZAP70, which upon TCR recruitment is directed to the TCR where it is phosphorylated , and facilitates subsequent signaling.

Briefly, human PBMCs were incubated at a suboptimal concentration of SEA peptide and 10 μg/ml isotype control antibody (IgG1) or anti-CTLA-4 antibody AGEN1884.H3 (IgG1), AGEN1884.H3 (IgG1 S239D/A330L/I332E), or AGEN1884.H3 ( IgG1 N297A). Cells were then incubated at 37°C for 0 (pre-period) 1, 5, 10, 30, or 60 min. At the end of incubation, cells were lysed with cold 1xRIPA buffer supplemented with phosphatase/protease inhibitor cocktail (Cell Signaling Technologies). After clarification of the supernatant, protein concentration was quantified using the bicinchoninic acid (BCA) assay (Pierce Biotechnology). Cell lysates (20 μg/lane) were prepared in Bolt LDS sample buffer diluted and heated for 10 min at 70°C before loading onto 4–12% Bolt Bis Tris gels (Novex). Proteins were separated in 1x Bolt MOPS buffer (ThermoFisher), and then the blots were transferred to a PVDF membrane. After blocking with 5% bovine serum albumin (BSA, 1 h), samples were incubated with rabbit anti-human phospho-ZAP70 (Tyr493)/Syk (Tyr526) primary antibody (Cell Signaling Technologies) in blocking buffer overnight at 4°C. Membranes were probed with secondary HRP-conjugated goat anti-rabbit antibody and visualized with SignalFire ECL reagent (Cell Signaling Technologies). Capture iso

- 73 044966 fermentations were carried out using a Chemidoc imaging system (BioRad). As a control, total ZAP70 protein was assessed after membrane stripping with Restore™ PLUS Western Blot Stripping Buffer. Densitometric analysis of phospho-ZAP70 normalized to total ZAP70 was performed using Image J (Wayne Rasband; National Institute of Mental Health, Bethesda, MD, USA) and results were expressed as fold change over treated isotype control samples that were incubated for 1 min.

As shown in FIG. 8A-8B, in the isotype control antibody sample, ZAP70 phosphorylation increased transiently within 10 min after stimulation and rapidly decreased, reaching undetectable levels after 15 min. In contrast, the addition of antibodies against CTLA-4 AGEN1884.H3 (IgG1) or AGEN1884.H3 (IgG1 S239D/A330L/i332E) prolonged the detectable activation of ZAP70 to 30 min, with the most pronounced activity and relative abundance observed with AGEN1884.H3 ( IgG1 S239D/A330L/I332E).

Effect of mouse anti-CTLA-4 antibodies on tumor growth and intratumoral regulatory T cell depletion in a mouse model.

In this example, the effect of Fc regions on the antitumor and intratumoral regulatory T cell (Treg) depleting activity of anti-CTLA-4 antibodies was analyzed using a mouse model of colon cancer (CT26 tumor-bearing mice).

Briefly, 5 x 10 ⁴ CT26 tumor cells were suspended in 100 ml PBS and injected subcutaneously into 6-8 week old female BALB/cJ mice (Jackson Laboratories). After transplantation and until the tumor reached a volume of 50-80 ^mm3 , mice were treated with a single dose of 100 μg of mouse anti-CTLA-4 antibody 9D9 (mIgG2a), an Fc-silent variant of anti-CTLA-4 antibody 9D9 (mIgG2aN297A), an Fc-variant antibody against CTLA-4 9D9 (mIgG2a-S239D/A330L/I332E) or isotype control antibody (mIgG2a). The amino acid sequences of the tested mouse antibodies are presented in table. 7.

