EA039322B1 - Anti-ctla-4 antibodies and methods of use thereof - Google Patents

Abstract

The invention provides antibodies that specifically bind to human CTLA-4 and antagonize CTLA-4 function. Also provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject using these antibodies.

Description

Related Applications

The present application claims the priority of U.S. Provisional Applications: 62/168391, filed May 29, 2015; 62/182363, filed June 19, 2015; 62/190,653, filed July 9, 2015; 62/257202, filed November 18, 2015; 62/280263, filed January 19, 2016; 62/292500, filed February 8, 2016; 62/294558, filed February 12, 2016; 62/323226, filed April 15, 2016, each of which is incorporated herein by reference in its entirety.

1. Field of technology to which the invention belongs

The invention relates to antibodies that specifically bind to human CTLA-4, and methods for their use.

2. State of the art

T lymphocytes play a central role in the adaptive immune response against an 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, ensures the interaction of the T-cell receptor (TCR) with the MHC/peptide complex on the antigen-presenting cell (APC). The second, costimulatory signal mediates interactions between receptors on the T cell and their ligands on the antigen presenting cell (APC). TCR/MHC uptake and co-stimulatory interactions result in T cell activation via a number of intracellular pathways, including calcium-calcineurin and RAS mitogen-activated protein kinase, and subsequent activation of transcription factors for a number of effector compounds, including cytokines such as IL-2. Such events lead to the proliferation of T cells, the formation of a pool of CD4 ⁺ T helper (TH) cells, and the proliferation of activated CD8+ cytotoxic T cells. Not only is costimulation essential for full T cell activation, but its absence on TCR/MHC binding leads to anergy and/or apoptosis.

Many positive and negative costimulatory pathways are involved in T cell regulation, but the most important are the interactions between CD28 on T cells and B7-1 (CD80) and B7-2 (CD86) on APC. CD28 induces T cell differentiation into the TH1 phenotype and also increases 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 upregulated by APC simultaneously with interaction with TCR/MHC (signal 1). Expression of B7-1 on a resting cell is very low, but is usually induced after prolonged stimulation by T cells. Such differences suggest that while B7-2 may be important in initiating T cell activation, B7-1 may play a more important role in maintaining the immune response.

Upon T cell activation, cytotoxic T lymphocyte negative regulatory receptor antigen 4 (CTLA-4) is upregulated on T cells (Alegre et al., 2001, Nat. Rev. Immunol. 1:220-8). CTLA-4 is structurally homologous to CD28 but binds more strongly to ligands B7-1 and B7-2. CTLA-4 inhibits the immune response in several ways: it competes with CD28 for binding to B7 ligands and thus blocks costimulation; it gives a negative signal that inhibits T-cell activation; and it can capture CD80 and CD86 from antagonistic cells in transendocytosis resulting in impaired costimulation via CD28 (Krummel and Allison, 1995, J. Exp. Med. 182:459-465; Walunas et al., 1994, Immunity 1:405 -413; Qureshi et al., 2011, Science 332:600-603).

Given the critical role of the B7 costimulatory pathway in stimulating and maintaining an immune response, therapeutics designed to block this pathway hold promise for the treatment of autoimmune diseases and disorders.

3. The essence of the invention

Provided herein are antibodies that specifically bind to human CTLA-4 and antagonize CTLA-4 functions, such as CTLA-4 mediated immune suppression. Also provided are pharmaceutical compositions comprising such antibodies, nucleic acids encoding such antibodies, expression vectors, and host cells for producing such antibodies, and methods of treating a subject with such antibodies. The antibodies disclosed herein are particularly useful for enhancing T cell activation in response to an antigen (eg, tumor antigen) and/or reducing Treg-mediated immune suppression, and therefore for treating cancer in a subject.

Thus, in one aspect of the present description, an isolated antibody is provided, comprising a heavy chain variable region, including the complementarity determining regions of CDRH1, CDRH2, and CDRH3, and a light chain variable region, including the complementarity determining regions of CDRL1, CDRL2, and CDRL3, where:

(a) CDRH1 includes the amino acid sequence SYX1MX2 (SEQ ID NO: 22), where X ₁ is S or A; and X ₂ is N or S;

(b) CDRH2 includes the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 2);

(c) CDRH3 includes the amino acid sequence VGLMGPFXI (SEQ ID NO: 23), where X is D or N;

(d) CDRL1 includes the amino acid sequence RASQSVX1X2YLX3 (SEQ ID NO: 24),

- 1 039322 where X ₁ represents S or G; X ₂ represents R, S or T; and X ₃ is G or A;

(e) CDRL2 includes the amino acid sequence X ₁ X ₂ SX ₃ RAT (SEQ ID NO: 25), where X ₁ is G or A; X2 is A or T; and X ₃ is T, S, R or N; and (f) CDRL3 includes the amino acid sequence QQYGX1SPX2T (SEQ ID NO: 26), where X ₁ is S or T; and X2 is W or F.

In some embodiments, CDRH1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 27. In some embodiments, CDRH3 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 28. In some embodiments, implementation CDRL1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 29 and 30. In some embodiments, CDRL2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 3135. In some embodiments, CDRL3 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 36, and 37. In some embodiments, CDRH1, CDRH2, and CDRH3 include the amino acid sequences CDRH1, CDRH2, and CDRH3, respectively, shown in SEQ ID NOs: 1, 2, and 3 ; 27, 2 and 3; or 27, 2, and 28. In some embodiments, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences of CDRL1, CDRL2, and CDRL3, respectively, as shown in SEQ ID NOs: 4, 5, and 6; 29, 32 and 36; 29, 33 and 37; 30, 31 and 6; 29, 34 and 6; or 29, 35, and 37. In some embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

In another aspect of the present description, an isolated antibody is provided that specifically binds to the human CTLA-4 protein, comprising a heavy chain variable region, including the complementarity determining regions of CDRH1, CDRH2, and CDRH3, and a light chain variable region, including the complementarity determining regions of CDRL1, CDRL2, and CDRL3. , where:

(a) CDRH1 includes the amino acid sequence SYX ₁ MX ₂ (SEQ ID NO: 22), where X ₁ is S or A; and X ₂ is N or S;

(b) CDRH2 includes the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 2);

(c) CDRH3 includes the amino acid sequence VGLMGPFXI (SEQ ID NO: 23), where X is D or N;

(d) CDRL1 includes the amino acid sequence RASQSVX ₁ X ₂ YLX ₃ (SEQ ID NO: 24), where X ₁ is S or G; X ₂ represents R, S or T; and X ₃ is G or A;

(e) CDRL2 includes the amino acid sequence X ₁ X ₂ SX ₃ RAT (SEQ ID NO: 25), where X ₁ is G or A; X2 is A or T; and X3 is T, S, R or N; and (f) CDRL3 includes the amino acid sequence QQYGX ₁ SPX ₂ T (SEQ ID NO: 26), where X ₁ is S or T; and X2 is W or F.

In some embodiments, CDRH1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 27. In some embodiments, CDRH3 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 28. In some embodiments, implementation CDRL1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 29 and 30. In some embodiments, CDRL2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 3135. In some embodiments, CDRL3 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 36, and 37. In some embodiments, CDRH1, CDRH2, and CDRH3 include the amino acid sequences CDRH1, CDRH2, and CDRH3, respectively, shown in SEQ ID NOs: 1, 2, and 3; 27, 2 and 3; or 27, 2, and 28. In some embodiments, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences of CDRL1, CDRL2, and CDRL3, respectively, as shown in SEQ ID NOs: 4, 5, and 6; 29, 32 and 36; 29, 33 and 37; 30, 31 and 6; 29, 34 and 6; or 29, 35 and 37.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to the human CTLA-4 protein, comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2 and CDRL3 where CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively.

In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72. 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 or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 7 and 38-42. In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 38-42. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7. In some

- 2 039322 embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 93. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 94. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 76. In some embodiments, the antibody comprises a heavy chain, comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence derived from a human IGHV3-21 germline sequence (e.g., IGHV3-21*01, e.g. having the amino acid sequence of SEQ ID NO: 9).

In some embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 73. 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 or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 43-47. In some embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 43-47. In some embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a light chain comprising the amino acid sequence SEQ ID NO: 15. In some embodiments, the antibody comprises a light chain variable region having an amino acid sequence derived from a human IGKV320 germline sequence (e.g., IGKV3-20*01, e.g., having the amino acid sequence of SEQ ID NO: 10) or sequences human germline IGKV3-11 (eg IGKV3-11*01, eg having the amino acid sequence of SEQ ID NO: 11).

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human CTLA-4 protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 38-42.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human CTLA-4 protein, comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 43-47.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to the human CTLA-4 protein, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region, respectively, comprise the amino acid sequences set forth in SEQ ID NOs: 7 and 8; 7 and 44; 7 and 45; 38 and 8; 38 and 45; 39 and 43; 39 and 45; 39 and 46; 39 and 47; 40 and 43; 40 and 8; 40 and 44; 40 and 45; 41 and 8; 41 and 44; 41 and 45; 41 and 47; 42 and 43; or 42 and 45. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12; and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 93; and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14; and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 94; and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 76; and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 97; and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to human CTLA-4 protein, comprising a heavy chain variable region having an amino acid sequence derived from a human IGHV321 germline sequence; and a light chain variable region having an amino acid sequence derived from a human IGKV3-20 germline sequence or a human IGKV3-11 germline sequence.

- 3 039322

In another aspect, the present disclosure provides an isolated antibody that cross-competes for binding to human CTLA-4 protein with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 7 and 8, respectively.

In another aspect, the present disclosure provides an isolated antibody that binds to the same epitope on the human CTLA-4 protein as an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 7 and 8, respectively.

In another aspect of the present disclosure, an antibody is provided that binds to an epitope of human CTLA-4. In some embodiments, the antibody binds to a human CTLA-4 epitope comprising, consisting essentially of, or consisting of residues 140-141 of SEQ ID NO: 77. In some embodiments, the antibody binds to a human CTLA-4 epitope consisting of residues 140 -141 in SEQ ID NO: 77. In some embodiments, the antibody binds to at least one human CTLA-4 residue selected from the group consisting of residues 140 and 141 in SEQ ID NO: 77. In some embodiments, the antibody binds to a human CTLA-4 epitope comprising, consisting essentially of, or consisting of residues 140-143 of SEQ ID NO: 77. In some embodiments, the antibody binds to a human CTLA-4 epitope consisting of residues 140-143 of SEQ ID NO: 77. In some embodiments, the antibody binds to a human CTLA-4 epitope comprising, consisting essentially of, or consisting of residues 135-143 in SEQ ID NO: 77. In some In some embodiments, the antibody binds to a human CTLA-4 epitope consisting of residues 135-143 in SEQ ID NO: 77. In some embodiments, the antibody binds to a human CTLA-4 epitope comprising, consisting essentially of, or consisting of residues 140- 149 of SEQ ID NO: 77. In some embodiments, the antibody binds to a human CTLA-4 epitope consisting of residues 140-149 of SEQ ID NO: 77. In some embodiments, the antibody binds to a human CTLA-4 epitope comprising, consisting of essentially of or consisting of residues 135-149 of SEQ ID NO: 77. In some embodiments, the antibody binds to a human CTLA-4 epitope consisting of residues 135-149 of SEQ ID NO: 77. In some embodiments, the antibody binds to a human CTLA-4 epitope comprising, consisting essentially of, or consisting of residues 80-82 in SEQ ID NO: 77. In some embodiments, the antibody binds to the epitope human CTLA-4, consisting of residues 80-82 in SEQ ID NO: 77.

In another aspect of the present disclosure, an antibody is provided that, upon binding such antibody to a human CTLA-4 protein, followed by the addition of deuterium, exchanges hydrogen in the human CTLA-4 protein for deuterium in a region including residues 140-141 of SEQ ID NO: 77 is substantially reduced compared to the exchange of hydrogen in the human CTLA-4 protein for deuterium in the same region in the absence of antibody, as determined by hydrogen-deuterium exchange. In some embodiments, the human CTLA-4 region includes residues 140-143 of SEQ ID NO: 77. In some embodiments, the human CTLA-4 region includes residues 135-143 of SEQ ID NO: 77. In some embodiments, the human CTLA- 4 includes residues 140-149 of SEQ ID NO: 77. In some embodiments, the human CTLA-4 region includes residues 135-149 of SEQ ID NO: 77. In some embodiments, when an antibody binds to a human CTLA-4 protein, followed by by adding deuterium, the exchange of hydrogen in the human CTLA-4 protein for deuterium in the region including residues 80-82 of SEQ ID NO: 77 is substantially reduced compared to the exchange of hydrogen in the human CTLA-4 protein for deuterium in the same region in absence of antibody, as determined by hydrogen-deuterium exchange.

In some embodiments, the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG ₂ , IgG ₃ , IgG4, IgA1 and IgA ₂ . In some embodiments, the heavy chain constant region is IgG1. In some embodiments, the heavy chain constant region is IgG ₂ . In some embodiments, the antibody comprises a light chain constant region selected from the group consisting of human IgGK and IqGλ.

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 human Fcy receptors selected from the group consisting of FcyRIIB and FcyRIIA with greater than Higher affinity than the wild-type human IgG heavy chain constant region binds to human Fcy receptors. 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 human FcyRIIB with higher affinity than the wild-type human IgG heavy chain constant region. type binds to human FcyRIIB. In some embodiments, the human IgG heavy chain constant region variant is a human

- 4 039322 human IgG1, human IgG2 variant, or human IgG ₄ heavy chain constant region variant. In some embodiments, the human IgG heavy chain constant region variant comprises one or more of the following amino acid mutations according to the EU numbering system: G236D, P238D, S239D, S267E, L328F, and L328E. In some embodiments, the human IgG heavy chain constant region variant comprises a number of amino acid mutations selected from the group consisting of S267E and L328F; P238D and L328E; P238D and one or more replacements selected from the group consisting of E233D, G237D, H268D, P271G and A330R; P238D, E233D, G237D, H268D, P271G and A330R; G236D and S267E; S239D and S267E; V262E, S267E and L328F; and V264E, S267E and L328F, according to the EU numbering system. In some embodiments, the human IgG heavy chain constant region variant further comprises one or more amino acid mutations that reduce the affinity of the IgG for human FcyRIIIA, human FcyRIIA, or human FcyRI. In some embodiments, FcyRIIB is expressed on a cell selected from the group consisting of macrophages, monocytes, B cells, dendritic cells, endothelial cells, and activated T cells.

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 human Fcy receptors with higher affinity than the human IgG heavy chain constant region. wild type binds to human Fcy receptors. In some embodiments, the human Fcy receptor comprises an immunoreceptor tyrosine activating motif (ITAM). In some embodiments, the human Fcy receptor is selected from the group consisting of FcyRIIIA, FcyRIIA, and FcyRI. In some embodiments, the human Fcy receptor is FcyRIIIA. In some embodiments, the human IgG heavy chain constant region variant comprises one or more of the following amino acid mutations, according to the EU numbering system: G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T and P396L. In some embodiments, the human IgG heavy chain constant region variant comprises a number of amino acid mutations selected from the group consisting of: S239D; T256A; K290A; S298A; I332E; E333A; K334A; A339T; S239D and I332E; S239D, A330L and I332E; S298A, E333A and K334A; G236A, S239D and I332E; and F243L, R292P, Y300L, V305I and P396L according to the EU numbering system. In some embodiments, the human IgG heavy chain constant region variant includes the following amino acid mutations according to the EU numbering system: S239D, A330L, and I332E. In some embodiments, the human IgG heavy chain constant region variant includes the following amino acid mutations, according to the EU numbering system: S239D and I332E. In some embodiments, the human IgG heavy chain constant region variant is a human IgG1 heavy chain constant region variant comprising the following amino acid mutations according to the EU numbering system: S239D and I332E. In some embodiments, the antibody comprises an afucosylated Fc region. In some embodiments, the antibody is formulated for intratumoral delivery. In some embodiments, the antibody or pharmaceutical composition is administered intratumorally. In some embodiments, the antibody is formulated for delivery to a tumor-draining lymph node. In some embodiments, the antibody or pharmaceutical composition is delivered to the tumor-draining lymph node. In some embodiments, the antibody is formulated for subcutaneous delivery. In some embodiments, the antibody or pharmaceutical composition is delivered subcutaneously.

In some embodiments, the human Fcy receptor is FcyRIIc. In some embodiments, the human Fcy receptor is expressed on a cell selected from the group consisting of macrophages, monocytes, B cells, and dendritic cells. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is CTLA-4 antagonistic. In some embodiments, the antibody inhibits the binding of human CTLA-4 protein to human CD80 or to human CD86.

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

In one embodiment, the present invention relates to an antibody of the present invention for use as a drug.

In one embodiment, the present invention relates to the use of an antibody of the present invention for the manufacture of pharmaceutical compositions or medicaments for immunotherapy. Preferably, the immunotherapy is designed to increase T cell activation in response to an antigen in a subject, optionally for the treatment of cancer, or the treatment or prevention of infectious diseases.

In one embodiment, the present invention relates to an antibody according to the present invention.

- 5 039322 to the invention for use as a diagnostic tool.

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

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

In another aspect of the present description, an isolated polynucleotide is provided that encodes the heavy and/or light chain of an antibody disclosed in this application. In another aspect of the present description, a vector is provided that includes a polynucleotide. In another aspect of the present description, a recombinant host cell is provided, comprising a polynucleotide or a vector. In another aspect, the present disclosure provides a method for producing an antibody that binds to human CTLA-4, wherein the method comprises culturing a host cell such that a polynucleotide is expressed and producing the antibody.

In another aspect, the present disclosure provides a method of increasing T cell activation in response to an antigen in a subject, wherein the method comprises administering to the subject an effective amount of an anti-CTLA-4 antibody or pharmaceutical composition disclosed herein. In another aspect, the present disclosure provides a method of treating cancer in a subject, wherein the method comprises administering to the subject an effective amount of an anti-CTLA-4 antibody or pharmaceutical composition disclosed herein. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is administered subcutaneously or intravenously. In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is administered intravenously at 0.1, 0.3, 1, 3, 6, or 10 mg/kg, optionally at three week intervals. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is administered intratumorally. In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is administered intratumorally at 0.01, 0.03, 0.1, 0.3, 1, or 3 mg/kg, optionally at intervals of three weeks. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is administered intratumorally at 0.03, 0.1, or 0.3 mg/kg, optionally at three week intervals. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is administered intratumorally at a dose that is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 200 times lower than than the dose administered systemically. In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is administered intratumorally at a dose that is 10 times lower than the systemic dose. In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is administered intratumorally at a dose that is 100 times lower than the systemic dose. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is administered intratumorally, the method further comprising administering an additional therapeutic agent to the subject. In some embodiments, the 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-PD-1 antibody is pembrolizumab or nivolumab. In some embodiments, the subject has head and neck squamous cell carcinoma and the additional therapeutic agent is an anti-EGFR antibody. In some embodiments, the anti-EGFR 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, such 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 disclosed 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 disclosed herein is administered intratumorally and the subject has head and neck cancer (eg, relapsed/refractory head and neck squamous cell carcinoma). In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition disclosed 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 disclosed herein is delivered to a tumor-draining lymph node. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is delivered via localized administration (eg, subcutaneous administration). In some embodiments,

0.01, 0.03, 0.1, 0.3, 1, or 3 mg of an anti-CTLA-4 antibody or pharmaceutical composition disclosed in this application is delivered via localized administration (eg, subcutaneous administration) /kg. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is delivered via localized administration (e.g., subcutaneous administration) at a dose that is 5, 10, 20, 30, 40, 50, 60, 70, 80 , 90, 100 or 200 times lower than the dose administered with systemic administration. In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is delivered via localized administration (eg, subcutaneous administration) at a dose that is 10 times lower than the dose administered systemically. In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is delivered via localized administration (eg, subcutaneous administration) at a dose that is 100 times lower than the dose administered systemically. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is delivered via localized administration (eg, subcutaneous administration) and the method further comprises administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent is a vaccine. In some embodiments, the vaccine comprises a heat shock protein complex (HSPPC) comprising a heat shock protein in combination with an antigenic peptide. In one embodiment, the heat shock protein is a gp96 protein and is complexed with a tumor-associated antigenic peptide, wherein the HSPPC is derived from a tumor obtained from the subject. In some embodiments, the heat shock protein is selected from the group consisting of hsc70, hsp70, hsp90, hsp110, grp170, gp96, calreticulin, a mutant thereof, and a combination of two or more of them. 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 has an infectious disease. In some embodiments, such 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, an anti-LAG-3 antagonist antibody, an anti-CEACAM1 antagonist antibody, an anti-GITR agonist antibody, an anti-OX40 agonist antibody, and an anti-CD137 agonist antibody, an anti-DR3 agonist antibody, an anti-TNSF14 agonist antibody, and an anti-CD27 agonist antibody. In some embodiments, the additional therapeutic agent is radiation therapy. In some embodiments, the additional therapeutic agent is an indolamine-2,3-dioxygenase (IDO) inhibitor. Suitable IDO inhibitors include, without limitation, epacadostat, F001287, indoxymod, and NLG919. In some embodiments, the additional therapeutic agent is a vaccine. In some embodiments, the vaccine comprises a heat shock protein complex (HSPPC) comprising a heat shock protein in combination with an antigenic peptide. In one embodiment, the heat shock protein is a gp96 protein and is complexed with a tumor-associated antigenic peptide, wherein the HSPPC is derived from a tumor obtained from the subject.

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

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

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

In one aspect, the present application provides a pharmaceutical composition comprising an anti-CTLA-4 antibody disclosed herein and a pharmaceutically acceptable carrier or excipient for use as a medicament.

In one aspect, the present application provides a pharmaceutical composition comprising an anti-CTLA-4 antibody disclosed herein and a pharmaceutically acceptable carrier or excipient for use as a diagnostic agent.

In one aspect, the present application provides a pharmaceutical composition comprising an anti-CTLA-4 antibody disclosed herein, a polynucleotide of the invention, a vector

- 7 039322 according to the invention and/or a recombinant host cell according to the invention, and a pharmaceutically acceptable carrier or excipient. In one aspect, the pharmaceutical composition is for use as a drug 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 for 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 for 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 a pharmaceutical composition of the present invention, for use in a method for increasing T cell activation in response to an antigen in a subject, comprising administering to the subject an effective amount of the antibody, polynucleotide , a vector, a recombinant host cell and/or a pharmaceutical composition according to the invention.

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.

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 in a subject.

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

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

In one aspect, the present invention relates to a pharmaceutical composition, kit or set of components comprising: (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 of the present invention; and (b) an anti-EGFR antibody, and optionally (c) a chemotherapeutic agent, for use as a drug.

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

In one aspect, the present invention relates to a pharmaceutical composition, kit or set of components, comprising: (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention and (b) an anti-EGFR antibody, and optionally (c ) chemotherapeutic agent.

In one aspect, the present invention provides: (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention, and (b) an anti-HER2 antibody, and optionally (c) a chemotherapeutic agent, for use in as a medicinal product.

- 8 039322

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

In one aspect, the present invention relates to a pharmaceutical composition, kit or set of components, comprising: (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 ) 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, where the antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention administered intratumorally to the subject, preferably administered intratumorally to the subject in an amount of 0.01, 0.03, 0.1, 0.3, 1 or 3 mg/kg, optionally at intervals of three weeks.

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, where the antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention administered subcutaneously or intravenously to a subject, preferably administered intravenously to a subject in an amount of 0.1, 0.3, 1, 3, 6 or 10 mg/kg, optionally at intervals of three weeks.

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 medicine. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic or checkpoint targeting agent, or an indolamine-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, the additional therapeutic agent is a chemotherapeutic or checkpoint targeting agent, or an indolamine-2,3-dioxygenase (IDO) inhibitor, or a vaccine.

In one aspect, the present invention relates to a pharmaceutical composition, kit or set of components, including: (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, the additional therapeutic agent is a chemotherapeutic or checkpoint targeting agent, or an indolamine-2,3-dioxygenase (IDO) inhibitor, or a vaccine.