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Table 7

Amino acid sequences of mouse anti-CTLA-4 antibodies

Description Subsequence SEQ ID NO Mouse anti-CTLA-4 antibody (m!gG2a), heavy chain EAKLQESGPVLVKPGASVKMSCKASGYTFTDYYMN WVKQSHGKSLEWIGVINPYNGDTSYNQKFKGKATL TVDKSSSTAYMELNSLTSEDSAVYYCARYYGSWFA YWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSV TLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQ SDLYTLSSSVTVTSST WPSQSITCNVAHPASSTKV DKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPP KIKDVLMISLSPIVTCVWDVSEDDPDVQISWFVN NVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMS GKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYV LPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNG KTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVER NSYSCSVVHEGLHNHHTTKSFSRTPG 49 Mouse antibody against CTLA-4 (m!gG2aS239D/A330L/I33 2E) , heavy chain EAKLQESGPVLVKPGASVKMSCKASGYTFTDYYMN WVKQSHGKSLEWIGVINPYNGDTSYNQKFKGKATL TVDKSSSTAYMELNSLTSEDSAVYYCARYYGSWFA YWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSV TLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQ SDLYTLSSSVTVTSST WPSQSITCNVAHPASSTKV DKKIEPRGPTIKPCPPCKCPAPNLLGGPDVFIFPP KIKDVLMISLSPIVTCVWDVSEDDPDVQISWFVN NVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMS GKE FKCKVNNKDLPLPEERTISKPKGSVRAPQVYV LPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTN NG KTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVER NSYSCSVVHEGLHNHHTTKSFSRTPG 50 Mouse anti-CTLA-4 antibody (mIgG2a-N297A), heavy chain EAKLQESGPVLVKPGASVKMSCKASGYTFTDYYMN WVKQSHGKSLEWIGVINPYNGDTSYNQKFKGKATL TVDKSSSTAYMELNSLTSEDSAVYYCARYYGSWFA YWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSV TLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQ SDLYTLSSSVTVTSST WPSQSITCNVAHPASSTKV 51 DKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPP KIKDVLMISLSPIVTCVWDVSEDDPDVQISWFVN NVEVHTAQTQTHREDYASTLRWSALPIQHQDWMS GKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYV LPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNG KTELNYKNTEPVLDSDGSY FMYSKLRVEKKNWVER NSYSCSVVHEGLHNHHTTKSFSRTPG Mouse anti-CTLA-4 antibody (m!gG2a), light chain DIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNGN TYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGS GSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGG GTKLEIKRADAAPTVSIFPPSSEQLTSGGASWCF LNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKD STYSMSSTLTLTK DEYERHNSYTCEATHKTSTSPI VKSFNRNEC 52

In the first experiment, tumor growth in mice treated with antibodies was then measured once every two weeks. As shown in FIG. 9A, Fc variant of anti-CTLA-4 antibody 9D9 (provided by

- 75 044966 as mIgG2a-S239D/A330L/I332E) induced complete regression in all CT26 tumor-bearing mice (8 out of 8 mice tested). In contrast, other variants of the 9D9 antibody were unable to provide the same effectiveness: the 9D9 antibody itself (mIgG2a) induced complete regression in three of nine mice tested, and the Fc-silent variant of the 9D9 antibody (mIgG2a-N297A) was unable to induce regression in any of the mice tested. nine mice tested.

In the second experiment, CT26 tumor-bearing mice were treated as described above and then sacrificed 0, 24, 72, or 240 hours after treatment to collect tumor tissue, tumor-draining lymph nodes, and spleens. FoxP3 ⁺ Treg expansion was assessed in collected tissues using flow cytometry. Single cell suspensions were obtained by mechanical separation followed by filtration (70 μM cell sieve). To reduce nonspecific binding, cells were incubated with an FcyR blocking antibody (Biolegend) in FACS buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) for 15 min at ambient temperature. The samples were then washed twice in FACS buffer and stained for a panel of CD3, CD4, CD8, and CD25 cell lines and stained with a fixed live/dead cell marker for 30 min at 4°C. For Treg detection, samples were then washed twice, fixed, permeabilized, and then incubated with anti-FoxP3 antibody (FJK-16s) for 30 min at 4°C. Samples were analyzed using an LSRFortessa flow cytometer (BD Biosciences). FACS plots were analyzed using a combination of FACS DIVA and WEHI Weasel software. As shown in FIG. 9B, both antibodies, anti-CTLA-4 antibody 9D9 (mIgG2a) and the Fc variant of anti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E), reduced the number of intratumoral FoxP3 + Tregs compared with the isotype control antibody, while Fc -anti-CTLA-4 antibody variant 9D9 (mIgG2a-S239D/A330L/I332E) most significantly reduced the number of intratumoral FoxP3+ Tregs. The Fc-silenced variant of anti-CTLA-4 antibody 9D9 (mIgG2a-N297A) did not substantially reduce the number of intratumoral FoxP3+ Tregs compared with the isotype control antibody. There were no significant changes in the numbers of intratumoral CD45+ leukocytes or CD4+ non-Treg cells in either treatment group. The Fc variant of the anti-CTLA-4 antibody 9D9 (mIgG2aS239D/A330L/I332E) induced the greatest increase in the intratumoral CD8/Treg ratio over time, followed by the 9D9 antibody (mIgG2a), followed by the Fc silent variant of the 9D9 antibody (mIgG2a-N297A) and isotype control antibody (mIgG2a).

As shown in FIG. 9C, anti-CTLA-4 antibody 9D9 (mIgG2a), Fc variant of anti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E) and Fc-silent variant of anti-CTLA-4 antibody 9D9 (mIgG2a-N297A) had no significant effect on the number of FoxP3+ Tregs in tumor-draining lymph nodes (TDLN) compared with isotype control antibody. Likewise, as shown in FIG. 9D, anti-CTLA-4 antibody 9D9 (mIgG2a), Fc variant anti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E) and Fc silent variant anti-CTLA-4 antibody 9D9 (mIgG2a-N297A) had no significant effect on the numbers of splenic FoxP3+ Tregs compared with isotype control antibody.

Effect of mouse anti-CTLA-4 antibodies in combination with a tumor vaccine on tumor growth.

In this example, the effect on tumor growth of a combination of mouse anti-CTLA-4 antibodies and an antitumor HPV vaccine was tested in a mouse model of HPV+ TC-1 syngeneic tumor.

The TC-1 cell line was developed by cotransforming primary lung epithelial cells (C57BL/6) with c-Ha-ras and HPV-16 (E6/E7) oncogenes as described in Lin et al. (1996, Cancer Res. 56(1): 21 26). For tumor implantation, 2x105 TC-1 cells were injected subcutaneously into 6-8 week old female C57BL/6 mice (Jackson Laboratories). On days 5, 10, and 15 after tumor implantation, mice were injected with 100 μg of anti-CTLA-4 9D9 antibody (mIgG2a), Fc variant of anti-CTLA-4 9D9 antibody (mIgG2a-S239D/A330L/I332E), or isotype control antibody (mIgG2a) in combination with a dose of HPV vaccine (HPV ⁺ tumor, E6/E7 viral antigens) or without additional treatment. Each dose of HPV vaccine contained 30 μg of HSP protein (0.4 nM) complexed with the HPV peptide pool (1.2 nM) with the addition of 10 μg of QS-21 Stimulon® adjuvant. After treatment, tumor growth in mice was assessed once every two weeks and they were sacrificed when tumors reached a size of 2000 mm ³ or when ulcerated.

As shown in FIG. 10, there was an increase in the antitumor efficacy of both the anti-CTLA-4 antibody 9D9 (mIgG2a) and the Fc variant of the anti-CTLA-4 antibody 9D9 (mIgG2aS239D/A330L/I332E) when administered in combination with an antitumor HPV vaccine. This effect was more pronounced in the case of the Fc variant of the antibody against CTLA-4 (mIgG2aS239D/A330L/I332E). In particular, the Fc variant of the anti-CTLA-4 antibody 9D9 (mIgG2aS239D/A330L/I332E) induced a significant additional reduction in TC-1 tumor growth when used in combination with an antitumor HPV vaccine compared to the administration of this antibody as a separate agent. This additional reduction in tumor growth was greater than that observed for combinations of the 9D9 antibody (mIgG2a) or the isotype control antibody (mIgG2a) with the HPV tumor vaccine.

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Characterization of expanded and activated T cell populations.

In this example, the characteristics of expanded and activated T cell populations were determined in relation to gene expression and CpG methylation. Briefly, natural CD4 ⁺ CD25 + FOXP3 + regulatory T cells or CD4 ⁺ CD25 ^+/− FOXP3 ⁻ nonregulatory T cells were isolated from the peripheral blood of a healthy human donor, expanded, and activated. These T cells were then characterized for F0XP3 and CTLA 4 expression by flow cytometry, and lineage stability was assessed by examining DNA CpG methylation at CpG regions within the FOXP3 and CTLA4 loci. As is known in the art, hypomethylation at these CpG sites can be used to accurately distinguish between effector and regulatory T cell lineages (Waight et al., 2015, J. Immunol. 194(3): 878-882).