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

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

In one aspect, the present invention relates to the use of an antibody, a polynucleotide, a vector, a recombinant host cell, and/or a pharmaceutical composition according to the present invention for the preparation of immunotherapy medicaments, for example, to increase the active

- 9 039322 T-cells in response to an antigen in a subject, cancer treatment, or treatment or prevention of infectious diseases, wherein the 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: (a) an antibody, a polynucleotide, a vector, a recombinant host cell and/or a pharmaceutical composition according to the present invention, and (b) an anti-HER2 antibody, and optionally (c) a chemotherapeutic agent for the preparation of a drug. means for immunotherapy, for example, to increase the activation of T cells in response to an antigen in a subject, the treatment of cancer, or the treatment or prevention of infectious diseases.

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

4. Brief description of the drawings

Fig. 1A is a table showing the binding affinities (K _D ) of anti-CTLA-4 antibody AGEN1884 and reference anti-CTLA-4 IgG1 antibody to dimeric and monomeric recombinant human, cynomolgus, mouse and rat recombinant CTLA-4 as measured by surface plasmon resonance. Fig. 1B is a graph showing the binding of AGEN1884w, an anti-CTLA-4 IgG1 reference antibody and an IgG1 isotype control, to CTLA-4 expressing cells as measured by flow cytometry. The mean fluorescence intensity (MFI) was calculated and plotted against a range of antibody concentrations. In FIG. 1C and 1D show the selectivity of AGEN1884 and a reference anti-CTLA-4 IgG1 antibody for binding to recombinant human (rh) and cynomolgus monkey (rc) CTLA-4 relative to family members (recombinant human CD28, ICOS, BTLA and PD-1 and recombinant cynomolgus monkey PD-1) shown in comparison with an isotype control IgG1 antibody. Binding was measured using suspension chip technology and the mean fluorescence intensity (MFI) at three different antibody concentrations (10, 100, and 1000 ng/mL) is shown in FIG. 1C. Fig. 1D is a summary table of relative binding affinity based on the data shown in FIG. 1C;

in fig. 2A shows the binding of AGEN1884w, a reference anti-CTLA-4 antibody, and an isotype control IgG1 antibody to activated primary human CD4+ T cells. Briefly, newly isolated CD4+ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for five days prior to analysis of antibody binding by flow cytometry. The mean fluorescence intensity (MFI) was calculated over a range of antibody concentrations. In FIG. 2B shows intracellular binding of AGEN1884 and a reference anti-CTLA-4 antibody compared to an isotype control IgG1 antibody when concentration titrated using activated (anti-CD3 and anti-CD28 with recombinant IL-2 for three days) primary cynomolgus monkey CD8+ T cells. . Data were obtained using flow cytometry and are shown as the percentage of CTLA-4+ T cells from the total population of CD8+ T cells. In FIG. 2C shows the results of a flow cytometry analysis that examined the binding of AGEN1884w and an isotype control IgG1 antibody to phytohemagglutinin-stimulated human, cynomolgus, rat, or mouse CD4+ T cells;

fig. 3A is a diagram of a ligand blocking assay using suspension chip technology. In FIG. 3B and 3C show the percentage of recombinant CD80-Fc (FIG. 3B) and CD86-Fc (FIG. 3C) proteins binding to CTLA-4 conjugated spheres in the absence (100%) or in the presence of a titratable dose of AGEN1884, a reference anti-CTLA IgG1 antibody. -4 or isotype control (IgG1) antibody. In FIG. 3D shows the percentage of recombinant CD80-Fc and CD86-Fc binding to CTLA-4 expressing T cells (Jurkat) in the presence of increasing concentrations of AGEN1884w, reference anti-CTLA-4 IgG1 antibody, or isotype control (IgG1) antibody. In FIG. 3E shows the results of an assay in which CTLA-4 expressing T cells (Jurkat) were incubated with a titratable dose of CD80-Fc or CD86-Fc prior to the addition of a fixed concentration of fluorochrome-directly conjugated AGEN1884w or a reference anti-CTLA-4 IgG1 antibody. The mean fluorescence intensity (MFI) was calculated and plotted against the CD80-Fc or CD86-Fc concentration range;

in fig. 4A shows the functional activity of anti-CTLA-4 antibodies against cultures of primary human PBMCs when stimulated with staphylococcal enterotoxin A (SEA). In particular, in FIG. 4A shows the fold change in IL-2 production in response to a dose-dependent effect of AGEN1884w and a reference anti-CTLA-4 IgG1 antibody compared to an isotype control IgG1. Error bars indicate the standard deviation for wells in triplicate. Fig. 4B and 4C before

- 10 039322 are the results of the analysis of IL-2 with reporter luciferase, which show that the blockade of CTLA-4 leads to the activation of T cells. A line of co-expressing human CD3 and CD28 T cells (Jurkat), genetically engineered to constitutively express CTLA-4 on the cell surface in addition to the luciferase reporter gene under the control of the IL-2 promoter, was co-cultured with an antigen-presenting cell line (Raji) that expressed CD80 and CD86. Fig. 4B is a pair of histograms showing CD80 and CD86 expression on Raji cells as measured by flow cytometry. TCR signaling was initiated using an anti-CD3 antibody in the presence of increasing concentrations of AGEN1884w or an isotype control IgG1 antibody. In FIG. 4C shows a fold increase in the expression of luciferase, a surrogate marker for IL-2 gene activation, in response to elevated concentrations of AGEN1884w compared to an isotype control antibody. Fig. 4D and 4E are the results of an assay examining the ability of the anti-CTLA-4 antibody, AGEN1884w, to reduce IFN-y production by primary CD8+ T cells in the presence of high concentrations of anti-CD3 antibody. Primary human T cells were stimulated with anti-CD3 antibody (10 μg/ml) in the presence of increasing concentrations of soluble (FIG. 4D) or plate bound (FIG. 4E) AGEN1884w or isotype control IgG1 antibody. The percentage of CD8+ IFNy+ T cells was plotted against antibody concentrations tested. The dotted line indicates the frequency of CD8+ IFNy+ T cells in the absence of stimulation with anti-CD3 antibody. Fig. 4F is a graph showing the functional activity of anti-CTLA-4 antibody, AGEN1884w, alone or in combination with anti-LAG-3 antibody, AGEN1746, anti-PD-1 antibody, nivolumab, or anti-PD-1 antibody, pembrolizumab, on cultures. primary human PBMCs stimulated with staphylococcal enterotoxin A (SEA). The mean and standard deviation of IL-2 concentration are shown. IC stands for isotype control;

in fig. 5A - Binding affinities (K _D ) of AGEN2041w, a reference anti-CTLA4 IgG1 antibody, and a reference anti-CTLA-4 IgG2 antibody to human and cynomolgus monkey recombinant CTLA-4 dimeric and monomeric as measured by surface plasmon resonance. Fig. 5B is a graph showing the binding of AGEN2041w, a reference anti-CTLA-4 IgG ₂ antibody, and an isotype control IgG ₂ to CTLA-4 expressing cells as measured in a flow cytometry assay. The mean fluorescence intensity (MFI) was calculated and plotted against various antibody concentrations tested. Fig. 5C is a graph showing the percentage of CD80 and CD86 binding to CTLA-4 in the presence of increasing concentrations of AGEN2041w or reference anti-CTLA-4 IgG ₂ antibody (12, 37, 111, 333, 1000, 3000 and 9000 ng/mL before adding spheres) when measured with suspension chip technology. Fig. 5D and 5E are tables that are similar to those shown in FIG. 1C and 1D, respectively, where the selectivity of AGEN2041w and a reference anti-CTLA-4 IgG ₂ antibody for binding to recombinant human (rh) and cynomolgus monkey (rc) CTLA-4, compared to related family members (recombinant human CD28, ICOS, BTLA, and PD-1; and recombinant cynomolgus monkey PD-1) were tested against an isotype control IgG2 antibody. In FIG. 5D shows MFI values as measured by suspension chip technology, and FIG. 5E is a summary table based on the data shown in FIG. 5D;

in fig. 6A shows the functional activity of anti-CTLA-4 antibodies against cultures of primary human PBMCs upon stimulation with staphylococcal enterotoxin A (SEA). In FIG. 6A shows the fold change in IL-2 production in response to a dose-dependent effect of AGEN1884w and AGEN2041w compared to isotype control IgG1 and IgG ₂ antibodies. Error bars indicate the standard deviation for wells in triplicate. Fig. 6B is a graph showing the results of a luciferase reporter IL-2 assay that tested AGEN2041w and an isotype control IgG ₂ antibody. A fold increase in the expression of luciferase, a surrogate marker for IL-2 gene activation, has been shown for elevated doses of AGEN2041w and an isotype control antibody;

fig. 7A is a graph showing the binding of AGEN1884w-105 (AGEN1884w produced by stable clone 105), a reference anti-CTLA-4 IgG1 antibody, and an isotype control IgG1 to CTLA-4 expressing cells as measured in a flow cytometry assay. The mean fluorescence intensity (MFI) was calculated and plotted against the titratable antibody. Fig. 7B is the result of an assay that examined IL-2 production increased by AGEN1884w-105, an anti-CTLA-4 reference IgG1 antibody, and an isotype control IgG1 after staphylococcal enterotoxin A (SEA) stimulation. In FIG. 7B shows six biological replicates and means (whiskers) of IL-2 fold change;

fig. 8A, 8B, and 8C are antibody dose titration binding curves with CHO cells expressing FcyRIIAH131 (FIG. 8A), FcyRIIB (FIG. 8B), and FcyRIIIAV158 (FIG. 8C), respectively. The curves show the mean fluorescence intensity (MFI) of the secondary detection antibody. Error bars indicate the standard deviation at triplicate. Testiro

- 11 039322 Valid the following antibodies: AGEN1884w-FortiCHO, AGEN1884w-PowerCHO, AGEN2041w, reference IgG1 antibody against CTLA-4 and isotype control IgG1 antibody;

fig. 9A and 9B are graphs showing the mean response (response units, RU) of triplicate samples over a range of AGEN1884w-FortiCHO, AGEN1884w-PowerCHO, AGEN2041w antibodies and a reference anti-CTLA-4 IgG1 antibody associated with human FcyRIIA (Fig. 9A) or human FcyRIIIA (FIG. 9B) as determined by surface plasmon resonance;

fig. 10A is a schematic of the Fcy IIIA receptor (FcyRIIIA) reporter gene assay. Fig. 10B10G are the results of a reporter assay in which the ability of AGEN1884W-105 or AGEN1884w (AGEN1884w-105, in FIGS. 10B, 10C, 10f, and 10G; AGEN1884w, in FIGS. 10D and 10E) to activate a reporter cell line expressing either variant FcyRIIIA V158 or FcyRIIIA F158 variant was compared with that of the reference IgG1 antibody (FIGS. 10B and 10C) against CTLA-4, AGEN2041w (FIGS. 10D and 10E) and AGEN1884w N297A variant (FIGS. 10F and 10G). Relative light units (RLU) are plotted against a range of antibody concentrations;

fig. 11 is the result of a reporter assay where AGEN2041w, an anti-CTLA-4 IgG ₂ reference antibody, and an IgG ₂ isotype control antibody were tested for the ability to activate a reporter cell line expressing the FcyRIIA H131 variant when antibodies bind to CTLA-4 expressing target cells. . Relative light units (RLU) are plotted against a range of antibody concentrations;

fig. 12A is a graph showing the binding of AGEN1884w-105, AGEN1884w-N297A variant and isotype control IgG1 to CTLA-4 expressing cells as measured in flow cytometry analysis. The mean fluorescence intensity (MFI) was calculated and plotted against various antibody concentrations tested. Fig. 12B is the result of an assay in which IL-2 production induced by AGEN1884w-105, AGEN1884w-N297A and isotype control IgG1 after staphylococcal enterotoxin A (SEA) stimulation was examined. In FIG. 12B shows six biological replicates and means (whiskers) of IL-2 fold change;

fig. 13A and 13B are the results of an analysis that examined the effect of blocking FcR binding on the antagonistic activity of anti-CTLA-4 antibodies. In FIG. 13A shows seven biological replicates and means (whiskers) of IL-2 fold change. In FIG. 13B shows seven biological replicates of IL-2 production (pg/ml);

fig. 14 is a graph showing IL-2 production by human PBMC when stimulated with staphylococcal enterotoxin A (SEA) in response to 10 μg/ml AGEN1884w,

AGEN1884w-N297A, AGEN1884w-S267E/L328F, AGEN1884wS239D/A330L/I332E, AGEN2041w, isotype control IgG1 antibody, or isotype control IgG ₂ antibody. Mean values (whiskers) of IL-2 production are shown for each antibody tested;

fig. 15 is a graph showing percent lysis of target cells in the presence of effector cells along with dose titration of AGEN1884w, reference anti-CTLA-4 IgG1 antibody, or isotype control antibody. Jurkat cells genetically engineered to constitutively express CTLA-4 on the cell surface were used as target cells, and NK92-FcyRIIIA cells were used as effector cells;

in fig. 16 compares the binding affinities (K _D ) of CD80-Fc-His, CD86-Fc-His, anti-CTLA-4 reference IgG1 antibody, and AGEN1884 for CTLA-4. Walker and Sansom Nat. Rev. Immunology 2011 corresponds to Walker and Sansom Nat. Rev. Immunol. (2011) 11(12):852-63. Xu et al., JI 2012 corresponds to Xu et al., J. Immunol. (2012) 189(9):4470-7;

fig. 17A is a sequence alignment of human CTLA-4 (SEQ ID NO: 77), cynomolgus monkey CTLA-4 (SEQ ID NO: 84), mouse CTLA-4 (SEQ ID NO: 85), and rat CTLA-4 (SEQ ID NO: : 86). The dots represent remnants identical to the corresponding human remnants. The * (asterisk) sign indicates positions that contain one completely conservative residue. The sign : (colon) indicates conservatism between groups with strong similarity. Sign . (dot) indicates conservatism between groups with little similarity. Fig. 17B and 17C are sequence alignments of human CTLA-4 (SEQ ID NO: 77), cynomolgus monkey CTLA-4 (SEQ ID NO: 84), human CD28 (SEQ ID NO: 87), human ICOS (SEQ ID NO: 88 ), human BTLA (SEQ ID NO: 89) and human PD-1 (SEQ ID NO: 90). Two areas showing a strong decrease in deuterium uptake upon binding of human CTLA-4 to AGEN1884w-Fab were underlined in FIG. 17A-C: Q ⁸⁰ VT ⁸² and Y ¹³⁵ PPPYYLGIGNGTQI ¹⁴⁹ . Fig. 17D is a layout of X-ray coordinates in files 3osk and 1i8l of the Protein Structure Bank. This combined structural analysis indicates a daisy chain clustering of CTLA-4 and B7-1 clusters. This gives a model of the human B7-1 dimer together with the human CTLA-4 dimer, which is thought to be the simplest form of the oligomeric signaling competent immune com.

- 12 039322 plex. The contact area between CTLA-4 and B7-1 includes the M ¹³⁴ YPPPYY ¹⁴⁰ region, but is not limited to this particular motif. Fig. 17E is a model of AGEN1884w-Fab binding to human CTLA-4. All of the CTLA-4 residues mentioned in the previous description of FIG. 17A-17E are numbered according to SEQ ID NO: 77;

fig. 18A and 18B are results of a T-cell dependent antigen response (TDAR) study in cynomolgus monkeys. AGEN1884w (10 mg/kg), anti-CTLA-4 IgG1 reference antibody (3 mg/kg) (Fig. 18A), AGEN2041w (10 mg/kg) (Fig. 18B), or control product (Figs. 18A and 18B) were administered intravenously (slow bolus) along with HBsAg vaccines (30 μg per hind limb) on Days 1 and 29. Samples in duplicate were analyzed for serum anti-hepatitis B virus (IgG) antibody titers by ELISA. Data points represent means±SEM in each control group;

fig. 19A and 19B are variable heavy chain sequence alignments BADD411-2354 (SEQ ID NO: 7), BADD412-2356 (SEQ ID NO: 38), BADD412-2357 (SEQ ID NO: 39), BADD412-2358 (SEQ ID NO: 39). : 40), BADD412-2359 (SEQ ID NO: 41) and BADD412-2360 (SEQ ID NO: 42) and variable light chains BADD412-2367 (SEQ ID NO: 43), BADD412-2376 (SEQ ID NO: 8) , BADD4122382 (SEQ ID NO: 44), BADD412-2384 (SEQ ID NO: 45), BADD412-2390 (SEQ ID NO: 46), and BADD4122393 (SEQ ID NO: 47), respectively. The heavy and light chain CDR regions shown are defined according to Kabat;

fig. 20A-20E are graphs showing the percentage of recombinant CD80-PE or CD86-PE binding to CTLA-4 conjugated spheres in the presence of titratable doses of 19 anti-CTLA-4 antibodies (AGEN1884-AGEN1902) as measured by suspension chip technology. .

5. Detailed description

Provided herein are antibodies that specifically bind to human CTLA-4 and antagonize CTLA-4 function, such as CTLA-4 mediated immune suppression. Also provided are pharmaceutical compositions comprising such antibodies, nucleic acids encoding such antibodies, expression vectors, and host cells for producing such antibodies, as well as methods of treating a subject with such antibodies. The antibodies disclosed herein are particularly useful for increasing T cell activation in response to an antigen (eg, tumor antigen) and hence for treating cancer or preventing or treating infectious diseases in a subject. The antibodies disclosed in this application can also be used in pharmaceutical compositions, or in the manufacture of drugs, for the treatment of cancer or the prevention or treatment of infectious diseases.

5.1. Definitions.

As used herein, the terms approximately, and approximately when used in defining a numerical value or numerical range, mean that deviations of 5 to 10% above and 5 to 10% below the value or range remain within the implied definition of the indicated value or range.

As used herein, the term CTLA-4 refers to the cytotoxic T lymphocyte protein 4. The nucleotide and polypeptide sequences of CTLA-4 are known in the art. An exemplary human CTLA-4 amino acid sequence is listed on GenBank as GI: 15778585 and an exemplary murine CTLA-4 amino acid sequence is listed on GenBank as GI: 15778586.

As used herein, the terms antibody and antibodies include full-length antibodies, antigen-binding fragments of antibodies, and molecules comprising 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 comprising two heavy chain molecules and two light chain molecules, monomer antibody light chain, antibody heavy chain monomer, antibody light chain dimer, antibody heavy chain dimer, antibody heavy chain antibody light chain pair, intrabodies, antibody heteroconjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv fragments (scFv), camelized antibodies, affibodies, Fab fragments, F(ab') ₂ fragments, disulfide-linked Fv fragments (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, antibodies against anti-Id antibodies), and antigen-binding fragments of any from those listed. In some embodiments, the implementation of the antibodies described in this application, refer to populations of polyclonal antibodies. 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 IgA2) or any subclass (eg IgG2a or IgG2b) immunoglobulin molecules. In some embodiments, the implementation of the antibodies described in this application are IgG antibodies or their class (eg, human IgG1 or IgG ₄ ) or subclass. In a specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody. In some embodiments, the implementation of the antibody described in this application is an IgG1 or IgG2 antibody.

- 13 039322

As used herein, the terms VH region and VL region refer to single antibody heavy and light chain variable regions, respectively, comprising FRs (framework regions) 1, 2, 3, and 4 and CDRs (complementarity determining regions) 1, 2, and 3 (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest. (NIH Publication No. 91-3242, Bethesda).

As used herein, the term CDR or complementarity determining region refers to non-continuous antigen-binding regions present in the variable region of heavy and light chain polypeptides. These defined regions 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), as well as in Chothia et al., J. Mol. Biol. 196:901-917 (1987) and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping amino acid residues or subsets of amino acid residues when compared with each other. Amino acid residues that are included in the CDR region, as defined in each of the above cited sources, are presented for comparison. Preferably, the CDR term is a CDR region determined according to Kabat based on sequence comparisons. CDRH1, CDRH2 and CDRH3 are heavy chain CDR regions, and CDRL1, CDRL2 and CDRL3 are light chain CDR regions.

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

As used herein, the term framework (FR) amino acid residues refers to such amino acids in the framework region of an immunoglobulin chain. The term framework regions or FR region as used herein includes amino acid residues that are part of the variable region but not part of the CDR region (eg, using the Kabat definition of CDR regions).

As used herein, the term afucosylation or afucosylation in relation to Fc refers essentially to the absence of fucose covalently attached, directly or indirectly, to residue 297 of the Fc region of human IgG1, when numbered according to the EU index (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), or to the corresponding residue in non-IgG1 or non-human IgG1 immunoglobulins. Thus, in a composition comprising a plurality of afucosylated antibodies, at least 70% of the antibodies will not be fucosylated, either directly or indirectly (e.g., through sugar intermediates), at the 297 residue of the antibody Fc region, and, in some embodiments, at least 80%, 85%, 90%, 95%, or 99% will not be fucosylated, directly or indirectly, at the 297 Fc region residue.

As used herein, the term specifically binds to refers to the ability of an antibody to bind to an antigen with a dissociation constant (Kd) of at least about 1x10'6M, ^1x10'7M , ^1x10'8M , ^1x10'9M , ^1x10'10M . , 1x10 ^-11 M, 1x10' ¹² M or less, and/or bind to the antigen with an affinity that is at least twice its affinity for a non-specific antigen.

As used herein, epitope is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or continuous epitope), or an epitope can be, for example, formed by joining two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous or discontinuous epitope). In some embodiments, the epitope to which the antibody binds can be determined, for example, using NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen-deuterium exchange in combination with mass spectrometry (for example, liquid chromatography/mass spectrometry with electrospray), microarray-based oligopeptide scanning and/or mutagenesis mapping (eg, site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be carried out using any of the methods known in the art (for example, Giege R. et al. (1994) Acta Crystallogr. D Biol. Crystallogr. 50(Pt 4): 339-350; McPherson A. (1990) Eur J Biochem 189: 123; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody:antigen crystals can be examined using known X-ray diffraction techniques and can be refined using a program such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Meth. Enzymol. (1985), issues 114 and 115, eds Wyckoff H.W. et al.; U.S. 2004/0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr. D Biol. Crystallogr. 49(Pt 1): 37-60; Bricogne G. (1997) Meth Enzymol 276A: 361-423, ed Carter C.W.; Roversi P. et al (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323). Mutagenesis mapping studies can be performed using any method known to one of skill in the art. See, for example, Champe M. et al. (1995) Biol. Chem. 270 J: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085 for describing methods of mutagenesis, including methods of mutagenesis using

- 14 039322 alanine scan. In a specific embodiment, an antibody epitope or antigen-binding fragment is determined using alanine scanning mutagenesis assays.

As used herein, the terms treat and treatment refer to the therapeutic or prophylactic procedures described herein. Methods of treatment employ administering an antibody to a subject having a disease or disorder or predisposed to developing such disease or disorder in order to prevent, treat, delay, reduce the severity or alleviate one or more symptoms of the disease or disorder, or recurrence of the disease or disorder, or to increase survival of the subject beyond that expected in the absence of such treatment.

When used in the present description, the term effective amount in relation to the use of therapy in a subject refers to the amount of therapy that achieves the desired prophylactic or therapeutic effect.

As used herein, the term subject includes any human or non-human animal.

Hydrogen-deuterium exchange is assessed using an assay involving the following steps: (a) taking a solution of CTLA-4 alone or CTLA-4 in combination with antibody in aqueous buffer and diluting this solution in approximately 90% volume/excess volume of deuterium oxide labeling buffer , for, for example, 0, 60, 300, 1800 and 7200 s; (b) stopping the exchange of hydrogen for deuterium by lowering the pH; (c) treating the samples with protease digestion and performing a mass spectrometric analysis; and (d) calculating deuterium incorporation in CTLA-4 proteolysis peptides and comparing deuterium incorporation in the presence or absence of antibody. Hydrogen-deuterium exchange in the CTLA-4 proteolysis peptide is reduced if hydrogen-deuterium exchange in the presence of an anti-CTLA-4 antibody is reduced by at least 10% compared to hydrogen-deuterium exchange in the absence of antibody, as assessed in a previous assay.

5.2. Antibodies against CTLA-4.

In one aspect, the present disclosure provides antibodies that specifically bind to human CTLA-4 and antagonize CTLA-4 functions. Amino acid sequences of exemplary antibodies are presented in table. 1-6 below.