PBMCs were isolated on a Ficoll gradient from buffy coats from healthy donors (Research Blood Components, LLC) and then enriched for effector T cells (Teff) or natural regulatory T cells (Treg) using magnetic bead isolation (MACS, Miltenyi). Enriched Teff or Tregs were activated with CD3-CD28 microbeads (bead:cell ratio 1:1; Invitrogen) and recombinant human IL-2 for seven days in RPMI medium supplemented with 10% heat-inactivated PBS at 37°C and 5% CO ₂ . After stimulation, the expression of FOXP3 and CTLA-4 in cells was assessed using flow cytometry. To reduce nonspecific binding, cells were preincubated with an FcyR blocking antibody (Biolegend) in FACS buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) for 15 min at ambient temperature. Then the samples were washed twice in FACS buffer and stained for a panel of CD3, CD4, CD8, CD25 lines, and also stained with a fixed cell death marker for 30 min at 4°C. To assess membrane expression of CTLA-4, CTLA-4 staining was performed at 37°C. For intracellular staining of FOXP3 and CTLA-4, samples were washed twice, fixed, permeabilized, and incubated with anti-FOXP3 antibody (PCH101) and anti-CTLA-4 antibody (BNI3), respectively, for 30 min at 4°C. Samples were then washed twice and analyzed using an LSRFortessa flow cytometer (BD Biosciences). FACS plots were analyzed using a combination of FACS DIVA and WEHI Weasel software. For CpGmethylation analysis, total DNA from approximately 1x105 naïve Teff-activated or Treg-activated CD4+ T cells was isolated and subjected to pyrosequencing.

As shown in FIG. 11A, high levels of FOXP3 expression were detected on activated Tregs, as well as high levels of both intracellular and membrane expression of CTLA-4. In contrast, decreased levels of FOXP3, intracellular CTLA-4, and membrane CTLA-4 were observed on activated Teff compared to activated Tregs. In particular, essentially lower levels of membrane CTLA-4 expression were observed on activated Teff compared to activated Tregs. In fig. Figure 11B also shows that activated Tregs also showed hypomethylation of the CpG regions of FOXP3 and CTLA4 compared to untrained and activated Teff from the same donor.

Effect of anti-CTLA-4 antibodies on antibody-dependent cellular cytotoxicity of human CTLA-4-expressing T cells.

In this example, the effect of the anti-CTLA-4 antibody AGEN1884.H3 (IgG1) or its Fc variants on antibody-dependent cellular cytotoxicity (ADCC) of human CTLA-4-expressing T cells was assessed using a multiparametric microscopic caspase 3/7 activation assay to quantify ADCC activity.

Briefly, CTLA-4-expressing target cells were co-cultured with FcyRIIIA-expressing NK-92 cells after opsonization with 10 μg/ml antibody against CTLA-4 or its Fc variants as described below. In the first experiment, Jurkat cells engineered to constitutively express human surface CTLA-4 were used as target cells. CTLA-4 expressing Jurkat cells were generated by transducing the Jurkat cell line with trCTLA4 (with the intracellular domain removed) under the control of the EF1a promoter as described in Nakaseko et al. (1999, J. Exp. Med. 190(6): 765-774). In the second experiment, primary activated human effector and regulatory T cells were used as target cells. CTLA-4-expressing target cells and FcyRIIIA-158V-expressing NK-92 cells were differentially stained using red and blue live cell indicators (Thermo Fisher) and cocultured at a 1:1 cell ratio (1.5 x 103 cells/well in 384-well tablets). Samples were treated with 10 μg/ml AGEN1884.H3 (IgG1), AGEN1884.H3 (IgG1 N297A), AGEN1884.H3 (IgG1 S239D/A330L/I332E), AGEN1884.H3 (IgG1 S267E/L328F), afucosylated AGEN1884. H3 (IgG1) or isotype control antibody (IgG1). The induction of apoptosis over time in the samples was then assessed by direct confocal imaging of the caspase 3/7 substrate, which fluoresces after cleavage by the activated caspase. Images of the samples were captured every 20 minutes for six hours. The percentage of ADCC activity is measured by the number of apoptotic cells relative to the total number of cells under each condition.