- 15 039322

Table 1. Sequences of variable regions, CDR regions and framework regions (FR) of exemplary anti-CTLA-4 antibodies

SEQID NO: Description Amino acid sequence one Kabat CDRH1 (AGEN1884w) SYSMN 2 Kabat CDRH2 (AGEN1884w) SISSSSSYIYYADSVKG 3 Kabat CDRH3 (AGEN1884w) VGLMGPFDI four Kabat CDRL1 (AGEN1884w) RASQSVSRYLG 5 Kabat CDRL2 (AGEN1884w) GASTRAT 6 Kabat CDRL3 (AGEN1884w) QQYGSSPWT 7 VH (AGEN1884w) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSS eight VL (AGEN1884w) EIVLTQSPGTLSLSPGERATLSCRASQSVSRYLGWYQQKPGQAPR LLIYGASTRATGIPDRFSGSGSGTDFTLTITRLEPEDFAVYYCQQ YGSSPWTFGQGTKVEIK 9 Germline sequence: IGHV3-21*01 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCAR ten Germline sequence: IGKV3-20*01 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAP RLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ QYGSSP eleven Germline sequence: IGKV3-ll*01 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPR LLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQ RSNWPP 12 AGEN1884w full length heavy chain (with C-terminated lysine) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 93 AGEN1884W full length heavy chain (without C-terminal lysine) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

- 16 039322

13 AGEN1884w full length light chain sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSRYLGWYQQKPGQAPR LLIYGASTRATGIPDRFSGSGSGTDFTLTITRLEPEDFAVYYCQQ YGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSTKLSSTLT LSKADYEKTHQNSVYACE fourteen AGEN2041W full-length heavy chain sequence (with C-terminal lysine) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSSASTKGPSVFPLAPCSRS TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECP PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE YKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 94 AGEN2041W full length heavy chain sequence (no C-terminal lysine) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSSASTKGPSVFPLAPCSRS TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECP PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE YKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG fifteen AGEN1884 full length light chain sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSRYLGWYQQKPGQAPR LLIYGASTRATGIPDRFSGSGSGTDFTLTITRLEPEDFAVYYCQQ YGSSPWTFGQGTKVEIKRSVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSTKLSSTLT LSKADYEKTHQNSVYACE 16 IMGT CDRH1 (AGEN1884w) GFTFSSYS 17 IMGT CDRH2 (AGEN1884w) ISSSSSYI eighteen IMGT CDRH3 (AGEN1884w) ARVGLMGPFDI 19 IMGT CDRL1 (AGEN1884w) QSVSRY twenty IMGT CDRL2 (AGEN1884w) GAS 21 IMGT CDRL3 (AGEN1884w) QQYGSSPWT 22 CDRH1 consensus sequence SYX _x MX ₂ where X _x is S or A; and X ₂ is N or S 23 CDRH3 consensus sequence VGLMGPFXI, where X is D or N 24 CDRL1 consensus sequence RASQSVX _x X ₂ YLX ₃ where X _x is S or G; X ₂ represents R, S or T; and X ₃ is G or A 25 CDRL2 consensus sequence X _x X ₂ SX ₃ RAT, where X _x is G or A; X ₂ is A or T; and X ₃ is T, S, R or N

- 17 039322

26 CDRL3 consensus sequence QQYGX ₂ SPX ₂ T, where X _x is S or T; and X ₂ is W or F 27 CDRH1 SYAMS 28 CDRH3 VGLMGPFNI 29 CDRL1 RASQSVSSYLA thirty CDRL1 RASQSVGTYLA 31 CDRL2 GASRRAT 32 CDRL2 ATSSRAT 33 CDRL2 GASSRAT 34 CDRL2 AASTRAT 35 CDRL2 GASNRAT 36 CDRL3 QQYGTSPWT 37 CDRL3 QQYGSSPFT 38 vh EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL VWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSS 39 vh EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNTLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSS 40 vh EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSS 41 vh EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL VWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFNIWGQGTMVTVSS 42 vh EVQLVESGGGLVQPGGSLTLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSS 43 VL EIVLTQSPATLSLSPGERATLSCRASQSVGTYLAWYQHKVGQAPR LLIYGASRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ YGSSPWTFGQGTKVEIK 44 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPS LLIYATSSRATGIPDRFSGSVSGTDFTLTISRLEPEDFAVYYCQQ YGTSPWTFGQGTKVEIK 45 VL EIVLTQSPATLSFSPGERATLSCRASQSVSSYLAWYQQKPGQAPR LLIYGASSRATGIPDRFSGSGSGTDFTISRLEPEDFAVYYCQQ YGSSPFTFGPGTKVDIK 46 VL EIVLTQSPATLSVSPGERATLSCRASQSVSSYLAWYQQKPGQAPR LLIYAASTRATGIPDRFSGSASGTDFTLTISRLEPEDFAVYYCQQ YGSSPWTFGQGTKVEIK 47 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPR LLIYGASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQ YGSSPFTFGPGTKVDIK 48 VH-FR1 EVQLVESGGGLVKPGGSLRLSCAASGFTFS 49 VH FR2 WVRQAPGKGLEWVS fifty VH FR3 RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR 51 VH FR4 WGQGTMVTVSS 52 VH-FR1 EVQLLESGGGLVQPGGSLRLSCAASGFTFS 53 VH FR2 WVRQAPGKGLVWVS

- 18 039322

54 VH FR3 R FTIS RDNAKNTLYLQMNS LRAE DTAVYY CAR 55 VH-FR1 EVQLLESGGGLVKPGGSLRLSCAASGFTFS 56 VH-FR1 EVQLVESGGGLVQPGGSLTLSCAASGFTFS 57 VL-FR1 EIVLTQSPGTLSLSPGERATLSC 58 VL FR2 WYQQKPGQAPRLLIY 59 VL FR3 GIPDRFSGSGSGTDFTLTITRLEPEDFAVYYC 60 VL FR4 FGQGTKVEIK 61 VL-FR1 EIVLTQSPATLSLSPGERATLSC 62 VL FR2 WYQHKVGQAPRLLIY 63 VL FR3 GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC 64 VL FR2 WYQQKPGQAPSLLIY 65 VL FR3 GIPDRFSGSVSGTDFTLTISRLEPEDFAVYYC 66 VL-FR1 EIVLTQSPATLSFSPGERATLSC 67 VL FR3 GIPDRFSGSGSGTDFTFTISRLEPEDFAVYYC 68 VL FR4 FGPGTKVDIK 69 VL-FR1 EIVLTQSPATLSVSPGERATLSC 70 VL FR3 GIPDRFSGSASGTDFTLTISRLEPEDFAVYYC 71 VL FR3 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC 72 VH consensus sequence EVQLX _x E SGGGLVX ₂ PGGSLX ₃ L SCAASG FT FS S YX ₄ MX ₅ WVRQAPG kglx ₆ wvssisssssyiyyadsvkgrftisrdnaknx ₇ lylqmnsl RAEDTAVYYCARVGLMGPFXglWGQGTMVTVSS, where X ₄ is V or L; X ₂ represents K or Q; X ₃ represents R or T; X ₄ is S or A; X ₅ is N or S; X ₆ is E or V; X ₇ is S or T; and X ₈ is D or N

- 19 039322

73 VL consensus sequence EIVLTQSPXXTLSX2SPGERATLSCRASQSVX3X4YLX5WYQX6KX7G QAPX ₈ LLIYX ₉ X ₁₀ SX ₁₁ RATGIPX ₁ 2RFSGSX ₁ 3SGTDFTX ₁ 4TIX ₁₅ X _{1 6} LEPEDFAVYYCQQYGX ₁₇ SPX ₁₈ TFGX ₁₉ GTKVX, where X is ₂₀ ; X ₂ is L, V or F; X ₃ is S or G; X ₄ represents R, T or S; X ₅ is G or A; X ₆ is Q or H; X ₇ is P or V; X ₈ is R or S; X ₉ is G or A; X ₁₀ is A or T; Xn is T, R, S or N; X ₁₂ is D or A; X ₁₃ is G, V or A; X ₁₄ is L or F; X ₁₅ is T or S; X ₁₆ is R or S; X ₁₇ is S or T; X ₁₈ is W or F; X ₁₉ is Q or P; and X ₂ o is E or D 74 AGEN1884w N2 97A full length heavy chain (with C-terminal lysine) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 95 AGEN1884w N297A full length heavy chain (no C-terminal lysine) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 75 AGEN1884W S267E/L328F full length heavy chain (with C-terminal lysine) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

- 20 039322

96 AGEN1884w S267E/L328F full length heavy chain (no C-terminal lysine) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 76 AGEN1884W S239D/A330L/I332E full length heavy chain (with C-terminated lysine) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 97 AGEN1884W S239D/A330L/I332E full length heavy chain (without C-terminal lysine) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVGLMGPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Table 2. Heavy chain CDR sequences of exemplary anti-CTLA-4 antibodies ¹

Antibody CDRH1 CDRH2 (SEQ ID NO:) CDRH3 (SEQ ID NO:) (SEQ ID NO:) AGEN1884W SYSMN (one) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3) AGEN1885 SYSMN (I) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3) AGEN1886 SYSMN (I) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3) AGEN1887 SYAMS (27) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3) AGEN1888 SYAMS (27) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3) AGEN1889 SYSMN (one) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3) AGEN1890 SYSMN (one) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3) AGEN1891 SYSMN (one) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3) AGEN1892 SYSMN (one) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3) AGEN1893 SYSMN (one) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3) AGEN1894 SYSMN (one) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3) AGEN1895 SYSMN (one) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3) AGEN1896 SYSMN (one) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3) AGEN1897 SYAMS (27) SISSSSSYIYYADSVKG (2) VGLMGPFNI (28) AGEN1898 SYAMS (27) SISSSSSYIYYADSVKG (2) VGLMGPFNI (28) AGEN1899 SYAMS (27) SISSSSSYIYYADSVKG (2) VGLMGPFNI (28) AGEN1900 SYAMS (27) SISSSSSYIYYADSVKG (2) VGLMGPFNI (28) AGEN1901 SYSMN (one) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3) AGEN1902 SYSMN (one) SISSSSSYIYYADSVKG (2) VGLMGPFDI (3)

¹ CDR region VH in the table. 2 defined according to Kabat.

- 21 039322

Table 3 Light chain CDR sequences of exemplary anti-CTLA-4 ² antibodies

Antibody CDRL1 (SEQ ID NO:) CDRL2 (SEQ ID NO:) CDRL3 (SEQ ID NO:) AGEN1884W RASQSVSRYLG (four) GASTRAT (5) QQYGSSPWT (6) AGEN1885 RASQSVSSYLA (29) ATSSRAT (32) QQYGTSPWT (36) AGEN1886 RASQSVSSYLA (29) GASSRAT (33) QQYGSSPFT (37) AGEN1887 RASQSVSRYLG (four) GASTRAT (5) QQYGSSPWT (6) AGEN1888 RASQSVSSYLA (29) GASSRAT (33) QQYGSSPFT (37) AGEN1889 RASQSVGTYLA (thirty) GASRRAT (31) QQYGSSPWT (6) AGEN1890 RASQSVSSYLA (29) GASSRAT (33) QQYGSSPFT (37) AGEN1891 RASQSVSSYLA (29) AASTRAT (34) QQYGSSPWT (6) AGEN1892 RASQSVSSYLA (29) GASNRAT (35) QQYGSSPFT (37) AGEN1893 RASQSVGTYLA (thirty) GASRRAT (31) QQYGSSPWT (6) AGEN1894 RASQSVSRYLG (four) GASTRAT (5) QQYGSSPWT (6) AGEN1895 RASQSVSSYLA (29) ATSSRAT (32) QQYGTSPWT (36) AGEN1896 RASQSVSSYLA (29) GASSRAT (33) QQYGSSPFT (37) AGEN1897 RASQSVSRYLG (four) GASTRAT (5) QQYGSSPWT (6) AGEN1898 RASQSVSSYLA (29) ATSSRAT (32) QQYGTSPWT (36) AGEN1899 RASQSVSSYLA (29) GASSRAT (33) QQYGSSPFT (37) AGEN1900 RASQSVSSYLA (29) GASNRAT (35) QQYGSSPFT (37) AGEN1901 RASQSVGTYLA (thirty) GASRRAT (31) QQYGSSPWT (6) AGEN1902 RASQSVSSYLA (29) GASSRAT (33) QQYGSSPFT (37)

² CDR region VL in the table. 3 defined according to Kabat.

Table 4 Framework (FR) VH sequences of exemplary anti-CTLA-4 antibodies ³

Antibody VH FR1 (SEQ ID NO:) VH FR2 (SEQ ID NO:) VH FR3 (SEQ ID NO:) VH FR4 (SEQ ID NO:) AGEN1884w EVQLVESGGGLVKPGGSLR LSCAASGFTFS (48) WVRQAPGKGLEW VS ( 4 9) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51) AGEN1885 EVQLVESGGGLVKPGGSLR LSCAASGFTFS (48) WVRQAPGKGLEW VS ( 4 9) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51) AGEN1886 EVQLVESGGGLVKPGGSLR LSCAASGFTFS (48) WVRQAPGKGLEW VS ( 4 9) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51) AGEN1887 EVQLLESGGGLVQPGGSLR LSCAASGFTFS (52) WVRQAPGKGLVW VS (53) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51) AGEN1888 EVQLLESGGGLVQPGGSLR LSCAASGFTFS (52) WVRQAPGKGLVW VS(53) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51) AGEN1889 EVQLVESGGGLVKPGGSLR LSCAASGFTFS (48) WVRQAPGKGLEW VS ( 4 9) RFTISRDNAKNTLYLQMNS LRAEDTAVYYCAR (54) WGQGTMVTVSS (51) AGEN1890 EVQLVESGGGLVKPGGSLR LSCAASGFTFS (48) WVRQAPGKGLEW VS ( 4 9) RFTISRDNAKNTLYLQMNS LRAEDTAVYYCAR (54) WGQGTMVTVSS (51)

- 22 039322

AGEN1891 EVQLVESGGGLVKPGGSLR LSCAASGFTFS (48) WVRQAPGKGLEW VS (4 9) RFTISRDNAKNTLYLQMNS LRAEDTAVYYCAR (5 4) WGQGTMVTVSS (51) AGEN1892 EVQLVESGGGLVKPGGSLR LSCAASGFTFS (48) WVRQAPGKGLEW VS (4 9) RFTISRDNAKNTLYLQMNS LRAEDTAVYYCAR (5 4) WGQGTMVTVSS (51) AGEN1893 EVQLLESGGGLVKPGGSLR LSCAASGFTFS (55) WVRQAPGKGLEW VS (4 9) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51) AGEN1894 EVQLLESGGGLVKPGGSLR LSCAASGFTFS (55) WVRQAPGKGLEW VS (4 9) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51) AGEN1895 EVQLLESGGGLVKPGGSLR LSCAASGFTFS (55) WVRQAPGKGLEW VS (4 9) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51) AGEN1896 EVQLLESGGGLVKPGGSLR LSCAASGFTFS (55) WVRQAPGKGLEW VS (4 9) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51) AGEN1897 EVQLLESGGGLVQPGGSLR LSCAASGFTFS (52) WVRQAPGKGLVW VS (53) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51) AGEN1898 EVQLLESGGGLVQPGGSLR LSCAASGFTFS (52) WVRQAPGKGLVW VS (53) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51) AGEN1899 EVQLLESGGGLVQPGGSLR LSCAASGFTFS (52) WVRQAPGKGLVW VS (53) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51) AGEN1900 EVQLLESGGGLVQPGGSLR LSCAASGFTFS (52) WVRQAPGKGLVW VS (53) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51) AGEN1901 EVQLVESGGGLVQPGGSLT LSCAASGFTFS (56) WVRQAPGKGLEW VS (4 9) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51) AGEN1902 EVQLVESGGGLVQPGGSLT LSCAASGFTFS (56) WVRQAPGKGLEW VS (4 9) RFTISRDNAKNSLYLQMNS LRAEDTAVYYCAR (5 0) WGQGTMVTVSS (51)

³ Frame regions VH, described in Table. 4 are determined based on the limitations of the Kabat numbering system for CDR regions. In other words, the VH CDRs are defined according to Kabat, and the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format FR1, CDRH1, FR2, CDRH2, FR3, CDRH3, and FR4.

Table 5 Framework (FR) VL sequences of exemplary anti-CTLA-4 antibodies ⁴

Antibody VL FR1 (SEQ ID NO:) VL FR2 (SEQ ID NO:) VL FR3 (SEQ ID NO:) VL FR4 (SEQ ID NO: ) AGEN1884w EIVLTQSPGTLSLSP GERATLSC (57) WYQQKPGQAPRLL IY (58) GIPDRFSGSGSGTDFTLTITR LEPEDFAVYYC (59) FGQGTKVEIK (60) AGEN1885 EIVLTQSPATLSLSP GERATLSC(61) WYQQKPGQAPSLL II (64) GIPDRFSGSVSGTDFTLTISR LEPEDFAVYYC (63) FGQGTKVEIK (60) AGEN1886 EIVLTQSPATLSFSP GERATLS(66) WYQQKPGQAPRLL IY (58) GIPDRFSGSGSGTDFTFTISR LEPEDFAVYYC (67) FGPGTKVDIK(68) AGEN1887 EIVLTQSPGTLSLSP GERATLSC (57) WYQQKPGQAPRLL IY (58) GIPDRFSGSGSGTDFTLTITR LEPEDFAVYYC (59) FGQGTKVEIK (60) AGEN1888 EIVLTQSPATLSFSP GERATLS(66) WYQQKPGQAPRLL IY (58) GIPDRFSGSGSGTDFTFTISR LEPEDFAVYYC (67) FGPGTKVDIK(68) AGEN1889 EIVLTQSPATLSLSP GERATLSC (61) WYQHKVGQAPRLL II (62) GIPDRFSGSGSGTDFTLTISR LEPEDFAVYYC (63) FGQGTKVEIK (60) AGEN1890 EIVLTQSPATLSFSP GERATLS(66) WYQQKPGQAPRLL IY (58) GIPDRFSGSGSGTDFTFTISR LEPEDFAVYYC (67) FGPGTKVDIK(68) AGEN1891 EIVLTQSPATLSVSP GERATLSC (69) WYQQKPGQAPRLL IY (58) GIPDRFSGSASGTDFTLTISR LEPEDFAVYYC (70) FGQGTKVEIK (60) AGEN1892 EIVLTQSPATLSLSP GERATLSC (61) WYQQKPGQAPRLL IY (58) GIPARFSGSGSGTDFTLTISS LEPEDFAVYYC (71) FGPGTKVDIK(68) AGEN1893 EIVLTQSPATLSLSP GERATLSC (61) WYQHKVGQAPRLL II (62) GIPDRFSGSGSGTDFTLTISR LEPEDFAVYYC (63) FGQGTKVEIK (60) AGEN1894 EIVLTQSPGTLSLSP GERATLSC (57) WYQQKPGQAPRLL IY (58) GIPDRFSGSGSGTDFTLTITR LEPEDFAVYYC (59) FGQGTKVEIK (60) AGEN1895 EIVLTQSPATLSLSP GERATLSC (61) WYQQKPGQAPSLL II (64) GIPDRFSGSVSGTDFTLTISR LEPEDFAVYYC (63) FGQGTKVEIK (60) AGEN1896 EIVLTQSPATLSFSP GERATLS(66) WYQQKPGQAPRLL IY (58) GIPDRFSGSGSGTDFTFTISR LEPEDFAVYYC (67) FGPGTKVDIK(68) AGEN1897 EIVLTQSPGTLSLSP GERATLSC (57) WYQQKPGQAPRLL IY (58) GIPDRFSGSGSGTDFTLTITR LEPEDFAVYYC (59) FGQGTKVEIK (60) AGEN1898 EIVLTQSPATLSLSP GERATLSC(61) WYQQKPGQAPSLL II (64) GIPDRFSGSVSGTDFTLTISR LEPEDFAVYYC (63) FGQGTKVEIK (60) AGEN1899 EIVLTQSPATLSFSP GERATLS(66) WYQQKPGQAPRLL IY (58) GIPDRFSGSGSGTDFTFTISR LEPEDFAVYYC (67) FGPGTKVDIK(68) AGEN1900 EIVLTQSPATLSLSP GERATLSC (61) WYQQKPGQAPRLL IY (58) GIPARFSGSGSGTDFTLTISS LEPEDFAVYYC (71) FGPGTKVDIK(68) AGEN1901 EIVLTQSPATLSLSP GERATLSC(61) WYQHKVGQAPRLL II (62) GIPDRFSGSGSGTDFTLTISR LEPEDFAVYYC (63) FGQGTKVEIK (60) AGEN1902 EIVLTQSPATLSFSP GERATLS(66) WYQQKPGQAPRLL IY (58) GIPDRFSGSGSGTDFTFTISR LEPEDFAVYYC (67) FGPGTKVDIK(68)

⁴ Frame regions VL, described in Table. 5, determined based on number system restrictions

- 23 039322 Kabat's instructions for CDR regions. In other words, the VL CDRs are defined according to Kabat, and the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format FR1, CDRL1, FR2, CDRL2, FR3, CDRL3, and FR4.

Table 6. VH and VL sequences of exemplary anti-CTLA-4 antibodies

Antibody Heavy chain variable region SEQID NO: Light chain variable region SEQID NO: AGEN1884W BADD411-2354 7 BADD412-2376 eight AGEN1885 BADD411-2354 7 BADD412-2382 44 AGEN1886 BADD411-2354 7 BADD412-2384 45 AGEN1887 BADD412-2356 38 BADD412-2376 eight AGEN1888 BADD412-2356 38 BADD412-2384 45 AGEN1889 BADD412-2357 39 BADD412-2367 43 AGEN1890 BADD412-2357 39 BADD412-2384 45 AGEN1891 BADD412-2357 39 BADD412-2390 46 AGEN1892 BADD412-2357 39 BADD412-2393 47 AGEN1893 BADD412-2358 40 BADD412-2367 43 AGEN1894 BADD412-2358 40 BADD412-2376 eight AGEN1895 BADD412-2358 40 BADD412-2382 44 AGEN1896 BADD412-2358 40 BADD412-2384 45 AGEN1897 BADD412-2359 41 BADD412-2376 eight AGEN1898 BADD412-2359 41 BADD412-2382 44 AGEN1899 BADD412-2359 41 BADD412-2384 45 AGEN1900 BADD412-2359 41 BADD412-2393 47 AGEN1901 BADD412-2360 42 BADD412-2367 43 AGEN1902 BADD412-2360 42 BADD412-2384 45

- 24 039322

Table 7. Exemplary sequences of CTLA-4 and family members

SEQID NO: Description Amino acid sequence CTLA-4 human (P16410) MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVV LASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMG 77 NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPP YYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAV SLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN 78 CTLA-4 epitope QVT 79 CTLA-4 epitope YPPPYYLGIGNGTQI 80 CTLA-4 epitope YLGI 81 CTLA-4 epitope MYPPPYY 82 CTLA-4 epitope YPPPYYLGI 83 CTLA-4 epitope YLGIGNGTQI MACLGFQRHKARLNLATRTRPYTLLFSLLFIPVFSKAMHVAQPAVV LANSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMG 84 (G7PL88) CTLA-4 mice (P09793) NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPP YYMGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAV SLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN MACLGLRRYKAQLQLPSRTWPFVALLTLLFIPVFSEAIQVTQPSVV LASSHGVASFPCEYSPSHNTDEVRVTVLRQTNDQMTEVCATTFTEK 85 NTVGFLDYPFCSGTFNESRVNLTIQGLRAVDTGLYLCKVELMYPPP YFVGMGNGTQIYVIDPEPCPDSDFLLWILVAVSLGLFFYSFLVSAV SLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN MACLGLQRYKTHLQLPSRTWPFGVLLSLLFIPIFSEAIQVTQPSVV 86 CTLA-4 rat (Q62859) Human CD28 (P10747) LASSHGVASFPCEYASSHNTDEVRVTVLRQTNDQVTEVCATTFTVK NTLGFLDDPFCSGTFNESRVNLTIQGLRAADTGLYFCKVELMYPPP YFVGMGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLVTAV SLNRTLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNL FSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGN 87 ESVT FYLQNLYVNQTDIY FCKIEVMY PPPYLDNEKSNGT11HVKGK HLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRS RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPD IVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSV Human ICOS (Q9Y6W8) 88 SFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQ LKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMR AVNTAKKSRLTDVTL MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEH SILAGDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEE KNISFFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTDVKS Human BTLA (Q7Z6A9) 89 ASERPSKDEMASRPWLLYRLLPLGGLPLLITTCFCLFCCLRRHQGK QNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDP DLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVK EAPTEYASICVRS MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTE GDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQD CRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESL Human PD-1 (Q15116) 90 RAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLL VWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQW REKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQP LRPEDGHCSWPL

In some embodiments, provided herein is an isolated antibody that specifically binds to human CTLA-4 protein, comprising:

(a) CDRH1 comprising the amino acid sequence SYX ₁ MX ₂ (SEQ ID NO: 22), where X1 is S or A; and X2 is N or S; and/or (b) a CDRH2 comprising the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 2); and/or (c) a CDRH3 comprising the amino acid sequence VGLMGPFXI (SEQ ID NO: 23), where X is D or N; and/or (d) a CDRL1 comprising the amino acid sequence RASQSVX1X2YLX3 (SEQ ID NO: 24), where X1 is S or G; X2 is R, S or T; and X ₃ is G or A; and/or (e) a CDRL2 comprising the amino acid sequence X1X2SX3RAT (SEQ ID NO: 25), where X ₁ is G or A; X ₂ is A or T; and X ₃ is T, S, R or N;

- 25 039322 and/or (f) CDRL3 comprising the amino acid sequence QQYGX1SPX2T (SEQ ID NO: 26), where X ₁ represents S or T; and X ₂ is W or F.

In some embodiments, the antibody comprises one, two, or all three of the VH CDR regions identified above. In some embodiments, the antibody comprises the CDRH1 of one of the antibodies in Table. 2. In some embodiments, the implementation of the antibody includes the CDRH2 of one of the antibodies in table. 2. In some embodiments, the implementation of the antibody includes the CDRH3 of one of the antibodies in table. 2. In some embodiments, the antibody comprises one, two, or all three VH CDR regions of one of the antibodies in Table. 2 (eg VH CDRs in one row of Table 2, eg all VH CDRs are from AGEN1884w). In some embodiments, the implementation of the antibody includes VH frame regions described in this application. In some embodiments, the antibody comprises the VH framework regions of the antibody shown in Table 1. 4 (for example, one, two, three, or four frame regions in one row of Table 4).

In some embodiments, the antibody comprises one, two, or all three of the VL CDRs identified above. In some embodiments, the antibody comprises CDRL1 of one of the antibodies in Table. 3. In some embodiments, the implementation of the antibody includes CDRL2 of one of the antibodies in table. 3. In some embodiments, the implementation of the antibody includes CDRL3 of one of the antibodies in table. 3. In some embodiments, the antibody comprises one, two, or all three VL CDR regions of one of the antibodies in Table. 3 (eg VL CDRs in one row of Table 3, eg all VL CDRs from AGEN1884w). In some embodiments, the implementation of the antibody includes VL frame regions described in this application. In some embodiments, the antibody comprises the VL framework regions (FR) of the antibody shown in Table 1. 5 (for example, one, two, three, or four frame regions in one row of Table 5).

In some embodiments, provided herein is an isolated antibody that specifically binds to human CTLA-4 protein, comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3 where:

(a) CDRH1 includes the amino acid sequence SYX1MX2 (SEQ ID NO: 22), where X ₁ is S or A; and X ₂ is N or S;

(b) CDRH2 includes the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 2);

(c) CDRH3 includes the amino acid sequence VGLMGPFXI (SEQ ID NO: 23), where X is D or N;

(d) CDRL1 includes the amino acid sequence RASQSVX1X2YLX3 (SEQ ID NO: 24), where X1 is S or G; X ₂ represents R, S or T; and X ₃ is G or A;

(e) CDRL2 includes the amino acid sequence X1X2SX3RAT (SEQ ID NO: 25), where Xi is G or A; X ₂ is A or T; and X ₃ is T, S, R or N; and (f) CDRL3 includes the amino acid sequence QQYGX1SPX2T (SEQ ID NO: 26), where X1 is S or T; and X ₂ is W or F.

In some embodiments, CDRH1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 27. In some embodiments, CDRH3 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 28. In some embodiments, implementation CDRL1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 29 and 30. In some embodiments, CDRL2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 3135. In some embodiments, CDRL3 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 36, and 37. In some embodiments, CDRH1, CDRH2, and CDRH3 include the amino acid sequences of CDRH1, CDRH2, and CDRH3, respectively, of the antibodies in Table. 2. In some embodiments, CDRL1, CDRL2, and CDRL3 include the amino acid sequences of CDRL1, CDRL2, and CDRL3, respectively, of the antibodies in Table. 3.

In some embodiments, provided herein is an isolated antibody that specifically binds to the human CTLA-4 protein, comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein CDRH1, CDRH2, and CDRH3 comprise the amino acid sequences CDRH1, CDRH2, and CDRH3, respectively, presented in SEQ ID NO: 1, 2 and 3; 27, 2 and 3; or 27, 2 and 28.

In some embodiments, provided herein is an isolated antibody that specifically binds to the human CTLA-4 protein, comprising a light chain variable region including the complementarity determining regions of CDRL1, CDRL2, and CDRL3, wherein CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences CDRL1, CDRL2, and CDRL3, respectively, presented in SEQ ID NO: 4, 5 and 6; 29, 32 and 36; 29, 33 and 37; 30, 31 and 6; 29, 34 and 6; or 29, 35 and 37.

In some embodiments, provided herein is an isolated antibody,

- 26 039322 which specifically binds to the human CTLA-4 protein, including the heavy chain variable region, including the complementarity-determining regions CDRH1, CDRH2 and CDRH3, and the light chain variable region, including the complementarity-determining regions of CDRL1, CDRL2 and CDRL3, where CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 include the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, respectively, shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6; 1, 2, 3, 29, 32 and 36; 1, 2, 3, 29, 33 and 37; 27, 2, 3, 4, 5 and 6; 27, 2, 3, 29, 33 and 37; 1, 2, 3, 30, 31 and 6; 1, 2, 3, 29, 34 and 6; 1, 2, 3, 29, 35 and 37; 27, 2, 28, 4, 5 and 6; 27, 2, 28, 29, 32 and 36; 27, 2, 28, 29, 33 and 37; or 27, 2, 28, 29, 35 and 37 respectively.

In some embodiments, the antibody comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions and a light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise amino acid sequences, presented in SEQ ID NO: 1, 2, 3, 4, 5 and 6, respectively.

In some embodiments, the CDR regions of an antibody can be defined according to the Chothia numbering scheme, which refers to the position of the structural loops of the immunoglobulin (see, for example, Chothia C. & Lesk A.M. (1987), J. Mol. Biol. 196: 901-917 Al-Lazikani B. et al (1997) J Mol Biol 273: 927-948 Chothia C et al (1992) J Mol Biol 227: 799-817 Tramontano A et al (1990) J Mol Biol 215(1): 175-82 and U.S. Patent 7,709,226). Typically, when using the Kabat numbering rule, Chothia's CDRH1 loop is present at heavy chain amino acids 26-32, 33, or 34, Chothia's CDRH2 loop is present at heavy chain amino acids 52-56, and Chothia's CDRH3 loop is present at heavy chain amino acids 95-102, while the Chothia CDRL1 loop is present at light chain amino acids 2434, the Chothia CDRL2 loop is present at light chain amino acids 50-56, and the Chothia CDRL3 loop is present at light chain amino acids 89-97. Chothia's CDRH1 loop end when numbered using Kabat's numbering rule changes between H32 and H34 depending on the length of the loop (this is because Kabat's numbering scheme places inserts at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32 ; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).

In some embodiments, provided herein is an isolated antibody that specifically binds to human CTLA-4 protein, wherein the antibody comprises the Chothia VL CDR region from the VL of the antibody disclosed in Table 1. 6 in this application (for example, AGEN1884w or AGEN2041w). In some embodiments, provided herein is an isolated antibody that specifically binds to human CTLA-4 protein, wherein the antibody comprises the VH CDRs of the Chothia antibody disclosed in Table 1. 6 in this application (for example, AGEN1884w or AGEN2041w). In some embodiments, provided herein is an isolated antibody that specifically binds to human CTLA-4 protein, wherein the antibody comprises the VH Chothia CDR region and the VL Chothia CDR region of the antibody disclosed in Table 1. 6 in this application (for example, AGEN1884w or AGEN2041w). In some embodiments, antibodies that specifically bind to the human CTLA-4 protein include one or more CDR regions, where the Chothia and Kabat CDR regions share the same amino acid sequence. In some embodiments, provided herein is an isolated antibody that specifically binds to human CTLA-4 protein and includes combinations of Kabat CDR regions and Chothia CDR regions.

In some embodiments, the CDR regions of an antibody can 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. According to the IMGT numbering scheme, CDRH1 is present at positions 26-35, CDRH2 is present at positions 51-57, CDRH3 is present at positions 93-102, CDRL1 is present at positions 27-32, CDRL2 is present at positions 50-52, and CDRL3 is present at positions 89-97. In a specific embodiment, provided herein are antibodies that specifically bind to human CTLA-4 protein and include the CDR regions of the antibody disclosed in Table 1. 6 in this application (eg, AGEN1884w or AGEN2041w), when defined using the IMGT numbering system, for example, as described in Lefranc M-P (1999) above and Lefranc M-P. et al. (1999) above.

In some embodiments, the CDR regions of an antibody can be determined according to MacCallum R.M. et al. (1996) J. Mol. Biol. 262:732-745. See also, for example, Martin A. Protein Sequence and Structure Analysis of Antibody Variable Domains, in Antibody Engineering, Kontermann and Dubel, eds., chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In a specific embodiment, provided herein are antibodies that specifically bind to human CTLA-4 protein and include the CDR regions of the antibody disclosed in Table 1. 6 in this application (for example, AGEN1884w or AGEN2041w), when determined using the method in MacCallum R.M. et al.

In some embodiments, antibody CDR regions can be defined according to the AbM numbering scheme, which refers to AbM hypervariable regions that are a compromise between Kabat CDR regions and Chothia structural loops, and is used in

- 27 039322 grams of modeling antibodies Oxford Molecular AbM (Oxford Molecular Group, Inc.). In a specific embodiment, provided herein are antibodies that specifically bind to human CTLA-4 protein and include the CDR regions of the antibody disclosed in Table 1. 6 in this application (for example, AGEN1884w or AGEN2041w), when defined using the AbM numbering scheme.

Thus, in some embodiments, provided herein is an isolated antibody that specifically binds to the human CTLA-4 protein, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequences of the CDRH1, CDRH2, and CDRH3 regions shown in SEQ ID NO: 7, and the amino acid sequences of the CDRL1, CDRL2, and CDRL3 regions shown in SEQ ID NO: 8, where each CDR is defined according to the Kabat definition, the Chothia definition, the combination of the Kabat definition and the Chothia definition, the IMGT numbering system, the AbM definition, or the contact CDR definition.

In some embodiments, provided herein is an isolated antibody that specifically binds to the human CTLA-4 protein, wherein the antibody comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions and a light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions. where CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 regions comprise the amino acid sequences shown in SEQ ID NOs: 16, 17, 18, 19, 20 and 21, respectively.

In some embodiments, provided herein is an isolated antibody that specifically binds to human CTLA-4 protein, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72.

In some embodiments, provided herein is an isolated antibody that specifically binds to human CTLA-4 protein, comprising a heavy chain variable region having an amino acid sequence derived from a human IGHV3-21 germline sequence. One or more regions selected from framework 1, framework 2, framework 3, CDRH1, and CDRH2 (e.g., two, three, four, or five of these regions) can be derived from a human IGHV3-21 germline sequence. In one embodiment, framework 1, framework 2, framework 3, CDRH1 and CDRH2 are derived from a human IGHV3-21 germline sequence.

In some embodiments, provided herein is an isolated antibody that specifically binds to human CTLA-4 protein, comprising a heavy chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% ( for example, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and 38-42. In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 38-42. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 93. In some embodiments, the antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 14. in SEQ ID NO: 94. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in SEQ ID NO: 76. In some embodiments, the antibody includes a heavy chain having the amino acid sequence shown in nuyu in SEQ ID NO: 97.

In some embodiments, provided herein is an isolated antibody that specifically binds to human CTLA-4 protein, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 73.

In some embodiments, provided herein is an isolated antibody that specifically binds to human CTLA-4 protein, comprising a light chain variable region having an amino acid sequence derived from a human IGKV3-20 or IGKV3-11 germline sequence. One or more regions selected from framework 1, framework 2, framework 3, CDRL1, and CDRL2 (e.g., two, three, four, or five of these regions) can be derived from the germline sequence IGKV3-20 or IGKV3- 11 people. In one embodiment, framework 1, framework 2, framework 3, CDRL1 and CDRL2 are derived from a human IGKV3-20 or IGKV3-11 germline sequence.

In some embodiments, provided herein is an isolated antibody that specifically binds to human CTLA-4 protein, comprising a light chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% ( e.g. at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%)

- 28 039322 is identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 4347. In some embodiments, the antibody comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 43- 47. In some embodiments, the antibody comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the antibody includes a light chain having the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the antibody comprises a light chain having the amino acid sequence shown in SEQ ID NO: 15.

In some embodiments, provided herein is an isolated antibody that specifically binds to the human CTLA-4 protein, comprising a heavy chain variable region having an amino acid sequence derived from a human IGHV3-21 germline sequence and a light chain variable region having the amino acid sequence derived from a human germline IGKV3-20 or IGKV3-11 sequence. One or more regions selected from framework 1, framework 2, framework 3, CDRH1, and CDRH2 (e.g., two, three, four, or five of these regions) can be derived from a human IGHV3-21 germline sequence. In one embodiment, framework 1, framework 2, framework 3, CDRH1 and CDRH2 are derived from a human IGHV3-21 germline sequence. One or more regions selected from framework 1, framework 2, framework 3, CDRL1, and CDRL2 (e.g., two, three, four, or five of these regions) can be derived from the germline sequence IGKV3-20 or IGKV3- 11 people. In one embodiment, framework 1, framework 2, framework 3, CDRL1 and CDRL2 are derived from a human IGKV3-20 or IGKV3-11 germline sequence.

In some embodiments, provided herein is an isolated antibody that specifically binds to human CTLA-4 protein, comprising a heavy chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., , at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 7 and 38-42, and a light chain variable region comprising an amino acid sequence that is at least 75, 80, 85, 90, 95, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 43-47. In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 38-42 and a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs. : 8 and 43-47. In some embodiments, the antibody comprises the heavy chain variable region and light chain variable region amino acid sequences shown in SEQ ID NOs: 7 and 8; 7 and 44; 7 and 45; 38 and 8; 38 and 45; 39 and 43; 39 and 45; 39 and 46; 39 and 47; 40 and 43; 40 and 8; 40 and 44; 40 and 45; 41 and 8; 41 and 44; 41 and 45; 41 and 47; 42 and 43; or 42 and 45 respectively. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12; and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 93; and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14; and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 94; and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 76; and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 97; and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

In some embodiments, provided herein is an isolated antibody that cross-competes for binding to human CTLA-4 protein with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 7 and 8, respectively.

In some embodiments, provided herein is an isolated antibody that binds to the same epitope on the human CTLA-4 protein as an antibody comprising the heavy and light chain variable region amino acid sequences shown in

- 29 039322

SEQ ID NO: 7 and 8, respectively.

Any Ig constant region can be used in the antibodies disclosed in this application. In some embodiments, the Ig region is a heavy chain constant region of human IgG ₁ or human IgG2.

In some embodiments, the IgG regions of the antibodies described herein have increased affinity for CD32B (also known as FcyRIIB or FCGR2B), for example, compared to an antibody with a wild-type Fc region, for example, an IgGi Fc. In some embodiments, the implementation of the antibodies described in this application, have a selectively increased affinity for CD32B (FcyRIIB) compared to CD32A (FcyRIIA) and CD16 (FcyRIIIA). Sequence changes that result in increased affinity for CD32B are known in the art, for example, in Mimoto et al., Protein Engineering, Design & Selection 10: 589-598 (2013), Chu et al., Molecular Immunology 45: 39263933 ( 2008) and Strohl, Current Opinion in Biology 20: 685-691 (2009), 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, comprising a mutation selected from the group consisting of G236D, P238D, S23D, S267E, L328F, and L328E, and combinations thereof, when numbered according to the EU index (Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, Bethesda (1991)). In some embodiments, the antibody comprises a heavy chain constant region, eg, an IgG ₁ constant region or fragment thereof, comprising substitutions S267E and L328F. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or a fragment thereof, comprising the P238D and L328E substitutions. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, comprising a P238D substitution and a substitution selected from the group consisting of E233D, G237D, H268D, P271G, A330R, and combinations thereof. In some embodiments, the antibody comprises a heavy chain constant region, for example, an IgG1 constant region or fragment thereof, including substitutions P238D, E233D, G237D, H268D, P271G, and A330R. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region or fragment thereof, including G236D and S267E. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, including S239D and S267E. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, including V262E, S267E, and L328F. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, including V264E, S267E, and L328F.

In some embodiments, the implementation of the IgG region of the antibodies described in this application, have increased affinity for FcyRIIIA, for example, compared with an antibody with a wild-type Fc region, for example, Fc IgG ₁ . Sequence changes that result in increased affinity for FcyRIIIA are known in the art, for example, in Kellner et al., Methods 65: 105-113 (2014), Lazar et al., Proc. Natl. Acad. sci. 103: 4005-4010 (2006), Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001), each of which is incorporated herein by reference in its entirety. In some embodiments, the antibody comprises a heavy chain constant region, e.g., an IgG1 constant region or fragment thereof, comprising a mutation selected from the group consisting of G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E , E333A, K334A, A339T, and P396L, and combinations thereof, when numbered according to the EU index (Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, Bethesda (1991)). In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, comprising S239D. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, including T256A. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, including K290A. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region or fragment thereof, comprising S298A. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region or fragment thereof, including I332E. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region or fragment thereof, including E333A. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, including K334A. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, including A339T. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, including S239D and I332E. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, including S239D, A330L, and I332E. In some embodiments, the antibody

- 30 039322 includes a heavy chain constant region, for example an IgG1 constant region or a fragment thereof, including S298A, E333A and K334A. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG ₁ constant region or fragment thereof, including G236A, S239D, and I332E. In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1 constant region or fragment thereof, including F243L, R292P, Y300L, V305I, and P396L.

In some embodiments, the implementation of the antibodies described in this application, demonstrate activity in the form of antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the antibodies described herein initiate natural killer cell-mediated cellular depletion. In some embodiments, the antibodies described herein are used to treat a tumor infiltrated by natural killer cells. In some embodiments, the implementation of the antibodies described in this application, demonstrate activity in the form of antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the antibodies described herein initiate macrophage-mediated cellular 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, the antibody disclosed herein is an activated antibody that, when activated, binds the human CTLA-4 protein. In some embodiments, the activated antibody comprises a masking molecule that inhibits the binding of the antibody in its uncleaved state to human CTLA-4 protein, and at least one cleavage molecule coupled to the antibody, for example, where the cleavage molecule is a polypeptide that functions as a substrate for a protease. , which enriches the tumor microenvironment. Exemplary activated antibodies are described, for example, in US Pat. 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 higher affinity than the human IgG heavy chain constant region. wild type binds to human FcyRIIIA.

5.3. pharmaceutical compositions.

The present application provides compositions comprising an anti-CTLA-4 antibody of the present application 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 doses and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); 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 comprise an effective amount of an antibody or antigen-binding fragment thereof as described herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. In some embodiments, the antibody is the only active ingredient included in the pharmaceutical composition.

The pharmaceutical compositions described in this application can be used in the inhibition of CTLA-4 activity and the treatment of a condition such as cancer or an infectious disease.

In one aspect, the present application provides a pharmaceutical composition comprising an

- 31 039322 an anti-CTLA-4 antibody according to the invention and a pharmaceutically acceptable carrier or excipient for use as a medicament.

In one aspect, the present application provides a pharmaceutical composition comprising an anti-CTLA-4 antibody of the invention and a pharmaceutically acceptable carrier or excipient for use in a method of treating cancer.

Pharmaceutically acceptable carriers for use in parenteral formulations include aqueous solvents, non-aqueous solvents, antimicrobials, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, complexing or chelating agents, and other pharmaceutically acceptable agents. Examples of aqueous solvents include sodium chloride solution for injection, Ringer's solution for injection, isotonic dextrose solution for injection, sterile water for injection, dextrose solution and lactated Ringer's solution for injection. Non-aqueous solvents for parenteral use include fixed vegetable oils, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobials at bacteriostatic or fungistatic concentrations may be added to parenteral preparations packaged in multi-dose containers, which include phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, p-hydroxybenzoic acid methyl and propyl 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). Metal ion complexing or chelating agents include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol in the case of water-miscible solvents; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid to adjust the pH.

The pharmaceutical composition may be formulated for any route of administration to a subject. Certain examples of routes of administration include intranasal, oral, polmonal, transdermal, dermal, and parenteral. Parenteral administration, which is characterized by subcutaneous, intramuscular or intravenous injection, is also contemplated in this application.

Formulations for injection may be prepared in standard forms, either as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in a liquid prior to injection, or as emulsions. Preparations for injection, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, sodium chloride solution, dextrose, glycerol or ethanol. In addition, if necessary, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic excipients such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubilizers and other such substances, such as, for example, sodium acetate. , sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

Formulations for parenteral administration of antibodies include sterile solutions ready for injection, sterile dry soluble products such as lyophilized powders ready to be combined with a solvent immediately before use, including subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products, ready to be combined with a diluent immediately before use, and sterile emulsions. Solutions may be aqueous or non-aqueous.

In the case of intravenous administration, suitable carriers include saline or phosphate buffered saline (PBS) and solutions containing thickeners and solubilizers such as glucose, polyethylene glycol and polypropylene glycol and mixtures thereof.

Topical mixtures containing the antibody are prepared as described for topical and systemic administration. The finished mixture may be a solution, suspension, emulsion, and the like. and may be formulated as creams, gels, ointments, emulsions, solutions, tinctures, lotions, suspensions, tinctures, pastes, foams, aerosols, washes, sprays, suppositories, dressings, transdermal patches, or any other preparation suitable for topical application. applications.

The anti-CTLA-4 antibody described herein can be formulated into an aerosol for topical use such as inhalation (see, for example, US Pat. diseases, especially asthma). Such respiratory dosage forms may be in the form of an aerosol or nebulizer solution, or as a microparticulate insufflation powder, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the dosage form, in one embodiment, will have a diameter of less than 50 microns, in one embodiment, less than 10 microns.

- 32 039322

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

Transdermal patches, including iontophoretic and electrophoretic devices, are known to those skilled in the art and can be used to administer an antibody. For example, such patches are disclosed in US Pat. Nos. 6,267,983; 6,261,595;

In some embodiments, a pharmaceutical composition comprising an antibody or antigen-binding fragment described herein is a lyophilized powder that can be reconstituted for administration as solutions, emulsions, and other mixtures. It can also be reconstituted and made into solids or gels. The lyophilized powder is made by dissolving the antibody or antigen-binding fragment described herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain a stability aid, or another pharmacological component of the powder, or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate, or another such buffer known to those skilled in the art, in one embodiment, at approximately neutral pH. Subsequent sterilization of the solution by filtration, followed by lyophilization under standard conditions known to those skilled in the art, yields the desired dosage form. In one embodiment, the finished solution is dosed into vials for lyophilization. Each vial will contain a single dose or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions, such as at a temperature of from about 4° C. to room temperature. Reconstitution of such a lyophilized powder with water for injection provides a dosage form for parenteral use. For reconstitution, the lyophilized powder is added to sterile water or other suitable vehicle. The exact amount depends on the selected compound. Such an amount can be determined empirically.