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As shown in FIG. 12A, all antibodies: Fc variant antibody AGEN1884.H3 (IgG1 S239D/A330L/I332E), afucosylated antibody AGEN1884.H3, and antibody AGEN1884.H3 (IgG1) induced substantially higher ADCC activity in Jurkat cells engineered to express surface CTLA-4 compared with the AGEN1884.H3 variant (IgG1 N297A), the AGEN1884.H3 variant (IgG1 S267E/L328F) and the isotype control antibody (IgG1). The Fc variant of the AGEN1884.H3 antibody (IgG1 S239D/A330L/I332E) and the afucosylated antibody aGeN1884.H3 induced a greater increase in ADCC activity compared to the AGEN1884.H3 antibody (IgG1). As shown in FIG. 12B, Fc variant antibody AGEN1884.H3 (IgG1 S239D/A330L/I332E) induced maximum levels of ADCC in both primary activated human effector T cells (left panel) and primary activated human regulatory T cells (right panel), this was followed by the afucosylated antibody AGEN1884.H3. Antibody AGEN1884.H3 (IgG1) also induced slightly higher levels of ADCC compared to controls. The remaining antibodies tested induced little or no ADCC activity in both effector and regulatory T cells. It should be noted that both the Fc variant of the AGEN1884.H3 antibody (IgG1 S239D/A330L/I332E) and the afucosylated AGEN1884.H3 antibody induced substantially greater ADCC in regulatory T cells compared to effector T cells.

Effect of anti-CTLA-4 antibodies in combination with anti-PD-1 antibody on T cell functionality.

In this example, the effect of anti-CTLA-4 antibodies in combination with an anti-PD-1 antibody on the function of primary human T cells was examined.

Briefly, PBMCs were isolated on a Ficoll gradient from buffy platelets from two healthy human donors (Research Blood Components, LLC). This experiment was performed twice on PBMCs collected from each donor, with a total of two replicates for each donor. For each replicate, isolated PBMCs were incubated for four days under stimulating culture conditions with dose titration of anti-CTLA-4 antibody AGEN1884.H3 (IgG1), Fc variant of anti-CTLA-4 antibody aGeN1884.H3 (IgG1 S239D/A330L/I332E) or isotype control antibody (IgG1) in combination with a fixed dose (5 μg/ml) of a reference anti-PD-1 antagonist antibody or isotype control antibody (IgG ₄ ). Stimulating culture conditions were defined as suspension of cells in RPMI medium supplemented with 100 ng/ml superantigen SEA (Sigma-Aldrich), 10% heat-inactivated PBS at 37°C, and 5% CO ₂ . After incubation, IL-2 production in cell-free supernatants was analyzed using the AlphaLISA immunoassay (Perkin-Elmer). Data were collected using an EnVision® Multilabel Plate Reader (Perkin-Elmer) and IL-2 concentrations were determined using an IL-2 standard curve. Values were interpolated and plotted using Graphpad Prism software.

As shown in FIG. 13A-13D, both the anti-CTLA-4 antibody AGEN1884.H3 (IgG1) and the Fc variant of the anti-CTLA-4 antibody AGEN1884.H3 (IgG1 S239D/A330L/I332E) induced increased IL-2 production compared to isotype control or reference anti-PD-1 antibody alone. IL-2 production was further increased when AGEN1884.H3 or AGEN1884.H3 (IgG1 S239D/A330L/I332E) was combined with the reference anti-PD-1 antibody. When administered with an isotype control antibody or in combination with a reference anti-PD-1 antibody, the Fc variant of the anti-CTLA-4 antibody AGEN1884.H3 (IgG1 S239D/A330L/I332E) induced a greater increase in IL-2 production compared to AGEN1884.H3 (IgG1 ). This effect was stable across replicates for the first donor (FIGS. 13A and 13B) and the second donor (FIGS. 13C and 13D).

Example 3 Epitope mapping of anti-CTLA-4 antibody.