The anti-CTLA-4 antibodies described herein and other compositions provided herein can also be formulated to target a specific tissue, receptor, or other area of the body of the subject being treated. A variety of such targeting methods are known to those skilled in the art. All such targeting methods are contemplated herein for use in the present compositions. В отношении неограничивающих примеров способов направленного воздействия см., например, патенты США 6316652, 6274552, 6271359, 6253872, 6139865, 6131570, 6120751, 6071495, 6060082, 6048736, 6039975, 6004534, 5985307, 5972366, 5900252, 5840674, 5759542 и 5709874. In a certain embodiment, the antibodies or antigen-binding fragment described in this application, directed to the impact on the tumor.

Compositions to be used for in vivo administration may be sterile. This is easily achieved by filtration through, for example, sterile filter membranes.

5.4. Application methods.

In another aspect of the present description provides a method of treating a subject using antibodies against CTLA-4 disclosed in this application. Any disease or disorder in a subject for which inhibition of CTLA-4 function is effective can be treated using the anti-CTLA-4 antibodies disclosed herein. The anti-CTLA-4 antibodies disclosed herein are particularly useful for inhibiting immune system tolerance to tumors and, accordingly, can be used as immunotherapy for subjects with cancer. For example, in some embodiments, provided herein is a method of increasing T cell activation in response to an antigen in a subject, wherein the method comprises administering to the subject an effective amount of an anti-CTLA-4 antibody, or a pharmaceutical composition thereof, as disclosed herein. In some embodiments, provided herein is a method of treating cancer in a subject, wherein the method comprises administering to the subject an effective amount of an antibody or pharmaceutical composition as disclosed herein. In some embodiments, the subject has previously received immunotherapy. In some embodiments, the subject has not previously received immunotherapy. In some embodiments, the cancer is an advanced or metastatic cancer. Cancers that can be treated with anti-CTLA-4 antibodies or pharmaceutical compositions disclosed

- 33 039322 terms in this application include, without limitation, solid cancer (for example, relapsed or refractory solid cancer and advanced or metastatic solid cancer), carcinoma, sarcoma, melanoma (for example, stage III or stage IV melanoma), small cell lung cancer , non-small cell lung cancer, urothelial cancer, ovarian cancer, prostate cancer (eg, metastatic hormone-resistant prostate cancer and advanced metastatic prostate cancer), pancreatic cancer, 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 metastasis, Merkel cell carcinoma, stomach cancer, gastroesophageal cancer , renal cell carcinoma, uveal melanoma, colon cancer, cervical cancer, whether mfoma (eg, relapsed or refractory lymphoma), non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, and multiple myeloma. In some embodiments, cancer is treated by intratumoral administration of anti-CTLA-4 antibodies or pharmaceutical compositions disclosed herein. Cancers that can be treated by intratumoral administration of anti-CTLA-4 antibodies or pharmaceutical compositions disclosed herein include, without limitation, solid tumors (e.g., spreading or metastatic solid tumors), head and neck cancers (e.g., relapsed/refractory head and neck squamous cell carcinoma (HNSCC)) and breast cancer (eg, HER2+ breast cancer (eg, relapsed/refractory HER2+ breast cancer)).

Additional cancers that can be treated with anti-CTLA-4 antibodies or pharmaceutical compositions disclosed herein include, without limitation, B-cell lymphomas (e.g., B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin's lymphoma, cutaneous B-cell lymphoma, diffuse large B-cell lymphoma), basal cell carcinoma, bladder cancer, blastoma, brain metastasis, breast cancer, Burkitt's lymphoma, carcinoma (eg, adenocarcinoma (eg, gastroesophageal junction)), cervical cancer, colon cancer, cancer colon and rectum (colon and rectal cancer), endometrial carcinoma, esophageal cancer, Ewing's sarcoma, follicular lymphoma, gastric cancer, gastroesophageal junction carcinoma, gastrointestinal cancer, glioblastoma (eg, glioblastoma multiforme, eg, newly diagnosed or recurrent), glioma, head and neck cancer (eg, squamous cell carcinoma of the head and neck), liver metastasis, Hodgkin's and non-Hodgkin's 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 (eg, multiple myeloma), neuroblastoma, ocular melanoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer (eg, pancreatic ductal adenocarcinoma), prostate cancer (eg, hormone-refractory (eg, castration-resistant), metastatic, metastatic hormone-refractory (eg, castration-resistant, androgen-independent)), renal cell carcinoma ( e.g. metastatic), salivary gland carcinoma, sarcoma (e.g. , rhabdomyosarcoma), skin cancer (eg, melanoma (eg, metastatic melanoma)), soft tissue sarcoma, solid tumor, squamous cell carcinoma, synovial sarcoma, testicular cancer, thyroid cancer, transitional cell carcinoma (urothelial cell carcinoma), uveal melanoma ( metastatic), verrucous carcinoma, vulvar cancer, and Waldenström's macroglobulinemia.

In some embodiments, the cancer being treated according to the methods described herein is a human sarcoma or carcinoma, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, 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 carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma , medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma (eg, metastatic), hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilms tumor, cervical cancer, testicular tumor, carcinoma lung noma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, retinoblastoma, or neuroblastoma. In some embodiments, the cancer being treated according to the methods described herein is acute lymphocytic leukemia or acute myelocytic leukemia (e.g., myeloblastic, promyelocytic, myelo

- 34 039322 monocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia or chronic lymphocytic leukemia); Hodgkin's disease; non-Hodgkin's lymphoma; acute myeloid leukemia; B-cell lymphoma; T-cell lymphoma; anaplastic large cell lymphoma; intraocular lymphoma; follicular lymphoma; lymphoma of the small intestine; or lymphoma of the marginal zone of the spleen. In some embodiments, the cancer being treated according to the methods described herein is multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, gastrointestinal stromal tumors, head and/or neck cancer (e.g., laryngopharyngeal squamous cell carcinoma, squamous cell carcinoma oropharyngeal cell carcinoma or larynx verrucous carcinoma), endometrial stromal sarcoma, mast cell sarcoma, adult soft tissue sarcoma, uterine sarcoma, Merkel cell carcinoma, urothelial carcinoma, melanoma with brain metastases, uveal melanoma, uveal melanoma with liver metastases , non-small cell lung cancer, rectal cancer, or myelodysplastic syndrome. In some embodiments, the cancer being treated according to the present methods is metastatic.

In some embodiments, the cancer being treated according to the methods described herein includes prostate cancer, breast cancer, lung cancer, colon and rectal cancer, melanoma, bronchial cancer, bladder cancer, brain or central cancer. 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/thymic carcinoma, glioblastoma, myelodysplastic syndrome, soft tissue sarcoma, DIPG, adenocarcinoma, osteogenic sarcoma, chondrosarcoma, leukemia, or pancreatic cancer. In some embodiments, the cancer being treated according to the methods described herein includes cancer (eg, adenocarcinoma), lymphoma, blastoma, melanoma, sarcoma, or leukemia. In some embodiments, the cancer being treated according to the methods described herein includes 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 (for example, liver and hepatoma carcinoma), bladder cancer, breast cancer, inflammatory breast cancer, Merkel cell carcinoma, colon cancer, colon and rectal cancer, gastric 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, the cancer being treated according to the methods described herein includes desmoplastic melanoma, inflammatory breast cancer, thymoma, rectal cancer, anal cancer, or surgically treatable or non-surgically treatable brainstem glioma. In a certain embodiment, the cancer is a solid tumor. In another specific embodiment, the cancer is glioblastoma multiforme. In some embodiments, glioblastoma multiforme is recurrent. In some embodiments, glioblastoma multiforme is newly diagnosed. In some embodiments, glioblastoma multiforme is in a subject having unmethylated MGMT promoters. In some embodiments, glioblastoma multiforme is refractory to bevacizumab therapy. In some embodiments, glioblastoma multiforme is in a subject who has not received bevacizumab therapy.

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

In some embodiments, provided herein is a method for preventing or treating an infectious disease in a subject, wherein the method comprises administering to the subject an effective amount of an anti-CTLA-4 antibody, or a pharmaceutical composition thereof, as disclosed herein. In one embodiment, the present application provides methods for preventing and/or treating an infection (e.g., a viral infection, a bacterial infection, mycosis, pro

- 35 039322 tozoic infection or parasitic infection). The infection that is prevented and/or treated in accordance with the present methods may be caused by the pathogen defined in this application. In a certain embodiment, the anti-CTLA-4 antibody described herein, or composition thereof, is the only active agent that is administered to the subject. In some embodiments, an anti-CTLA-4 antibody described herein or a composition thereof is used in combination with an anti-infective therapy (eg, antiviral, antibacterial, antifungal, or anthelmintic) to treat infectious diseases.

Infectious diseases that can be treated and/or prevented by the anti-CTLA-4 antibodies or pharmaceutical compositions disclosed herein are caused by pathogens including, but not limited to, bacteria, parasites, fungi, protozoa, and viruses. In a specific embodiment, the infectious disease being treated and/or prevented by the anti-CTLA-4 antibodies or pharmaceutical compositions disclosed herein is caused by a virus. Viral diseases or viral infections that can be prevented and/or treated according to the methods described herein include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, influenza (e.g., influenza A or influenza B), chickenpox, adenovirus, herpes simplex virus type I (HSV-I), herpes simplex type II (HSV-II), rinderpest virus, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, papovavirus , cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackievirus, mumps virus, measles virus, rubella virus, poliovirus, variola virus, Epstein-Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II ( HIV-II) and causative 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 (e.g., Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris, Staphylococcus viridans, and Pseudomonas aeruginosa) that can be prevented and/or treated according to the methods described herein include, without limited to those listed, Mycobacteria rickettsia, mycoplasma, neisseria, S.pneumonia, Borrelia burgdorferi (Lyme disease), Bacillus anthracis (anthrax), tetanus, streptococcus, staphylococcus, mycobacterium, whooping cough, cholera, plague, diphtheria, chlamydia, S. aureus and legionella.

Protozoal diseases or protozoal infections caused by protozoa that can be prevented and/or treated according to the methods described herein include, but are not limited to, leishmania, coccidiosis, trypanosoma, schistosome, or malaria. Parasitic diseases or parasitic infections caused by parasites that can be prevented and/or treated in accordance with the methods described in this application include, without limitation, chlamydia and rickettsia.

Fungal diseases or fungal infections that can be prevented and/or treated in accordance with the methods described in this application include, without limitation, infections caused by candida, zygomycosis, candidiasis of the mammary glands, progressive disseminated trichosporonosis with latent trichosporonemia, disseminated candidiasis, легочный паракокцидиоидомикоз, легочный аспергиллез, пневмонию Pneumocystis carinii, криптококковый менингит, кокцидиоидный менингоэнцефалит и спинномозговой васкулит, инфекцию Aspergillus niger, кератит Fusarium, микозы околоносовых пазух, эндокардит Aspergillus fumigatus, тибиальную дисхондроплазию, вагинит Candida glabrata, ротоглоточный кандидоз, X-сцепленную хроническую гранулематозную болезнь , ringworm of the foot, cutaneous candidiasis, fungal placentitis, disseminated trichosporonosis, allergic bronchopulmonary aspergillosis, fungal keratitis, Cryptococcus neoformans infection, fungal peritonitis, Curvularia geniculat infection a, staphylococcal endophthalmitis, sporotrichosis and ringworm.

In some embodiments, the infectious disease is acute. In some embodiments, the 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 Encephalitis Virus, TBE Virus, Mdengue 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 thereof, as disclosed herein, promotes virus control. In some embodiments, the anti-CTLA-4 antibody

- 36 039322 or its pharmaceutical composition, as disclosed in this application, eliminates the virus reservoirs.

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

The present invention relates in one aspect to an anti-CTLA-4 antibody of the invention and/or its use, in combination with pharmaceutically acceptable carriers or excipients, for the preparation of pharmaceutical compositions or medicaments for immunotherapy (e.g., immunotherapy to increase T cell activation in response to 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 of the invention and/or a pharmaceutical composition of the invention comprising an anti-CTLA-4 antibody of the invention and a pharmaceutically acceptable carrier or adjuvant, for use in a method of treating cancer.

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

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

In some embodiments, these 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-PDL2 antagonist antibody, an anti-CTLA-4 antagonist antibody, an anti-TIM-3 antagonist antibody. , an anti-LAG-3 antagonist antibody, an anti-CEACAM1 antagonist antibody, an anti-GITR agonist antibody, an anti-OX40 agonist antibody, an anti-CD137 agonist antibody, an anti-DR3 agonist antibody, an anti-TNFSF14 agonist antibody, and an anti-CD27 agonist antibody. 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 with respect to a member of the tumor necrosis factor receptor superfamily or a member of the tumor necrosis factor superfamily.

In some embodiments, the present invention relates to: (a) an anti-CTLA-4 antibody of the invention and/or a pharmaceutical composition of the invention comprising an anti-CTLA-4 antibody of the invention and a pharmaceutically acceptable carrier or excipient, and (b) an additional therapeutic agent , for use as a drug. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic agent or a checkpoint targeting agent.

In some embodiments, the present invention relates to: (a) an anti-CTLA-4 antibody of the invention and/or a pharmaceutical composition of the invention comprising an anti-CTLA-4 antibody of the invention and a pharmaceutically acceptable carrier or excipient, and (b) an additional therapeutic agent , for use in a method of treating cancer.

In some embodiments, the present invention relates to: (a) an anti-CTLA-4 antibody of the invention and/or a pharmaceutical composition of the invention comprising an anti-CTLA-4 antibody of the invention and a pharmaceutically acceptable carrier or excipient, and (b) an additional therapeutic agent , for use in a method of treating an infectious disease.

In some embodiments, an anti-CTLA-4 antibody disclosed herein is administered to a subject in combination with a compound that targets an immunomodulatory enzyme(s), such as IDO (indolamine-(2,3)-dioxygenase) and/or TDO (tryptophan-2,3-dioxygenase). In some embodiments, such a compound is selected from the group consisting of epa

- 37 039322 cadostat (Incyte Corp.; see, for example, WO 2010/005958, which is incorporated herein by reference in its entirety), F001287 (Flexus Biosciences), indoxymod (NewLink Genetics) and NLG919 (NewLink Genetics). In one embodiment, the compound is epacadostat. In another embodiment, the compound is F001287. In another embodiment, the compound is indoxymod. In another embodiment, the compound is NLG919.

In some embodiments, the present invention relates to: (a) an anti-CTLA-4 antibody of the invention and/or a pharmaceutical composition of the invention comprising an anti-CTLA-4 antibody of the invention and a pharmaceutically acceptable carrier or excipient, and (b) a compound that Directly affects the immunomodulatory enzyme, for use as a drug. In a preferred embodiment, the compound targets the IDO and/or the TDO.

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

In some embodiments, an anti-CTLA-4 antibody disclosed herein is administered to a subject in combination with a vaccine. In some embodiments, the vaccine is a heat shock protein antitumor vaccine or a heat shock protein antipathogenic vaccine. In a specific embodiment, an anti-CTLA-4 antibody disclosed herein is administered to a subject in combination with a heat shock protein based antitumor vaccine. Heat shock proteins (HSPs) are a family of highly conserved proteins found in all species. Their expression can be strongly induced to much higher levels due to heat shock or other forms of stress, including exposure to toxins, oxidative stress, or lack of glucose. Five families have been identified according to molecular weight: HSP-110, -90, -70, -60 and -28. HSP proteins deliver immunogenic peptides in a cross-presentation pathway in antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs), resulting in T-cell activation. HSP proteins function as chaperone carriers of tumor-associated antigenic peptides, forming complexes that can induce tumor-specific immunity. When released from dying tumor cells, HSP-antigen complexes capture antigen-presenting cells (APCs), in which antigens are processed to form peptides that bind class I and class II MHC molecules, which leads to the activation of antitumor CD8+ and CD4+ T cells. Immunity induced by HSP complexes derived from tumor preparations is specifically directed against the unique repertoire of antigenic peptides expressed by each subject's cancer.

The peptide-heat shock protein complex (HSPPC) is a peptide protein complex consisting of a heat shock protein non-covalently complexed with antigenic peptides. HSPPC complexes induce both innate and adaptive immune responses. In a particular embodiment, the antigenic peptide(s) demonstrates the antigenicity of the cancer being treated. HSPPC complexes are efficiently taken up by APC through membrane receptors (mainly CD91) or by binding to Toll-like receptors. Internalization of HSPPC leads to functional maturation of APC with production of chemokines and cytokines leading to activation of natural killer (NK) cells, monocytes, and Th1 and Th-2 mediated immune responses. In some embodiments, the HSPPC complexes used in the methods disclosed herein comprise one or more heat shock proteins from the hsp60, hsp70, or hsp90 stress protein family, in complex with antigenic peptides. In some embodiments, the HSPPC complexes include hsc70, hsp70, hsp90, hsp110, grp170, gp96, calreticulin, or combinations of two or more of these.

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

In some embodiments, the implementation of HSPPC, for example HSPPC-96, is obtained from the tumor tissue of the subject. In a particular embodiment, the HSPPC (eg, HSPPC-96) is derived from a tumor of the type of cancer, or metastasis thereof, that is being treated. In another specific embodiment, the HSPPC (e.g., HSPPC-96) is autogenous in the subject being treated.

- 38 039322 niyu. In some embodiments, the tumor tissue is non-necrotic tumor tissue. In some embodiments, at least 1 g (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 g) of non-necrotic tumor tissue are used to prepare the vaccine course. In some embodiments, non-necrotic tumor tissue after surgical resection is frozen prior to use for vaccine preparation. In some embodiments, HSPPCs, such as HSPPC-96, are 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 HSPPC, such as HSPCC-96. In such embodiments, doses of HSPPC, eg HSPPC-96, may be administered weekly for the first 4 doses, and then every two weeks for 2-8 additional doses.

Additional examples of HSPPC complexes that can be used in accordance with the methods described in this application are disclosed in the following patents and patent applications, which are incorporated into this application by reference in their entirety, US patents 6391306, 6383492, 6403095, 6410026, 6436404, 6447781 and 6610659.

In some embodiments, the present invention relates to: (a) an anti-CTLA-4 antibody of the invention and/or a pharmaceutical composition of the invention comprising an anti-CTLA-4 antibody of the invention and a pharmaceutically acceptable carrier or excipient, and (b) a vaccine, for use as a medicine. In a preferred embodiment, the vaccine is a heat shock protein antitumor vaccine or a heat shock protein antipathogenic vaccine. In a preferred embodiment, the vaccine is a heat shock protein-based antiviral vaccine.

In some embodiments, the invention relates to: (a) an anti-CTLA-4 antibody of the invention and/or a pharmaceutical composition of the invention comprising an anti-CTLA-4 antibody of the invention and a pharmaceutically acceptable carrier or excipient, and (b) a vaccine, for use in the treatment of cancer. In a preferred embodiment, the vaccine is a heat shock protein-based cancer vaccine.

The anti-CTLA-4 antibody and the additional therapeutic agent (eg, chemotherapeutic agent, checkpoint targeting agent, IDO inhibitor, and/or vaccine) may be administered separately, sequentially, or in parallel, as 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 disclosed herein is administered intratumorally to a subject. In some embodiments, an anti-CTLA-4 antibody disclosed herein is administered intratumorally to a subject in combination with an additional therapeutic agent. In some embodiments, the 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-EGFR antibody (eg, cetuximab). In some embodiments, the additional therapeutic agent that is administered systemically is an anti-HER2m antibody (eg, trastuzumab). In some embodiments, the 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 administered systemically is an anti-PD-1 antibody (eg, pembrolizumab or nivolumab). In some embodiments, the subject has head and neck squamous cell carcinoma (HNSCC) and the additional therapeutic agent administered systemically is an anti-EGFR 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 receives a chemotherapeutic agent (eg, gemcitabine). In one aspect, the present invention relates to an anti-CTLA-4 antibody and/or a pharmaceutical composition of the present invention, and optionally an additional therapeutic agent, for use in a method of treating cancer, wherein the anti-CTLA-4 antibody and/or the pharmaceutical composition of the present invention is administered subject intratumorally. In one preferred embodiment, the additional therapeutic agent is administered to the subject, more preferably the additional therapeutic agent is administered systemically to the subject.

In some embodiments, the implementation of the antibody against PD-1 is used in the methods disclosed in this application. In some embodiments, the anti-PD-1 antibody is nivolumab,

- 39 039322 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.

Other non-limiting examples of antibodies against PD-1 that can be used in the methods of treatment disclosed in this application are disclosed in the following patents and patent applications, which are fully incorporated in this application by reference in all respects: US patent 6808710; US Pat. No. 7,332,582; US patent 7488802; US Pat. No. 8,008,449; US patent 8114845; US Pat. No. 8,168,757; US Pat. No. 8,354,509; US Pat. No. 8,686,119; US Pat. No. 8,735,553; US Pat. No. 8,747,847; US Pat. No. 8,779,105; US Pat. No. 8,927,697; US Pat. No. 8,993,731; US patent 9102727; US patent 9205148; U.S. Patent Application Publication US 2013/0202623 A1; U.S. Patent Application Publication US 2013/0291136 A1; publication of US patent application US 2014/0044738 A1; U.S. Patent Application Publication US 2014/0356363 A1; U.S. Patent Application Publication US 2016/0075783 A1; and PCT Publication WO 2013/033091 A1; PCT Publication WO 2015/036394; 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, the implementation of the antibody against PD-L1 is used in the methods disclosed in this application. In some embodiments, the anti-PD-L1 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-PDL1 antibody is AMP-224 developed by Amplimmune and GSK.

Non-limiting examples of antibodies against PD-L1 that can be used in the methods of treatment disclosed in this application are disclosed in the following patents and patent applications, which are fully incorporated into this application by reference in all respects: US patent 7943743; US Pat. No. 8,168,179; US Pat. No. 8,217,149; US Pat. No. 8,552,154; US patent 8779108; US Pat. No. 8,981,063; US patent 9175082; publication of US patent application US 2010/0203056 A1; publication of US patent application US 2003/0232323 A1; U.S. Patent Application Publication US 2013/0323249 A1; U.S. Patent Application Publication US 2014/0341917 A1; publication of US patent application US 2014/0044738 A1; U.S. Patent Application Publication US 2015/0203580 A1; U.S. Patent Application Publication US 2015/0225483 A1; U.S. Patent Application Publication US 2015/0346208 A1; U.S. Patent Application 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 disclosed herein. In some embodiments, the anti-LAG-3 antibody is BMS986016 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 antibodies against LAG-3 that can be used in the treatments disclosed in this application are disclosed in the following patents and patent applications, which are fully incorporated into this application by reference in all respects: US patent 9,244,059; publication of US patent application US 2011/0150892 A1; publication of US patent application US 2014/0093511 A1; U.S. Patent Application Publication US 2014/0286935 A1; U.S. Patent Application 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-EGFR antibody is used in the methods disclosed herein. In some embodiments, the anti-EGFR antibody is cetuximab from Bristol-Myers Squibb and ImClone, panitumumab from Abgenix and Amgen, or

- 40 039322 motuzumab developed by CMI Cuba and BioSciences YM, 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, MM151 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.

In some embodiments, the implementation of the antibody against HER2 is used in the methods disclosed in this application. 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 by Pfizer, MM-302 by Merrimack, SB3 by Merck & Co, CMAB302 by Shanghai CP Guojian, TrasGEX by Glycotope, ARX788 by Ambrx and Zhejiang Medicine, SYD985 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 in this application can be delivered to the subject in a variety of ways. These include, but are not limited to, parenteral, intranasal, endotracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, and subcutaneous routes. Pulmonary administration, for example by means of an inhaler or nebulizer, and incorporation into the dosage form of a propellant for use as a spray may also be used. In some embodiments, the antibody or pharmaceutical composition described herein is delivered subcutaneously or intravenously. In some embodiments, the antibody or pharmaceutical composition described herein is delivered intratumorally. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is delivered to a tumor-draining lymph node. In some embodiments, the antibody or pharmaceutical composition described herein is delivered via localized administration (eg, subcutaneous administration). In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is delivered systemically. In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition disclosed herein is delivered locally.

In one aspect, the present invention provides an anti-CTLA-4 antibody and/or a pharmaceutical composition of the present invention, and optionally an additional therapeutic agent, for use in a method of treating cancer, wherein the anti-CTLA-4 antibody and/or the pharmaceutical composition of the present invention deliver the subject intratumorally, delivered to the subject's tumor-draining lymph node, or delivered by 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 will depend on the nature of the disease and can be determined using standard clinical methods.