The interaction of the Fab fragment of AGEN1884 (AGEN1884-Fab) with the extracellular domain of human CTLA-4 was studied using hydrogen-deuterium exchange (HDX) mass spectrometry. The extracellular domain of CTLA-4, alone or in combination with AGEN1884-Fab, in phosphate buffered saline at pH 7.4, was diluted with a 10-fold volume of deuterium oxide labeling buffer and incubated for varying periods of time (0. 60, 300, 1800 and 7200 s) at room temperature. The exchange of hydrogen for deuterium was stopped by adding one volume of 4 M guanidine hydrochloride and 0.85 M TCEP (tris(2-carboxyethyl)phosphine) buffer; the final pH value was 2.5. The samples were then subjected to on-column pepsin/protease type XIII digestion and LC-MS analysis. Mass spectra were recorded in MS-only mode. To calculate deuterium incorporation, mass spectra for a given peptide were pooled within the peak of the extracted ion chromatogram and the weighted average m/z ratio was calculated. The increase in mass from the natural peptide mass (0 min) to the weighted average mass corresponds to the level of deuterium incorporation. Deuterium accumulation curves over the exchange time were plotted for all indicated peptides for further analysis and compared using HDExaminer software.

Most CTLA-4 peptides showed identical or similar levels of deuterium in the presence and absence of the anti-human CTLA-4 Fab fragment. For several peptide segments, however, a significant reduction in deuterium incorporation upon Fab binding was found. All leftovers

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Claims (12)