The exact dose to be used in the composition will also depend on the route of administration and the severity of the infection or disease caused by it, and should be determined according to the judgment of the skilled person and the circumstances of each subject. For example, effective doses may also vary depending on the routes of administration, the site of interest, the physiological state of the patient (including age, body weight, and health status), whether the patient is a human or animal, other drugs used, or whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, may also be treated. Doses in treatment are optimally titrated to optimize safety and efficacy.

In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to the subject (e.g., by intravenous injection) in an amount of 0.1, 0.3, 1, 3, 6, 10, 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, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to the subject (eg, by intravenous injection) once every three weeks at the doses described above.

In one aspect, the present invention provides an anti-CTLA-4 antibody and/or a pharmaceutical composition of the present invention, and optionally an additional therapeutic

- 41 039322 to a cancer agent, for use in a method of treating cancer, wherein the anti-CTLA-4 antibody and/or the pharmaceutical composition of the present invention is administered to a subject in an amount of 0.1, 0.3, 1, 3, 6, 10, approximately 0, 1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 3 mg/kg, about 6 mg/kg, or about 10 mg/kg, more preferably every three weeks.

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

In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject via intratumoral injection in an amount of 0.01, 0.03, 0.1, 0.3, 1, 3, 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, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject via intratumoral injection once every three weeks at the doses described above.

In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject via intratumoral injection at 0.01 mg/kg or about 0.01 mg/kg every three weeks. In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject via intratumoral injection at 0.03 mg/kg or about 0.03 mg/kg every three weeks. In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject via intratumoral injection at 0.1 mg/kg or about 0.1 mg/kg every three weeks. In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject by intratumoral injection at 0.3 mg/kg or about 0.3 mg/kg every three weeks. In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject by intratumoral injection at 1 mg/kg or about 1 mg/kg every three weeks. In some embodiments, the anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject via intratumoral injection at 3 mg/kg or about 3 mg/kg every three weeks.

In some embodiments, an anti-CTLA-4 antibody or pharmaceutical composition described herein is administered to a subject via localized administration (e.g., subcutaneous administration) in an amount of 0.01, 0.03, 0.1, 0.3, 1, 3 , about 0.01 mg/kg, about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, or about 3 mg/kg.

The anti-CTLA-4 antibody described in this application can also be used to analyze the content of the CTLA-4 protein in a biological sample using classical immunohistochemical methods known to those skilled in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation or Western -blotting.

Suitable labels for antibody analysis 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 labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, as well as biotin. Such labels can be used to label the antibody or antigen-binding fragment described in this application. In the alternative, a second antibody that recognizes an anti-CTLA-4 antibody or antigen-binding fragment thereof as described herein can be labeled and can be used in combination with an anti-CTLA4 antibody or antigen-binding fragment thereof to determine CTLA-4 protein levels. In one embodiment, the present invention relates to the use of an anti-CTLA-4 antibody of the invention for the analysis and/or determination of CTLA-4 protein levels in a biological sample in vitro.

- 42 039322

Assaying the level of CTLA-4 protein expression is intended to involve a qualitative or quantitative measurement or assessment of the level of CTLA-4 protein in the first biological sample, either directly (for example, when determining or assessing the absolute protein content) or relatively (for example, when compared with the level of associated with protein disease in the second biological sample). The level of expression of the CTLA-4 polypeptide in the first biological sample can be measured or estimated and compared with a standard level of CTLA-4 protein, and the standard is obtained from a second biological sample obtained from an individual who does not have a disorder, or is determined by averaging levels in a group of individuals who do not having a violation. As will be known from the prior art, if the standard level of the CTLA-4 polypeptide is known, it can be repeatedly used as a standard for comparison.

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

The anti-CTLA-4 antibody or antigen-binding fragment thereof described herein can be used for prognostic, diagnostic, monitoring and screening applications, including in vitro and in vivo applications known and standard to those skilled in the art and based on the present disclosure. Prognostic, diagnostic, monitoring and screening assays and kits for assessing and evaluating the status of the immune system and/or immune response in vitro can be used for prognosis, diagnosis and monitoring to evaluate patient samples, including those known to have had or were suspected of having an immune system dysfunction, or compared to an expected or required immune system response, antigen response, or vaccine response. Evaluation and determination of the status of the immune system and/or immune response may also be used in determining the suitability of a patient for a clinical drug trial or for the administration of a particular chemotherapeutic agent or antibody, or antigen-binding fragment, including combinations thereof, against another agent or antibody, or its antigen-binding fragment. This type of prognostic and diagnostic monitoring and evaluation has already been applied in practice with antibodies against the HER2 protein in breast cancer (HercepTest™, Dako), where such an analysis is also used to evaluate patients in the case of antibody therapy using Herceptin®. In vivo applications include targeted cell therapy and immune system modulation, as well as radioimaging of immune responses.

In one aspect, the invention provides an anti-CTLA-4 antibody and/or a pharmaceutical composition of 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 of 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 invention for predicting, diagnosing and/or monitoring immune system dysfunction and/or cancer in a subject by analyzing and/or determining levels of CTLA-4 protein in a biological sample of the subject in vitro .

In one embodiment, the anti-CTLA-4 antibody, or antigen-binding fragment thereof, can be used in the immunohistochemistry of biopsy specimens. In another embodiment, an anti-CTLA-4 antibody, or antigen-binding fragment thereof, can be used to determine levels of CTLA-4 or levels of cells that contain CTLA-4 on their membrane surface, where such levels can then be correlated with symptoms of a particular disease. Anti-CTLA-4 antibodies or antigen-binding fragments described herein may carry a detectable or functional label. In the case of using fluorescent labels, currently available microscopy and fluorescent activated cell sorting (FACS) analysis, or a combination of both methods known in the art, can be used to identify and quantify specific binding participants. The anti-CTLA-4 antibodies, or antigen-binding fragments thereof, described herein 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, Cy dyes, and DyLight dyes. The anti-CTLA-4 antibody or antigen-binding fragment thereof may be radioactively labeled, 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, ⁿ¹ 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 techniques 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 label is an enzyme, detection can be performed using any of the colors currently in use.

- 43 039322 metric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric methods known in the art. This can be achieved by contacting the sample or control sample with the anti-CTLA-4 antibody or antigen-binding fragment thereof under conditions that allow the formation of a complex between the antibody or antigen-binding fragment and CTLA-4. Any complexes formed between the antibody or its antigen-binding fragment and CTLA-4 are detected and compared in sample and control. In view of the specific binding of the antibodies described in this application to CTLA-4, such antibodies or antigen-binding fragments can be used to specifically detect the expression of CTLA-4 on the surface of cells. The antibodies or antigen-binding fragments thereof described herein can also be used to purify CTLA-4 by immunoaffinity purification. Also included in the present application is an assay system that can be prepared in the form of a test kit for quantitative analysis of the degree of presence of, for example, CTLA-4 or CTLA-4/CTLA-4 ligand complexes. The system or test kit may include a labeled component, such as a labeled antibody, and one or more additional immunochemical reagents.

In one embodiment, the present invention provides a method for in vitro analysis and/or determination of CTLA-4 protein levels in a biological sample, comprising: (1) contacting the sample, and optionally a control sample, with an anti-CTLA-4 antibody or antigen-binding fragment thereof according to the invention under conditions that allow 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, control.

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

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

As used herein, an isolated polynucleotide or nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source (eg, mouse or human) of the 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 if produced by recombinant methods, or substantially free of chemical precursors or other chemical compounds if chemically synthesized. For example, the expression essentially does not include polynucleotide preparations or nucleic acid molecules containing less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular, less than about 10%) of other material, for example, cell material, culture medium, other nucleic acid molecules, chemical precursors, and/or other chemical compounds. In a certain embodiment, the nucleic acid molecule(s) encoding the antibody described in this application is isolated or purified.

In certain aspects, this application provides polynucleotides comprising nucleotide sequences encoding antibodies or antigen-binding fragments thereof that specifically bind to a CTLA-4 polypeptide (for example, human CTLA-4) and include an amino acid sequence as described in this application, 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 some aspects, this application provides polynucleotides comprising a nucleotide sequence encoding a light chain or a heavy chain of an antibody described in this application. Polynucleotides may include nucleotide sequences encoding a light chain, including VL FR regions and CDR regions of the antibodies described in this application (see, for example, table. 1).

Also provided herein are polynucleotides encoding an anti-CTLA-4 antibody that are optimized, for example, by codon/RNA optimization, replacement with heterologous signal sequences, and removal of mRNA instability elements. Methods for obtaining optimized nucleic acids encoding an anti-CTLA-4 antibody or fragment (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon substitutions and/or removing inhibitory regions in mRNA, can be carried out by adapting the optimization methods described, for example, in US patents 5965726; 6174666; 6291664; 6414132; and 6794498 respectively. For example, potential splicing sites and elements

- 44 039322 instabilities (eg A/T or A/U rich elements) in RNA can be mutated without changing the amino acids encoded by the nucleic acid sequences, increasing the stability of the RNA for recombinant expression. Such changes exploit the degeneracy of the genetic code, for example by using an alternative codon for an identical amino acid. In some embodiments, one or more codons encoding a conservative mutation may need to be changed, for example, a similar amino acid with similar chemical structure and properties and/or function as the original amino acid. Such methods can increase the expression of an anti-CTLA-4 antibody or fragment thereof by at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 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., VL domain and/or 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 some embodiments, an optimized nucleotide sequence encoding an anti-CTLA-4 antibody described herein, or a fragment thereof, is hybridized under highly stringent conditions to an antisense polynucleotide of a non-optimized polynucleotide sequence encoding an anti-CTLA-4 antibody described herein, or a fragment thereof. fragment. In a particular embodiment, an optimized nucleotide sequence encoding an anti-CTLA-4 antibody described herein, or a fragment thereof, hybridizes under high, intermediate, or lower stringency hybridization conditions to an antisense polynucleotide of a non-optimized nucleotide sequence encoding an anti-CTLA-4 antibody, described in this application, or a fragment thereof. Information regarding hybridization conditions has been described, for example, in US Patent Application Publication US 2005/0048549 (eg, paragraphs 72-73), which is incorporated herein by reference.

Polynucleotides can be obtained and the nucleotide sequence of the polynucleotides determined using any method known in the art. Nucleotide sequences encoding the antibodies described in this application, such as the antibodies described in table. 1, as well as modified variants of such antibodies, can be determined using methods known in the art, i. nucleotide codons known to code for particular amino acids are assembled in such a way as to produce a nucleic acid that codes for an antibody. Such an antibody-coding polynucleotide can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G. et al. (1994), BioTechniques 17: 242-6), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding an antibody, annealing and ligation of such oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

In the alternative, a polynucleotide encoding an antibody described in this application can be obtained from a nucleic acid from a suitable source (eg, hybridoma) using methods known in the art (eg, PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizing 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 techniques can be used to generate nucleic acids comprising a sequence encoding the light chain and/or heavy chain of an antibody. Such PCR amplification methods can be used to produce nucleic acids comprising a sequence encoding a light chain variable region and/or an antibody heavy chain variable region. The amplified nucleic acids can be cloned into vectors for expression in host cells and for subsequent cloning, for example to produce chimeric and humanized antibodies.

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

DNA encoding the anti-CTLA-4 antibodies described herein can be easily

- 45 039322 was isolated and sequenced using standard techniques (for example, using oligonucleotide probes that are able to specifically bind to the genes encoding the heavy and light chains of anti-CTLA-4 antibodies). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors that are then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells, which otherwise do not produce immunoglobulin protein, to obtain the synthesis of antibodies against CTLA-4 in recombinant host cells.

To obtain 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 γ4 constant region, and PCR amplified VL domains can be cloned into vectors expressing a light chain constant region, for example human k or λ constant regions. In some embodiments, vectors for expressing VH or VL domains include an EF-1a promoter, a secretion signal, a cloning site for the variable region, constant domains, and a selectable marker such as neomycin. Also, the VH and VL domains can be cloned into a single vector expressing the required constant regions. Heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to produce stable or transient cell lines that express full-length antibodies, such as IgG, using methods known to those skilled in the art.

The DNA can also be modified, for example, by replacing the murine sequences with the coding sequence for the human heavy and light chain constant domains, or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence.

Also provided are polynucleotides that hybridize under high, intermediate or lower stringency hybridization conditions with polynucleotides that encode an antibody described herein. In some embodiments, the polynucleotides described herein hybridize under high, intermediate, or lower stringency hybridization conditions with polynucleotides encoding the VH domain and/or VL domain provided herein.

Hybridization conditions have been described in the prior art and known to the person skilled in the art. For example, stringent hybridization may include hybridization to filter-bound DNA in 6x 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 high stringency conditions may include hybridization to the 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 hybridization conditions of a different stringency is known to those skilled in the art and has been described, for example, in Ausubel F.M. et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, at pp. 6.3.1-6.3.6 and 2.10.3.

In some aspects, the present application provides cells (e.g., host cells) that express (e.g., recombinantly) the antibodies described herein (or an antigen-binding fragment thereof) that specifically bind to CTLA-4 (e.g., human CTLA-4) , as well as the corresponding polynucleotides and expression vectors. The present application provides vectors (eg, expression vectors) comprising polynucleotides comprising nucleotide sequences encoding anti-CTLA-4 antibodies or a fragment for recombinant expression in host cells, preferably mammalian cells. Also provided herein are host cells comprising such vectors for recombinant expression of the anti-CTLA-4 antibodies described herein (eg, a human or humanized antibody). In a certain aspect, the present application provides methods for producing an antibody described in this application, including the expression of such an antibody from a host cell.

Recombinant expression of an antibody described herein (e.g., full length antibody, heavy and/or light chain antibody, or single chain antibody described herein) that specifically binds to CTLA-4 (e.g., human CTLA-4) involves constructing a vector expression containing a polynucleotide that encodes an antibody. After obtaining a polynucleotide encoding an antibody molecule, heavy and/or light chain of an antibody or a fragment thereof (for example, heavy and/or light chain variable regions) described in this application, a vector for the production of an antibody molecule can be obtained using recombinant DNA technology with using methods known in the art. Thus, methods for obtaining protein,

- 46 039322 expressing polynucleotide containing encoding an antibody or antibody fragment (eg light chain or heavy chain) nucleotide sequence are described in this application. Methods that are known to those skilled in the art can be used to construct expression vectors containing antibody or antibody fragment (eg light chain or heavy chain) encoding sequences and appropriate transcriptional and translational regulation signals. Such methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicating vectors comprising 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, international publications WO 86/05807 and WO 89/01036; and US patent 5122464), while the variable regions of the antibody can be cloned into such a vector for the expression of a full-length heavy, full-length light chain, or full-length heavy and light chains.

The expression vector can be transferred into a cell (eg, a host cell) using standard methods, and the resulting cells can then be cultured using standard methods to obtain an antibody described in this application, or a fragment thereof. Thus, this application provides host cells containing a polynucleotide encoding an antibody described in this application, or fragments thereof, or a heavy or light chain, or a fragment thereof, or a single-chain antibody described in this application, operably linked to a promoter to express such sequences in the host cell. In some embodiments, for the expression of double chain antibodies, vectors encoding the heavy and light chains separately can be co-expressed in a host cell to express the entire immunoglobulin molecule, as detailed below. In some embodiments, the implementation of the host cell contains a vector comprising a polynucleotide encoding the heavy chain and light chain of the antibody described in this application, or a fragment thereof. In some embodiments, the host cell comprises two different vectors, the first vector comprising a polynucleotide encoding the heavy chain or variable region of the antibody described herein, or a fragment thereof, and the second vector comprising a polynucleotide encoding the light chain or variable region of the light chain of the antibody described in this application, or a fragment thereof. In other embodiments, the first host cell comprises a first vector comprising 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 comprising a second vector comprising a polynucleotide encoding a light chain or the variable region of the light chain of the antibody described in this application. In certain embodiments, the heavy chain/heavy chain variable region expressed by the first cell associates with 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. In some embodiments, provided herein is a population of host cells comprising such a first host cell and such a second host cell.

In a specific embodiment, the present application provides a population of vectors comprising a first vector comprising a polynucleotide encoding the light chain/light chain variable region of an anti-CTLA-4 antibody described herein and a second vector comprising a polynucleotide encoding the heavy chain/variable region the heavy chain of the anti-CTLA-4 antibody described herein.

Many systems of host organisms/expression vectors can be used to express the antibody molecules described in this application (see, for example, US patent 5807715). Such expression systems in host organisms are vehicles by which coding sequences of interest can be generated and then purified, but are also cells which, when transformed or transfected with the appropriate coding nucleotide sequences, can express an antibody molecule as described herein. , in situ. These include, but are not limited to, microorganisms such as bacteria (eg, E. coli and B. subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody-coding sequences; yeast (eg Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; systems based on insect cells infected with recombinant viral expression vectors (eg, baculovirus) containing sequences encoding the antibody; plant cell systems (e.g. green algae such as Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g. cauliflower mosaic virus, CaMV; tobacco mosaic virus, BTM) or transformed with recombinant plasmid expression vectors (e.g. Ti plasmid ) containing sequences encoding the antibody; or

- 47 039322 mammalian cell systems (e.g. COS cells (e.g. COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa and NIH 3T3, HEK-293T , HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, and BMT10) carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (for example, the metallothionein promoter) or from mammalian viruses (for example, adenovirus late promoter; 7.5K vaccinia promoter). In a particular embodiment, the cells for expressing the antibodies described herein, or an antigen-binding fragment thereof, are CHO cells, eg CHO cells from the CHO GS System™ (Lonza). In a specific embodiment, the cells for expressing the antibodies described herein are human cells, eg, 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), especially for expression of an entire recombinant antibody molecule, are used to express the recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, in combination with a vector such as the major immediate early promoter gene element from human cytomegalovirus, are an efficient expression system for antibodies (Foecking M.K. & Hofstetter H. (1986) Gene 45: 101-5 and Cockett M. I. et al (1990) Biotechnology 8 (7): 6627). In some embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In a particular embodiment, 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.

In bacterial systems, a range of expression vectors can advantageously be selected depending on the intended use for the antibody molecule to be expressed. For example, if a large amount of such an antibody is to be produced, pharmaceutical formulations of the antibody molecule may require vectors that direct the expression of high levels of fusion protein products that can be readily purified. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794), wherein the antibody coding sequence can be separately ligated into the vector in frame with the coding region. lac Z, producing a fusion protein; pIN vectors (Inouye S & Inouye M. (1985) Nuc. Acids. Res. 13: 3101-3109; Van Heeke G. & Schuster S. M. (1989) J. Biol. Chem. 24: 5503-5509) and the like . For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione-5-transferase (GST). Typically, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding to a matrix of glutathione-agarose beads, followed by elution in the presence of free glutathione. The pGEX vectors include cleavage sites for thrombin or factor Xa proteases, whereby the cloned target gene product can be freed from the GST molecule.

In an insect cell system, Autographa californica nucleated polyhedrosis virus (AcNPV), for example, can be used as a vector for the expression of foreign genes. The virus replicates in the cells of Spodoptera frugiperda. An antibody coding sequence can be cloned individually into non-essential regions (eg, the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (eg, the polyhedrin promoter).

In mammalian cells, a variety of virus-based expression systems can be used. In cases where an adenovirus is used as the expression vector, the antibody sequence of interest can be ligated to an adenovirus transcription/translation control complex, such as a late promoter sequence and a triple leader sequence. Such a chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., E1 or E3 region) produces a recombinant virus that is viable and capable of expressing the antibody molecule in infected host cells (e.g., see Logan J. & Shenk T. (1984) PNAS 81 (12) : 3655-9). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. Such signals include the start codon ATG and adjacent sequences. In addition, the start codon must match the reading frame of the desired coding sequence to ensure translation of the entire insert. Such exogenous translation control signals and initiation codons may come from a variety of sources, both natural and synthetic. Expression efficiency can be increased by including suitable transcriptional enhancer elements, transcription terminators, etc. (See, for example, Bitter G. et al. (1987) Methods Enzymol. 153: 516-544).

In addition, a strain of host cells can be selected that modulate the expression of the introduced sequences or modify and process the gene product in the specific manner desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of a protein

- 48 039322 output products may be important for protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host cell 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 possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product. Such mammalian cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 cells (a mouse myeloma cell line that does not endogenously produce immunoglobulins), CRL7O3O, COS (eg 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 certain embodiment, the antibodies described in this application, or their antigennegative fragments have a reduced content of fucose or do not contain fucose. Such antibodies can be obtained using methods known to the person skilled in the art. For example, antibodies can be expressed in cells deficient or lacking the ability to fucosylate. In a specific example, cell lines that knock out both alleles of a.1,6-fucosyltransferase can be used to generate antibodies or antigen-binding fragments thereof with reduced fucose content. The Potelligent® system (Lonza) is an example of such a system that can be used to produce antibodies or antigen-binding fragments thereof with a reduced fucose content.

For long-term production of recombinant proteins in high yield, stable expressing cells can be obtained. For example, cell lines can be created that stably express an anti-CTLA-4 antibody described herein, or an antigen-binding fragment thereof. In some embodiments, the present application provides a cell that stably expresses a light chain/light chain variable region and a heavy chain/heavy chain variable region that are associated to form an antibody of the present application, or an antigen-binding fragment thereof.

In some aspects, instead of using expression vectors that contain viral origins of replication, host cells can be transformed with DNA under the control of appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and selective marker. After the introduction of foreign DNA/polynucleotide, recombinant cells can grow for 1-2 days in enriched media, and then the media is replaced with selective media. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form plaques, which in turn can be cloned and expanded into cell lines. This method can be advantageously used to create cell lines that express the anti-CTLA-4 antibody described in this application, or a fragment thereof. Such recombinant cell lines may be particularly useful 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, herpes simplex virus thymidine kinase (Wigler M. et al. (1977) Cell 11(1): 223-32), hypoxanthineguanine phosphoribosyltransferase (Szybalska EH & Szybalski W. (1962) PNAS 48(12): 2026-2034) and adenine phosphoribosyltransferase (Lowy I. et al. (1980) Cell 22(3): 817-23) in tk, hgprt or aprt cells, respectively. In addition, antimetabolite resistance can 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: 1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan R.C. & Berg P. (1981) PNAS 78(4): 2072-6); neo which confers resistance to the aminoglycoside G-418 (Wu G.Y. & Wu C.H. (1991) Biotherapy 3: 87-95; Tolstoshev P. (1993) Ann. Rev. Pharmacol. Toxicol. 32: 573-596; Mulligan R. C. (1993 ) Science 260: 926-932 and Morgan R. A. & Anderson W. F. (1993) Ann. Rev. Biochem. 62: 191-217; Nabel G. J. & Feigner P. L. (1993) Trends Biotechnol. 11(5): 211-5); and hygro, which confers resistance to hygromycin (Santerre R.F. et al. (1984) Gene 30(1-3): 147-56). Methods well known in the field of recombinant DNA technology can be routinely used to select the desired recombinant clone, and such methods are described, for example, in Ausubel F.M. et al. (eds.) Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M., Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and also in Chapters 12 and 13, Dracopoli N.C. et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbere-Garapin F. et al. (1981) J. Mol. Biol. 150:1-14, which are incorporated herein by reference in their entirety.

Expression levels of an antibody molecule can be increased by increasing the copy number of the vector (see the review by Bebbington C.R. & Hentschel C.C.G. The use of vectors based on gene amplification for the ex

- 49 039322 expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). If the marker in the antibody expression vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is linked to the antibody gene, antibody production will also increase (Crouse G.F. et al. (1983) Mol. Cell. Biol. 3: 257-66).

The host cell can be co-transfected with two or more expression vectors described herein, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. These two vectors may contain identical selectable markers that provide equal expression of the heavy and light chain polypeptides. Host cells can be co-transfected with varying amounts of these two or more expression vectors. For example, host cells can be transfected at any of the following ratios of first expression vector to second expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1: 8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 1:50.

Alternatively, a single vector can be used that encodes, and is capable of expressing, heavy and light chain polypeptides. In such situations, the light chain must be placed before the heavy chain to avoid excess toxic free heavy chain (Proudfoot N. J. (1986) Nature 322: 562565; and Kohler G. (1980) PNAS 77: 2197-2199). Heavy and light chain coding sequences may include cDNA or genomic DNA. The 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, or in the range of 2-5, 5-10, or 10-20, genes/nucleotide sequences. For example, a bicistronic nucleic acid construct may include, in the following 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 directed by a promoter, while translation of mRNA from the first gene can proceed according to a cap-dependent scanning mechanism, and translation of mRNA from the second gene can proceed according to a cap-independent mechanism, for example, using IRES.

Once an antibody molecule described herein has been obtained by recombinant expression, it can be purified by any method known in the art for purifying an immunoglobulin molecule, e.g. protein A antigen, and size-separated column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. In addition, the antibodies described in this application can be fused with heterologous polypeptide sequences described in this application or otherwise known in the art, to facilitate purification.

In some embodiments, the implementation of the antibody or antigennegative fragment described in this application, selected or purified. Typically, an isolated antibody is one that is substantially free of other antibodies with a different antigenic specificity than the isolated antibody. For example, in a certain embodiment, the preparation of the antibody described in this application, essentially does not contain cellular material and/or chemical precursors. The expression essentially does not contain cellular material includes antibody preparations in which the antibody is separated from the cellular components of the cells from which it is isolated or recombinantly obtained. Thus, an antibody that is substantially free of cellular material includes antibody preparations containing less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of the heterologous protein. (also referred to herein as a contaminant protein) and/or antibody variants, eg, other post-translationally modified forms of the antibody, or other distinct antibody variants (eg, antibody fragments). In the case where the antibody is produced recombinantly, it also typically contains essentially no culture medium, i.e. the culture medium is less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% by volume of the protein preparation. In the case where the antibody is obtained by chemical synthesis, it usually essentially does not contain chemical precursors or other chemical compounds, i. it is separated from chemical precursors or other chemical compounds that are involved in protein synthesis. Thus, such antibody formulations 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 certain embodiment, the antibodies described in this application are isolated or purified.

Antibodies or fragments thereof that specifically bind to CTLA-4 (eg, human CTLA-4) can be made by any method known in the art of antibody synthesis, eg, chemical synthesis or recombinant expression techniques. The methods described in this application apply, unless otherwise indicated, standard methods of molecular biology, microbiology, genetic analysis, genetic engineering, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, as well as from related fields in the level of technology. Such methods are described, for example, in the sources cited in this

- 50 039322 application, and fully disclosed 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 and annual editions); Current Protocols in Immunology, John Wiley & Sons (1987 and annual editions) 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.

In a particular embodiment, an antibody described herein is an antibody (e.g., a recombinant antibody) obtained, expressed, generated, or isolated by any method, which includes the creation, for example, through synthesis, genetic engineering, of DNA sequences. In some embodiments, such an antibody includes sequences (eg, DNA sequences or amino acid sequences) that do not normally exist in the animal or mammalian (eg, human) germline antibody repertoire in vivo.

In one aspect, the present application provides a method for producing an antibody or antigen-binding fragment that specifically binds to CTLA-4 (eg, human CTLA-4), comprising culturing a cell or host cell described in this application. In a certain aspect, this application provides a method for producing an antibody or antigen-binding fragment that specifically binds to CTLA-4 (for example, human CTLA-4), including expression (for example, recombinant expression) of the antibody or antigen-binding fragment using a cell or cell - the host described in this application (for example, cells or cells of the host, including polynucleotides encoding the antibody described in this application). In a specific embodiment, the cell is an isolated cell. In a particular embodiment, exogenous polynucleotides have been introduced into the cell. In a specific embodiment, the method further comprises the step of purifying an antibody or antigen-binding fragment thereof derived from a host cell or cell. Preferably the method is carried out in vitro.

Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 in Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel F. M. et al., eds., John Wiley and Sons, New York).

Monoclonal antibodies can be obtained using a variety of methods known in the art, including the use of hybridoma, recombinant technologies and phage display technologies, or combinations thereof. For example, monoclonal antibodies can be made using hybridoma technologies, 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). 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 some embodiments, a monoclonal antibody as used herein is an antibody produced by a single cell (e.g., a hybridoma or a host cell producing a recombinant antibody), wherein the antibody specifically binds to CTLA-4 (e.g., human CTLA-4) as defined, for example , using ELISA or other antigen binding assay or competitive binding assay known in the art or provided in the examples below. In some 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). Monoclonal antibodies described in this application can be, for example, obtained using the hybridoma method, as described in Kohler G. & Milstein C. (1975) Nature 256: 495, or can be, for example, isolated from phage libraries using methods , as described, for example, in this application. Other methods for obtaining clonal cell lines and monoclonal antibodies expressed in this way are known in the art (see, for example, Chapter 11 in Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

Methods for obtaining and screening specific antibodies using hybridoma technology are standard and 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 macaque is immunized to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to a protein (e.g., CTLA -4 (e.g. human CTLA-4)), using

- 51 039322 immunization wash. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable membrane fusion agent such as polyethylene glycol to form a hybridoma cell (Goding J.W. (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). In addition, RIMMS (multiple immunization at multiple sites) technology can be used to immunize an animal (Kilpatrick KE et al. (1997) Hybridoma 16:381-9, incorporated by reference in its entirety).

In some embodiments, mice (or other animals such as rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) may be immunized with an antigen (e.g., CTLA-4 (e.g., human CTLA-4)), and upon detection of immune response, for example, antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and the splenocytes are isolated. The splenocytes are then fused using known methods with the membranes of any suitable myeloma cells, for example, cells from the SP20 cell line available from the American Type Culture Collection (ATCC®) (Manassas, VA) to form hybridomas. Hybridomas are selected and cloned by serial dilution. In some embodiments, lymph nodes from immunized mice are harvested and fused with NS0 myeloma cells.

The hybridoma cells thus obtained are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused original myeloma cells. For example, if the original myeloma cells do not have the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas will typically include hypoxanthine, aminopterine, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells.

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

The culture medium in which the hybridoma cells grow is examined for the production of monoclonal antibodies directed against CTLA-4 (eg, human CTLA-4). The binding specificity of monoclonal antibodies produced by hybridoma cells is determined using methods known in the art, such as immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme immunoassay (ELISA).

Once hybridoma cells have been identified that produce antibodies with the desired specificity, affinity and/or activity, clones can be subcloned by serial dilution and grown using 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 medium. In addition, hybridoma cells can be grown in vivo as ascitic tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascitic fluid or serum by standard immunoglobulin purification techniques such as, for example, protein A-sepharose, 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 may be generated using any technique known to those skilled in the art. For example, Fab and F(ab') ₂ fragments described in this application can be obtained by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to obtain Fab fragments) or pepsin (to obtain F(ab') ₂ fragments). The Fab domain corresponds to one of two identical arms of the antibody molecule and contains the entire light chain linked to the VH and CH1 domains of the heavy chain. The F(ab') ₂ fragment contains two antigen-binding arms of the antibody molecule linked by disulfide bridges in the hinge region.

In addition, the antibodies described in this application, or their antigennegative fragments can also be obtained using various methods of phage display known in the prior art. In phage display methods, the functional domains of an antibody are exposed on the surface of phage particles that 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 cDNA libraries of diseased tissues). DNA encoding the VH and VL domains is recombined with the scFv linker by PCR and cloned into the phagemid

- 52 039322 vector. The vector is electroporated into E. coli and infected with E. coli helper phage. The phage used in such methods is typically filamentous phage, including fd and M13, and the VH and VL domains are typically recombinantly fused to either the phage III gene or the VIII gene. A phage expressing an antigen binding domain that binds to a particular antigen can be selected for or identified with the antigen, for example by using a labeled antigen or an antigen bound or fixed to a solid surface or sphere. Examples of phage display include methods that can be used to obtain antibodies described in this application, include the methods disclosed in Brinkman U. et al. (1995) J. Immunol. methods. 182:41-50; Ames R.S. et al. (1995) J. Immunol. Methods 184: 177-186; Kettleborough C.A. et al. (1994) Euro. J. Immunol. 24:952-958; Persic L. et al. (1997) Gene 187: 9-18; Burton D.R. & Barbas C.F. (1994) Advan. Immunol. 57:191-280; PCT Application PCT/GB91/001134; International Application Publications WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401 and WO 97/13844; and U.S. Patents 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,796, and, 5.8.

As described in the above references, once the phage has been selected, antibody coding regions from the phage can be isolated and used to generate complete 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 F(ab') ₂ fragments can also be used using methods known in the art, such as those disclosed in PCT publication WO 92/22324; Mullinax R.L. et al. (1992) BioTechniques 12(6): 864-9; Sawai H. et al. (1995) Am. J. Reprod. Immunol. 34:26-34; and Better M. et al. (1988) Science 240: 1041-1043.

In some embodiments, 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 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 a VH constant region, and PCR amplified VL domains can be cloned into vectors expressing a VL constant region, such as human k or λ constant regions. The VH and VL domains can also be cloned into a single vector expressing the desired constant regions. Heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to produce stable or transient cell lines that express full-length antibodies, such as 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 a constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison S.L. (1985) Science 229: 1202-7; Oi VT & Morrison S.L. (1986) BioTechniques 4: 214-221; Gillies S.D. et al. (1989) J. Immunol. methods. 125:191-202; and U.S. Patents 5,807,715, 4,816,567, 4,816,397 and 6,331,415.

A humanized antibody is capable of binding to a given antigen and includes a framework region essentially having the amino acid sequence of a human immunoglobulin and CDR regions essentially having the amino acid sequence of a non-human immunoglobulin (eg, mouse immunoglobulin). In certain embodiments, the humanized antibody also includes at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. The antibody may also include the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG ₂ , IgG ₃ and IgG4. Humanized antibodies can be obtained using a variety of techniques known in the art, including, but not limited to, CDR grafting (European patent EP 239400; International publication WO 91/09967; and US Pat. 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 shuffling (US Pat. No. 5,565,332), as well as the methods disclosed in, for example, US Pat. Tan P. et al. (2002) J. Immunol. 169:1119-25; Caldas C. et al. (2000) Protein Eng. 13(5): 35360; Morea V. et al. (2000) Methods 20(3): 267-79; Baca M. et al. (1997) J. Biol. Chem. 272(16): 10678-84; Roguska M.A. et al. (1996) Protein Eng. 9(10): 895 904; Couto JR et al. (1995) Cancer Res. 55 (23 Supp) : 5973s-5977s; Couto JR et al. (1995) Cancer Res. 55(8): 1717-22; Sandhu JS (1994) Gene 150(2): 409-10 and Pedersen JT et al. (1994) J. Mol. Biol. 235(3): 959-73. See also US Patent Application Publication US 2005/0042664 A1 (February 24, 2005), which is hereby incorporated by reference in its entirety.

- 53 039322

Methods for making multispecific (eg, bispecific) antibodies have been described, for example, in US Pat. Nos. 7,951,917; 7183076; 8227577; 5837242; 5989830; 5869620; 6132992 and 8586713.

Single domain antibodies, eg, antibodies lacking light chains, can be prepared using methods known in the art. See Riechmann L. & Muyldermans S. (1999) J. Immunol. 231:25-38; Nuttall S.D. et al. (2000) Curr. Pharm. Biotechnol. 1(3):253-263; Muyldermans S., (2001) J. Biotechnol. 74(4): 277-302; US patent 6005079; and International Publications WO 94/04678, WO 94/25591 and WO 01/44301.

In addition, antibodies that specifically bind to the CTLA-4 antigen can in turn be used to generate anti-idiotypic antibodies that mimic the antigen using techniques known to those skilled in the art. See, for example, Greenspan N.S. & Bona C.A. (1989) FASEB J. 7(5): 437-444; and Nissinoff A. (1991) J. Immunol. 147(8): 2429-2438.

In certain embodiments, the antibody described herein that binds to the same CTLA-4 epitope (e.g., human CTLA-4) as the anti-CTLA-4 antibody described herein is a human antibody or an antigen-binding fragment thereof. In certain embodiments, the 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 an antigen-binding fragment thereof. . Human antibodies can be obtained using any method known in the art. For example, transgenic mice that are not capable of expressing functional endogenous immunoglobulins but that can express human immunoglobulin genes can be used. In particular, complexes of human immunoglobulin heavy and light chain genes can be randomly or homologously recombined introduced into mouse embryonic stem cells. Alternatively, the human variable, constant and diversity regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. Mouse immunoglobulin heavy and light chain genes can be made non-functional alone or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, a homozygous deletion of the JH region interferes with the production of endogenous antibodies. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized with the selected antigen in the normal way, eg the whole antigen or part of the antigen (eg CTLA-4). Monoclonal antibodies directed against the antigen can be obtained from immunized transgenic mice using standard hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice are rearranged during B cell differentiation and then subjected to class switching and somatic mutation. Thus, using this technique, therapeutically useful IgG, IgA, IgM and IgE antibodies can be obtained. For an overview of this technology for producing human antibodies, see Lonberg N & Huszar D (1995) Int. Rev. Immunol. 13:65-93. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, as well as methods for producing such antibodies, see, for example, International publications WO 98/24893, WO 96/34096 and WO 96/33735; and US Pat. Mederex, Inc./Gen Pharm; US Patents 5,545,806 and 5,569,825), Trans Chromo Mouse™ (Kirin), and KM Mouse™ (Medarex/Kirin).

Human antibodies that specifically bind to CTLA-4 (eg, human CTLA-4) can be generated by a variety of methods known in the art, including the phage display methods described above, using antibody libraries derived from human immunoglobulin sequences. See also US Pat. Nos. 4,444,887, 4,716,111 and 5,885,793; 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.

In some embodiments, human antibodies can be generated 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 secreting human monoclonal antibodies, and such mouse-human hybridomas can be screened for hybridomas, which 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 prior art, for example, in Shinmoto H. et al. (2004) Cytotechnology 46: 19-23; Naganawa Y. et al. (2005) Human Antibodies 14:27-31.

5.6. Sets.

- 54 039322

Also provided are kits comprising one or more of the antibodies described herein, or a pharmaceutical composition or conjugate thereof. In a certain embodiment, this application provides a pharmaceutical package or kit comprising one or more containers filled with one or more components of the pharmaceutical compositions described in this application, such as one or more antibodies proposed in this application, or its antigen-binding fragment. In some embodiments, the kits contain a pharmaceutical composition as described herein 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 an antibody that stimulates the TKR complex, such as an anti-CD3 antibody and an anti-CD28 antibody. Optionally, such container(s) may be associated with a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, where such notice reflects the government's authorization of manufacture, use, or sale for human use.

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

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

6. Examples

The examples in this section (ie, Section 6) are offered by way of illustration and not limitation.

6.1. Example 1 Anti-CTLA-4 antibody assay.

This example describes the study of antibodies that bind to human CTLA-4. In particular, the antibody designated AGEN1884 was tested in a number of assays described below. The anti-CTLA-4 antibody, AGEN1884, comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 93 and a light chain comprising the amino acid sequence of SEQ ID NO: 15. AGEN1884 is a human IgG1 antibody containing a T109S substitution (i.e. replacement of threonine with serine at position 109 compared to the wild-type Fc sequence), when numbered according to Kabat, in the constant domain of the light chain, which facilitates the cloning of the variable region in reading frame with the constant region. This mutation is a conservative modification that does not affect antibody binding or function. A wild-type analog, designated AGEN1884w, was also generated, which contains a threonine at position 109, numbered according to Kabat. The AGEN1884w antibody is a human IgG1 antibody comprising the heavy chain of SEQ ID NO: 93 and the light chain of SEQ ID NO: 13.

6.1.1. Kinetic analysis using surface plasmon resonance.

Surface plasmon resonance was used to determine the affinity of anti-CTLA-4 antibody, AGEN1884 and reference anti-CTLA-4 IgG1 antibody (BIAcore® T100/T200 high sensitivity system (GE Healthcare) and Fab capture assay). All interactions were analyzed at 25°C using 1xDPBS (PAA, H15-002) plus P20 (0.05%, Pierce, 28320) as running buffer. Anti-CTLA-4 antibodies (8 μg/ml in running buffer) were captured on the surface of a CM5 sensor chip (GE Healthcare, Series S CM5, BR-1005-30) with an immobilized anti-human Fab antibody (GE Healthcare, Fab Capture Kit, 28958325) . To detect non-specific CTLA-4 antigen interactions, antibody capture was performed in flow cell 2 only, while in

- 55 039322 flow cell 1 was only immobilized capture antibody. Following capture of anti-CTLA-4 antibodies, CTLA-4 antigens were passed through both flow cells in varying amounts. In particular, recombinant human CTLA-4-Fc (R&D Systems, #7268-CT), recombinant human CTLA-4 (Sino Biological, #11159-H08H), recombinant cynomolgus monkey CTLA-4-Fc (Sino Biological, #90213- C02H) and recombinant cynomolgus monkey CTLA-4 (Sino Biological, # 90213-C08H) were skipped at 100, 25 and 6.25 nM; and recombinant mouse CTLA-4-Fc (R&D, #434-CT), recombinant rat CTLA-4-Fc (Sino Biological, # 81069-R02H) and recombinant rat CTLA-4 (Sino Biological, # 81069-R08H) were passed in amount of 400, 100 and 25 nM. A null curve (working buffer only) was also included in each assay. Association was performed for 90 s and dissociation for 600 s, at a flow rate of 10 μl/min. Each assay was followed by a regeneration step with 10 mM glycine, pH 2.0 (GE Healthcare, BR-1003-55) for 60 seconds at 30 μl/min. Binding curves were evaluated using the BIAcore® T200 scoring software version 2.0.1 using a 1:1 Langmuir model with a global Rmax fit. Based on these values, the affinities (K _D ) of AGEN1884 and the reference anti-CTLA-4 antibody for various antigens were calculated, which are shown in FIG. 1A. The recombinant cynomolgus monkey CTLA-4 monomer, mouse CTLA-4-Fc, and rat CTLA-4 monomer used in this study did not pass quality control evaluation.

The binding of anti-CTLA-4 antibodies to the recombinant mouse CTLA-4-Fc protein, which exhibited 50% aggregation, appeared to be due to non-specific interactions. Surface plasmon resonance analysis was later repeated twice using recombinant mouse CTLA-4-Fc (Sino Biological, # 81069-R02H), which passed the quality control, at 360, 120, 40 and 13.3 nM for AGEN1884w. AGEN1884w showed no detectable binding to mouse CTLA-4.

6.1.2. Binding of the antibody to cells overexpressing CTLA-4.

Jurkat cells overexpressing human (Promega) CTLA-4 were maintained in RPMI1640 (Life Technologies) containing 10% FBS (Gemini Bio Products), 100 μg/ml hygromycin B (Gibco) and 500 μg/ml G418 (Promega). Cells were washed once in FACS buffer (PBS with 2% FBS) before antibody staining. Serial dilutions of AGEN1884w, reference anti-CTLA-4 IgG1 antibody, and isotype control IgG1, starting at 10 µg/mL, were added to wells of 96-well round bottom well plates in duplicate, in a volume of 100 µL, and incubated for 30 min at 4°C. C. Cells were washed three times with 150 µl FACS buffer and stained in 100 µl FACS buffer containing 0.25 µl PE-conjugated mouse anti-human κ chain antibody (Invitrogen) for 30 min at 4°C. The cells were then washed twice and resuspended in 100 µl of FACS buffer. Samples were analyzed on LSRFortessa (BD Biosciences). Mean fluorescence intensity (MFI) values were analyzed using FlowJo software (FlowJo, LLC) and plotted using Prism 6 (GraphPad software). As shown in FIG. 1B, the human CTLA-4-coupled AGEN1884w antibody was expressed on the surface of Jurkat cells.

6.1.3. Analysis of the selectivity of antibodies to CTLA-4.

The selectivity of AGEN1884 for CTLA-4 was assessed over other members of the immunoglobulin superfamily using suspension chip technology as a multiplex analysis. A number of members of the immunoglobulin superfamily were chemically coupled to Luminex® microspheres using standard NHS ester chemistry. Purified AGEN1884, anti-CTLA-4 reference IgG1, and isotype control IgG1 materials were diluted in assay buffer (Roche 11112589001) to 10, 100, and 1000 ng/mL. Briefly, 25 μl of each dilution was incubated in the dark (20° C., 650 rpm) with 1500 Luminex® microspheres in 5 μl assay buffer, for 1 hour in 96-well filter membrane plates (Millipore, MABVN1250). Luminex® microspheres were crosslinked with recombinant human CTLA-4-Fc (R&D Systems, #7268-CT), recombinant cynomolgus monkey CTLA-4-Fc (Sino Biological, #90213-C02H), rhCD28-Fc (R&D, #342-CD -200), rhICOS-Fc (R&D, #169-CS-050), rhBTLA-Fc (Sino Biological, #11896H02H), rhPD-1-Fc (R&D Systems, #1086-PD) or recombinant PD-1-Fc cynomolgus macaque (obtained in our own laboratory) by binding amino groups to COOH on the surface of the spheres. Standard curves were generated using 25 µl human IgG1 standard (Sigma, I5154) in a 1:3 dilution series (0.08-540 ng/ml) in duplicate. Detections were performed using 60 μl of R-PE labeled goat anti-human F(ab) ₂ IgG (2.5 μg/ml; JIR 109-116-098, AbDSerotec Rapid RPE Antibody Conjugation Kit, LNK022RPE) when incubated for another one hour (20°C, 650 rpm). The plates were analyzed using the Luminex® 200 system (Millipore). A total of 100 spheres were counted in each well in a sample volume of 48 μl. PE MFI values were used to determine specific or non-specific binding to the recombinant proteins mentioned above.

As shown in FIG. 1C and 1D, the AGEN1884 antibody showed specific binding to human and cynomolgus monkey CTLA-4. No significant binding to the other listed members of the immunoglobulin superfamily was observed at the concentrations tested.

- 56 039322

6.1.4. Binding of the antibody to CTLA-4 expressed by activated T cells.

The next step was to investigate the binding of AGEN1884w to CTLA-4 expressed on the surface of activated human T cells. Human PBMCs isolated by ficoll density gradient separation from healthy donor films (Research Blood Components, LLC) were enriched with intact CD4+ T cells using magnetic beads (Stemcell Technologies). The enriched CD4+ T cell population was then resuspended in RPMI1640 medium supplemented with 10% human AB serum (Sigma) at a density of 1 x 10 ⁶ /ml. 1x105 cells in 100 µl were seeded into each well of 96 well flat-bottomed plates pre-coated with anti-CD3 antibody (3 µg/ml, BD Biosciences) and anti-CD28 antibody (10 µg/ml, BD Biosciences) and cultured at 37°C and 5% CO ₂ . On day 5, cells were pooled and counted on a Muse Cell Analyzer (Millipore EMD). 50,000 cells were stained with 100 μl of AGEN1884w, anti-CTLA-4 IgG ₁ reference antibody, and isotype control IgG ₁ in serial dilutions from 10 μg/ml in 96 well round bottom plates for 30 min at 4°C. Cells were washed three times with 150 µl FACS buffer and stained in 100 µl FACS buffer containing 0.25 µl PE-labeled mouse anti-human kappa chain antibody (Invitrogen) and 1 µl APC-CD4 (BD Biosciences) for 30 min at 4°C. The cells were then washed twice and resuspended in 100 µl of FACS buffer. Samples were analyzed on the FACSCanto II System (BD Biosciences). Mean fluorescence intensity (MFI) values of PE in CD4+ T cells were analyzed using the FlowJo software (FlowJo, LLC). As shown in FIG. 2A, AGEN1884w binds to CTLA-4 expressed on the surface of activated human CD4+ T cells.