in this paragraph are numbered in accordance with SEQ ID NO: 33. In two regions, residues 80-82 (QVT, SEQ ID NO: 39) and residues 135-149 (YPPPYYLGIGNGTQI, SEQ ID NO: 37), significant protection from deuterium was observed upon binding of human CTLA-4 to Fab. The greatest reduction in deuterium uptake was observed at residues 140–141 (YL), which is therefore believed to be the main distinguishing characteristic of the AGEN1884 epitope on CTLA-4. When examining the sequences of human and cynomolgus CTLA-4, both of which strongly bind AGEN1884 (data not shown), there is almost complete sequence identity between the two regions described above, with the exception of a methionine substitution of leucine at position 141 (Fig. 14A). In contrast, AGEN1884 does not bind to any significant extent to either mouse CTLA-4 or rat CTLA-4 (data not shown), which differ from human CTLA-4 at residues 140-143 (YLGI, SEQ ID NO: 34) in three of four positions (Fig. 14A). Additional selectivity data indicate that AGEN1884 binds with high specificity to human and cynomolgus CTLA-4, but not to other related members of the CD28 family, including CD28, ICOS, BTLA, and PD-1 (data not shown). Sequence comparison of these related proteins shows that all non-CTLA-4 proteins differ at residues 140-143 (YLGI, SEQ ID NO: 34) (Fig. 14B), further confirming the importance of this epitope for AGEN1884 binding. The scope of the present invention is not limited to the specific embodiments described herein. Thus, various modifications of the present invention other than those described will be apparent to those skilled in the art upon examination of the above description and the accompanying drawings. It is intended that such modifications be included within the scope of the appended claims. All sources (e.g., publications or patents or patent applications) referenced herein are incorporated herein in their entirety and for all purposes by reference to the same extent as if for each individual source (e.g., publication or patent , or patent application) has been specifically and individually stated to be incorporated by reference in its entirety and for all purposes. Other embodiments are covered by the claims below. CLAIM 1. An isolated antibody that specifically binds to human CTLA-4, wherein the antibody comprises a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein: (a) CDRH1 contains the amino acid sequence of SEQ ID NO: 10; (b) CDRH2 contains the amino acid sequence SISSSSSYIYYAXSVKG (SEQ ID NO: 18), where X is E or D; (c) CDRH3 contains the amino acid sequence VGLXGPFDI (SEQ ID NO: 19), where X represents F or M; (d) CDRL1 contains the amino acid sequence of SEQ ID NO: 15; (e) CDRL2 contains the amino acid sequence of SEQ ID NO: 16 and (f) CDRL3 contains the amino acid sequence of SEQ ID NO: 17, and wherein the antibody sequences CDRH1, CDRH2 and CDRH3 are not the sequences of SEQ ID NO: 10, 11 and 13, respectively. 2. Isolated antibody according to claim 1, where: (a) CDRH2 contains the amino acid sequence of SEQ ID NO: 12; (b) CDRH3 contains the amino acid sequence of SEQ ID NO: 14 or (c) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 contain the amino acid sequences of SEQ ID NO: 10, 12, 14, 15, 16 and 17, respectively. 3. Isolated antibody according to claim 1, where the antibody contains: (a) 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 ; and/or (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20; and/or (c) 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. 4. An isolated antibody that specifically binds to human CTLA-4, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region and light chain variable region comprise the amino acid sequences of 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. - 79 044966 5. An isolated antibody that specifically binds to human CTLA-4, wherein the antibody contains: (a) a heavy chain containing the amino acid sequence of SEQ ID NO: 23, and a light chain containing the amino acid sequence of SEQ ID NO: 27; (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 24, and a light chain containing the amino acid sequence of SEQ ID NO: 27; (c) a heavy chain containing the amino acid sequence of SEQ ID NO: 25, and a light chain containing the amino acid sequence of SEQ ID NO: 27; or (d) 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. 6. An isolated antibody according to any one of claims 1 to 5, wherein the antibody contains a heavy chain constant region selected from the group consisting of human IgG1, _IgG2 , _IgG3 , IgG4, _IgA1 and _IgA2 . 7. Isolated antibody according to claim 6, where: (a) the antibody contains a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 28, 29, 30 and 31; (b) the amino acid sequence of the heavy chain constant region of IgG _i contains the S239D/I332E mutations according to the EU numbering system; (c) the amino acid sequence of the IgG1 heavy chain constant region contains mutations S239D/A330L/I332E according to the EU numbering system; (d) the amino acid sequence of the IgG1 heavy chain constant region contains the mutations L235V/F243L/R292P/Y300L/P396L according to the EU numbering system; (e) the IgG1 heavy chain constant region is an afucosylated region of IgG1 and/or (f) the N-terminal amino acid residue of the heavy chain variable region and/or light chain variable region of the antibody has been converted to pyroglutamate. 8. An isolated antibody according to any of the preceding claims, wherein the antibody contains: (a) a human IgG heavy chain constant region that is a variant of a wild-type human IgG heavy chain constant region, wherein said variant of the human IgG heavy chain constant region binds to FcyRIIIA with a higher affinity than the wild-type human IgG heavy chain constant region binds with FcyRIIIA; (b) a light chain constant region selected from the group consisting of a human kappa light chain constant region and a human lambda light chain constant region; and/or (c) a light chain constant region comprising the amino acid sequence of SEQ ID NO: 32. 9. An isolated antibody according to any one of the preceding claims, wherein the antibody: (a) is a human antibody; (b) is antagonistic to human CTLA-4; (c) inactivates, reduces or inhibits human CTLA-4 activity; (d) inhibits the binding of human CTLA-4 to human CD80 or human CD86 and/or (e) induces the production of IL-2 by peripheral blood mononuclear cells (PBMCs) stimulated with staphylococcal enterotoxin A (SEA). 10. An isolated bispecific antibody, characterized in that it contains an antibody according to any of the preceding paragraphs. 11. A conjugate comprising an isolated antibody that specifically binds to human CTLA-4, wherein the antibody comprises a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region containing the complementarity determining regions CDRL1, CDRL2 and CDRL3 , Where: (a) CDRH1 contains the amino acid sequence of SEQ ID NO: 10; (b) CDRH2 contains the amino acid sequence SISSSSSYIYYAXSVKG (SEQ ID NO: 18), where X represents E or D; (c) CDRH3 contains the amino acid sequence VGLXGPFDI (SEQ ID NO: 19), where X represents F or M; (d) CDRL1 contains the amino acid sequence of SEQ ID NO: 15; (e) CDRL2 contains the amino acid sequence of SEQ ID NO: 16 and (f) CDRL3 contains the amino acid sequence of SEQ ID NO: 17, and wherein the antibody sequences CDRH1, CDRH2 and CDRH3 are not the sequences of SEQ ID NO: 10, 11 and 13, respectively; wherein the antibody is conjugated to a cytotoxic agent, cytostatic agent, toxin, radionuclide or detectable label. 12. A pharmaceutical composition containing an antibody according to any of the preceding claims and a pharmaceutically acceptable carrier or excipient. -