The binding characteristics of the AGEN1884 antibody to CTLA-4 of the cynomolgus monkey (Macaca fascicularis) were examined by flow cytometric analysis. Cynomolgus monkey PBMCs (Biomedical Research Center for Primates, The Netherlands) isolated in a ficoll gradient were activated using plate-bound anti-CD3 (8 µg/mL, BD, 557 052) and anti-CD28 (0.1 µg/mL, eBioscience, 16-0289-85) in the presence of 50 U/ml IL-2 (BioLegend, 589106) for 3 days in RPMI-1640 supplemented with 10% fetal bovine serum, 10 mM HEPES and 1x Pen/Strep glutamine at 37 °C and 5% CO2. After activation, cells were incubated with anti-CD8a antibody (eBioscience, 9043-0087-120) diluted 1:100 in FACS buffer (PBS with 2% FBS) for 30 minutes at 4°C. The cells were then washed twice with FACS buffer and the Cytofix/Cytoperm method for the BD Bioscience kit (51-2090KZ) was applied according to the manufacturer's protocol. Serial 1:3 dilutions of 9 to 0.037 μg/ml of AGEN1884, reference anti-CTLA-4 IgG ₁ antibody, and isotype control IgG ₁ were added to the cells. Samples were suspended in 200 μl FACS buffer and analyzed using a FACS Diva flow cytometer (BD Biosciences). When used for intracellular staining, the anti-CTLA-4 antibody AGEN1884 showed binding to activated cynomolgus monkey CD8+ T cells (FIG. 2B).

Human, cynomolgus (Indochinese), rat, or mouse peripheral blood mononuclear cells (PBMCs) were then plated into wells of a 96-well tissue culture plate with round bottom wells (1x105 cells/well) and stimulated for 4 days in RPMI-1640 supplemented with 10% fetal bovine serum, 15 mM HEPES, 100 IU/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 100 IU/ml IL-2 (Peprotech) and 1 µg/ml phytohemagglutinin (PHA) (Roche) at 37°C and 5% CO2. After stimulation, cells were washed and 5% heat-inactivated human AB serum (Sigma-Aldrich) was added to block Fc receptors and prevent non-specific antibody binding. Cells were washed again and resuspended in 100 μl of diluted anti-CD4 APC-conjugated antibody (clone OKT4, GK15 or W3/25; Biolegend). After incubation for 45 minutes in the dark, cells were washed three times and permeabilized using the FoxP3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer's instructions. After permeabilization, cells were incubated with serial dilutions of fluorescently labeled (Pacific Blue™) AGEN1884w or isotype control antibody at concentrations from 0.00003 μg/ml to 10 μg/ml for 45 min at 4°C in the dark. Cells were washed twice before flow cytometric analysis using Becton Dickinson LSRFortessa™. Data were analyzed using the FlowJo software (Version 10). As shown in FIG. 2C, AGEN1884w bound to PHA-stimulated human and cynomolgus CD4+ T cells, but not to rat or mouse T cells.

6.1.5. Ligand blocking activity.

To determine if anti-CTLA-4 antibodies block the binding of CD80 and CD86 ligands, preparation for a ranking analysis was performed using suspension chip technology (schematically shown in FIG. 3A). 1200 Luminex® spheres in 5 µl assay buffer (Luminex Corp., #14 LC10014-01) were added to each well of a 96-well half-well plate (Corning, Inc., 3884). The spheres were cross-linked with the CTLA-4 antigen (rhCTLA-4-Fc, R&D, #7268-CT) by coupling amino groups to COOH on the surface of the spheres. The crosslinking reaction was performed using 50 μg/ml CTLA-4 antigen and 1x10 ⁷ Luminex spheres per ml. Standard NHS ester chemistry

- 57 039322 was used to form carbodiimide bonds between the primary amino groups of the antigen and carboxyl groups on the surface of the spheres (chapter 3 of the Luminex Xmap manual).

Antigen binding in the case of proteins is a simple two-step carbodiimide procedure in which the carboxyl groups of the microspheres are first activated with an EDC reagent (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) in the presence of sulfo-NHS (N-hydroxysulfosuccinimide) to form an intermediate complex sulfo-NHS-ether. The reactive intermediate is then displaced by reaction with the primary amine of the target molecule (antibody, protein, or peptide) to form a covalent amide bond. Bound spheres were incubated with different concentrations of anti-CTLA-4 antibodies (concentrations ranging from 9000 to 12 ng/ml in 25 µl of assay buffer per well, prior to sphere addition) for 1 hour at 20° C. and 650 rpm. The following anti-CTLA-4 antibodies were used in the test: AGEN1884, a reference IgG1 anti-CTLA-4 antibody, and an isotype control IgG1 antibody. Thereafter, 30 µl of R-PE labeled with CD80 (R&D Systems, # 140-B1) or CD86 (R&D Systems, # 141-B2) at a concentration of 1 nM was added to each well for a total volume of 60 µl per well (1200 beads per well). well and a final concentration of 0.5 nM labeled CD80 or CD86). Ligand labeling was performed in-house using R-PE labeling kits (AbDSerotec, LYNX Rapid RPE Antibody Conjugation Kit, LNK023RPE) according to the manufacturer's protocol. The plates were analyzed using the Luminex® 200 system (Millipore). 100 spheres were counted per well in a sample volume of 50 μl. Ligand blocking activity was calculated using the MFI values of the non-competitive signal (100% binding) of the ligand-only control. A detectable PE signal showed binding of the ligand to the antigen. AGEN1884 inhibited the binding of CD80 (FIG. 3B) and CD86 (FIG. 3C) to CTLA-4 with an estimated IC _{50 of} 56 ng/mL.

The ability of anti-CTLA-4 antibodies to block the binding of recombinant CD80-Fc and CD86-Fc to cell surface-expressed CTLA-4 was then examined. Briefly, CTLA-4 expressing T cells (Jurkat) were incubated with increasing concentrations of AGEN1884w, a reference anti-CTLA-4 IgG1 antibody, or an isotype control IgG1 antibody (0, 0.001, 0.004, 0.012, 0.037, 0.11, 0.33, 0 .99 and 2.96 µg/ml) followed by the addition of a fixed concentration (0.625 µg/ml) of fluorescently labeled CD80-Fc or CD86-Fc. The percentage of cells bound to CD80-Fc or CD86-Fc was determined using flow cytometry. As shown in FIG. 3D, AGEN1884w and a reference anti-CTLA-4 IgG1 antibody inhibited the binding of CD80-Fc and CD86-Fc to Jurkat cells expressing CTLA-4.

Thereafter, the anti-CTLA-4 antibody, AGEN1884w, was tested for its ability to degrade preformed complexes of cell surface expressed CTLA-4 and recombinant CD80 or CD86. Briefly, recombinant Jurkat cells constitutively expressing CTLA-4 on the cell surface were incubated with serial dilutions of CD80-Fc or CD86-Fc (R&D Systems) (10 to 0.0002 µg/mL) for 30 min at 4°C, in a 96 well round bottom well plate. After washing, a fixed concentration (67 nM) of fluorescently labeled (PerCP-CY5.5) AGEN1884w or a reference anti-CTLA-4 IgG1 antibody was added and the plate was incubated for 30 min at 4°C. The plate was washed again and the cells were resuspended in 100 μl of 1% paraformaldehyde (Alfa Aear) in PBS. Samples were analyzed using a Becton Dickinson FACSCanto™ flow cytometer and data was analyzed using the FlowJo software (Version 10). The AGEN1884w antibody degraded pre-complexes CTLA-4 and CD80 or CD86 in a dose dependent manner (FIG. 3E).

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

The functional activity of antibodies against CTLA-4 against primary human T cells was evaluated after stimulation with staphylococcal enterotoxin A (SEA).

Cryopreserved PBMCs (105 cells/well) in RPMI1640 supplemented with penicillin, streptomycin and 10% FBS (Hyclone) were added to NUNCLON 96-well plates with Delta surface (NUNC™). Cells were cultured in the presence of increasing antibody concentrations of 3.2, 16, 80, 400, 2000, 10000 and 50000 and 100 ng/ml SEA (Toxin Technologies) for 5 days at 37°C, 5% CO ₂ and 97% humidity. The following antibodies were used in testing: AGEN1884w, an anti-CTLA-4 IgG1 reference antibody, and an IgG1 isotype control. The clarified supernatant was collected and stored at -80°C until analysis. Titers of IL-2 were obtained using electrochemiluminescence (MSD).

As shown in FIG. 4A, AGEN1884w increased IL-2 production by T cells in the presence of SEA stimulation.

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

The functional activity of the anti-CTLA-4 antibody AGEN1884w was further investigated using the IL-2-luciferase reporter assay. Briefly, a human T cell line (Jurkat) that endogenously expressed CD28 was designed to constitutively express CTLA-4 on the cell surface and the luciferase reporter gene under the control of the IL-2 promoter.

- 58 039322

The recombinant Jurkat cell line was co-cultured with an antigen presenting cell line (Raji) that expressed CD80 and CD86. T cell receptor (TCR) activation (Signal 1) was provided by mouse anti-human CD3 and goat anti-mouse IgG (H+L); and co-stimulatory signaling (Signal 2) was provided by trans-activation of CD80 and CD86 expressed on Raji cells. Expression of CD80 and CD86 on Raji cells was confirmed by flow cytometry (FIG. 4B). Cross-linking of TCR on the Jurkat T cell line elicited expression of IL-2 leading to the production of luciferase, a surrogate marker for T cell activation. Co-cultivation of these two cell lines led to the uptake of the inhibitory co-receptor CTLA-4 (expressed on Jurkat cells) by its natural ligands CD80 and CD86 (expressed on Raji cells), causing inhibition of T cell activation exhibited in the absence of luciferase expression. This inhibition was reversed by the addition of elevated concentrations of AGEN1884w (0 to 300 μg/mL) due to AGEN1884w blocking the interaction of CTLA-4 with its ligands, CD80 and CD86, and shifting the interaction of these ligands with the costimulatory T cell coreceptor, CD28. Luciferase expression was quantified using the Bio-Glo™ reagent and used to determine the maximum response folds (fold increase in the presence of AGEN1884w compared to the isotype control IgG1 antibody).

As shown in FIG. 4C, the anti-CTLA-4 antibody AGEN1884w dose-dependently abolished CTLA-4 mediated inhibition of T cells in this IL-2-luciferase reporter assay.

6.1.8. Lack of agonist activity in anti-CTLA-4 antibody.

The ability of AGEN1884w to mediate direct CTLA-4 signaling was evaluated. Primary CD3-expressing human T cells were isolated from peripheral blood mononuclear cells using a Miltenyi Pan T cell microsphere kit and cultured with plate-bound anti-CD3 antibody (clone SP34, 10 μg/mL) to activate TCR signaling. AGEN1884w or an IgG1 isotype control antibody was included in the TCR activation format in either soluble or plate-immobilized form (0.003-50 μg/mL). After four days of culture and a final 6-hour incubation with Brefeldin A (BD GolgiPlug™), cells were stained with fluorochrome-conjugated antibodies against lineage surface markers, including anti-CD3 antibody (APC Cy7, clone SP34), anti-CD8 antibody (PE Cy7, clone SK1) and anti-CD4 antibody (PercP Cy5.5, clone L200) followed by cell permeabilization using Cytofix-Cytoperm™ (Beckton Dickinson). To evaluate intracellular cytokine production, cells were stained with an anti-IFNy antibody (Alexa fluor 647, clone B27) and an anti-TNFa antibody (PE, clone Mab11). Cells were collected for flow cytometry using FACSCanto II and data analysis was performed using Flowjo version 10.

Elevated concentrations of soluble (FIG. 4D) or plate-immobilized (FIG. 4E) AGEN1884w had no effect on the percentage of CD8+ IFNy+ T cells compared to the isotype control IgG1 antibody. At concentrations up to 50 μg/ml, AGEN1884w did not act as an anti-CTLA-4 agonist antibody.

6.1.9. Combination with anti-LAG-3 antibody or anti-PD-1 antibody.

In the next step, the anti-CTLA-4 antibody, AGEN1884w, was tested for its synergy with anti-LAG-3 antagonist antibody or anti-PD-1 antagonist antibodies using suboptimally stimulated staphylococcal enterotoxin A (SEA) PBMCs. Briefly, cryopreserved human PBMCs (Research Blood Components) were seeded at a density of 105 cells/well, in RPMI1640 supplemented with Normocin™ (Invivogen #ant-nr) and 10% heat-inactivated FBS (Gibco, Invitrogen Corporation), in NUNCLON 96-well plates with Delta surface (NUNC™). Cells were cultured with 100 ng/ml SEA (Toxin Technologies) in the presence of 10 μg/ml soluble AGEN1884w or isotype control IgG1 together with 5 μg/ml anti-LAG-3 antibody AGEN1746, Nivolumab (lot AAB5719, Myoderm), Pembrolizumab (lot 7002688300 , Myoderm), isotype control IgG1 or isotype control IgG4 for 5 days at 37°C, 5% CO ₂ and 97% humidity. The antibodies tested and their respective concentrations are shown in FIG. 4F. The anti-LAG-3 antibody AGEN1746 used in this assay was derived from the variable regions of the 25F7 antibody proposed in US Patent Application Publication US 2011/0150892 (incorporated herein by reference). AGEN1746 includes the heavy chain of the amino acid sequence of SEQ ID NO: 91 and the light chain of the amino acid sequence of SEQ ID NO: 92 (Table 8). The clarified supernatants were collected and stored at -80°C until analysis. Detection of the cytokine IL-2 was performed using AlphaLISA (Perkin Elmer). The mean value and standard deviation for the concentration of IL-2 was calculated.

- 59 039322

Table 8. Anti-LAG-3 antibody sequences, AGEN1746

SEQID NO: Description Amino acid sequence 91 Heavy chain AGEN1746 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWI GEINHNGNTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCA FGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG 92 Light chain AGEN1746 EIVLTQSPATLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLI YDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPL TFGQGTNLEIKRSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVECSS ACEVNSFTHQGL

As shown in FIG. 4F, combination of anti-CTLA-4 antibody AGEN1884W with anti-LAG-3 antibody AGEN 1746, anti-PD-1 antibody Nivolumab, or anti-PD-1 antibody Pembrolizumab increased IL-2 production in PBMC suboptimally stimulated with SEA superantigen compared to processing with one tool.

6.2. Example 2 Comparison of anti-CTLA-4 antibodies with IgGi or IgG ₂ constant region.

In this example, AGEN1884w, which is a human IgGi antibody, was converted into a human IgG ₂ antibody, AGEN2041w. The AGEN2041w antibody has the same heavy chain variable region and the same light chain as AGEN1884w, but includes the human IgG ₂ constant region. The AGEN2041W antibody includes the heavy chain sequence of SEQ ID NO: 94 and the light chain sequence of SEQ ID NO: 13.

In addition, the AGEN1884w series with mutant Fc regions was also tested to investigate the effect of FcyR interaction on the antagonistic activity of anti-CTLA-4 antibodies. AGEN1884wN297A includes the heavy chain sequence of SEQ ID NO: 95 and the light chain sequence of SEQ ID NO: 13. AGEN1884w-S267E/L328F includes the heavy chain sequence of SEQ ID NO: 96 and the light chain sequence of SEQ ID NO: 13. AGEN1884w-S239D/A330L/ I332E includes the heavy chain sequence of SEQ ID NO: 97 and the light chain sequence of SEQ ID NO: 13. All residues are numbered according to the EU numbering system.

6.2.1. Binding, ligand blocking and selectivity analysis of anti-CTLA-4 antibodies with IgG ₂ constant region.

The affinity of the anti-CTLA-4 antibody, AGEN2041W, the reference anti-CTLA-4 IgGi antibody, and the reference anti-CTLA-4 IgG ₂ antibody were examined by surface plasmon resonance as described in Section 6.1.1. The following CTLA-4 antigens were tested: recombinant human CTLA-4-Fc (R&D Systems, #7268-CT), recombinant human CTLA-4 (Sino Biological, #11159-H08H), recombinant cynomolgus monkey CTLA-4-Fc (Sino Biological , # 90213-C02H) and recombinant cynomolgus monkey CTLA-4 (Sino Biological, # 90213-C08H). The affinity values are listed in FIG. 5A.

The binding of AGEN2041W to Jurkat cells overexpressing human CTLA-4 was then examined in a flow cytometric assay similar to that described in Section 6.1.2. Briefly, Jurkat cells overexpressing human CTLA-4 (Promega) were first stained with serial dilutions of AGEN2041W, a reference anti-CTLA-4 IgG ₂ antibody or isotype control IgG ₂ , and then with APC-conjugated mouse anti-human kappa chain antibody (Invitrogen). Samples were examined on FACSCanto II (BD Biosciences) and mean fluorescence intensity (MFI) values were analyzed using FlowJo software (FlowJo, LLC). AGEN2041w bound to Jurkat cells overexpressing human CTLA-4 (FIG. 5B).

Ligand blocking activity of AGEN2041W, a reference anti-CTLA-4 IgG ₂ antibody, and an isotype control IgG ₂ was examined using suspension chip technology as described in Section 6.1.5. As shown in FIG. 5C, AGEN2041W inhibited CD80 and CD86 binding to CTLA-4.

The selectivity of AGEN2041W and the reference anti-CTLA-4 IgG ₂ antibody was compared to the isotype control IgG ₂ antibody using suspension chip technology as described in Section 6.1.3. As shown in FIG. 5D and 5E, AGEN2041W specifically binds to human and cynomolgus monkey CTLA-4. No significant binding to related family members (recombinant human CD28, ICOS, BTLA and PD-1; and recombinant cynomolgus monkey PD-1) was observed.

6.2.2. Effect of anti-CTLA-4 antibodies with IgGi or IgG ₂ constant region on human T cells after stimulation with staphylococcal enterotoxin A (SEA).

The functional activities of antibodies against CTLA-4, AGEN1884w and AGEN2041W, were investigated in

-60039322 assay with primary human PBMCs for IL-2 induction. The analysis was performed as described in Section 6.1.6. Briefly, human PBMCs were cultured in the presence of increasing concentrations of antibodies: 0.016, 0.08, 0.4, 2.0, 10, 50 and 250 μg/ml, and 100 ng/ml SEA (Toxin Technologies) for 5 days. The following antibodies were tested: antibodies against CTLA-4, AGEN1884w and AGEN2041w, isotype control IgG1 and isotype control IgG ₂ . The titer of IL-2 was determined using electrochemiluminescence (MSD).

As shown in FIG. 6A, AGEN1884w and AGEN2041w stimulated IL-2 production after SEA stimulation.

6.2.3. Effect of anti-CTLA-4 antibody with IgG2 constant region on cell line with IL-2 luciferase reporter.

The next step was to evaluate the functional activity of the anti-CTLA-4 antibody, AGEN2041w, using an IL-2-luciferase reporter assay similar to the assay described above. Briefly, T cells (Jurkat) genetically engineered to express surface CTLA-4 and the luciferase reporter gene after the IL-2 promoter were added to wells containing increasing concentrations of AGEN2041w or isotype control IgG ₂ . Then, Raji cells that endogenously express CD80 and CD86 and were genetically engineered to express the T cell activator were added to the cell culture. T cell activator is a commercial cell surface receptor that binds T cell receptors (TCR) in an antigen-independent manner resulting in T cell activation. Stimulation of TCR in a recombinant Jurkat cell line induces the expression of IL-2, which leads to the production of luciferase. The addition of AGEN2041w in this assay blocks the binding of CTLA-4 (expressed on Jurkat cells) to its natural ligands CD80 and CD86 (expressed on Raji cells), which enhances luciferase production. Luciferase concentrations were quantified using Bio-Glo™ and the data used to determine the maximum response folds (fold increase in the presence of AGEN2041w compared to basal luciferase activity without antibodies).

As shown in FIG. 6B, increasing concentrations of AGEN2041w resulted in enhanced T cell activation in a dose-dependent manner. In contrast, the isotype control IgG ₂ did not increase luciferase expression even at the highest concentration tested.

6.2.4. Analysis of AGEN1884w produced by a stable clone.

In this example, AGEN1884w produced by stable clone 105 (AGEN1884w-105) was tested for binding to cell surface expressed CTLA-4 and functional activity in a primary human PBMC assay.

Binding of AGEN1884w-105 to Jurkat cells overexpressing human CTLA-4 was examined in a flow cytometric assay similar to that described in Section 6.1.2. Briefly, Jurkat cells overexpressing human (Promega) CTLA-4 were stained first with serial dilutions of AGEN1884w-105, an anti-CTLA-4 IgG1 reference antibody, or an IgG1 isotype control, and then with PE-conjugated mouse anti-human κ-chain antibody (Invitrogen). Samples were examined on FACSCanto II (BD Biosciences) and mean fluorescence intensity (MFI) values were analyzed using FlowJo software (FlowJo, LLC). AGEN1884w105 bound to Jurkat cells overexpressing human CTLA-4 (FIG. 7A).

The ability of AGEN1884w-105 to induce IL-2 production was examined in an assay similar to that described in Section 6.1.6. Briefly, human PBMCs were cultured for 5 days in the presence of 100 ng/ml SEA (Toxin Technologies) and 20 μg/ml AGEN1884w-105, an anti-CTLA-4 IgG1 reference antibody or an IgG1 isotype control. The titer of IL-2 was determined using electrochemiluminescence (MSD). As shown in FIG. 7B, AGEN1884w-105 caused IL-2 production after SEA stimulation.

6.2.5. Binding of anti-CTLA-4 antibodies to surface-expressed Fcy receptors.

The binding of anti-CTLA-4 antibodies to human FcyRIIA, FcyRIIB and FcyRIIIA expressed on the surface of CHO cells was measured by flow cytometry. Briefly, CHO cells were transfected with cDNA encoding human FcyRIIAH131, FcyRIIB or FcyRIIIAV158 to generate the following CHO cell lines: rCHO-huFcyRIIA ⁺ , rCHO-huFcyRIIB ⁺ and rCHO-huFcyRIIIA ⁺ . Binding with antibody dose titration ranging from 0.0056-1000 μg/mL was performed and primary antibodies were detected using a secondary, phycoerythrin (PE) conjugated anti-human F(ab') antibody (Jackson Immune Research). Samples were analyzed by flow cytometry and mean fluorescence intensity (MFI) PE values were analyzed and used to plot binding curves. The following antibodies were used in testing: AGEN1884w, AGEN2041w, an anti-CTLA-4 IgG1 reference antibody, and an IgG1 isotype control antibody. This study used AGEN1884w produced in two different cell growth media: Life Technologies Inc./Thermo CD FortiCHO™ Growth Medium (AGEN1884wFortiCHO) and PowerCHO™ 2 Growth Medium from Lonza (AGEN1884w-PowerCHO). The reference IgG1 antibody against CTLA-4 contains a different IgG1 allotype that differs from the allotype in

- 61 039322

AGEN1884w. The isotype control IgG1 antibody was only tested at a concentration of 1000 μg/ml.

As shown in FIG. 8A, AGEN1884w-FortiCHO, AGEN1884w-PowerCHO and AGEN2041w showed increased binding to FcγRIIAH131 expressing cells compared to the reference IgG1 anti-CTLA-4 antibody. Similarly, binding of AGEN1884w-PowerCHO and AGEN1884w-FortiCHO to FcγRIIB-expressing cells was stronger than for the reference anti-CTLA-4 IgG1 antibody (FIG. 8B). All IgG1 antibodies, AGEN1884w-FortiCHO, AGEN1884w-PowerCHO, and the reference anti-CTLA-4 IgG1 antibody, bound identically to FcyRIIIAV158 expressed on the cell surface (FIG. 8C). AGEN2041w showed weaker binding to FcyRIIB (FIG. 8B) and FcyRIIIAV158 (FIG. 8C) than IgG1 antibodies.

6.2.6. Binding of anti-CTLA-4 antibodies to recombinant Fcy receptors.

The binding of anti-CTLA-4 antibodies, AGEN1884w-FortiCHO, AGEN1884w-PowerCHO, AGEN2041w and reference anti-CTLA-4 IgG1 antibody, to human FcyRIIA and FcyRIIIA was measured using surface plasmon resonance (BIAcore® 3000).

For binding to FcyRIIA, a direct immobilization method on a CM5 chip using standard amine chemistry was used. Recombinant human FcyRIIA-His protein was diluted in sodium acetate solution, pH 4.0, to a concentration of 10 μg/ml and immobilized in flow cell 2 and 4 using an injection for 8 min at a flow rate of 5 μl/min. Flow cells 1 and 3 used as reference flow cells were spurious, i. amine condensation reagents were passed through flow cells as in flow cells 2 and 4, without FcyRIIA. The functionality of the FcyRIIA-immobilized surfaces was tested using 1 mg/mL Trastuzumab in BiaCore HBS-EP running buffer through each flow cell at 0.5 min injection and a flow rate of 100 µl/min.

For binding to FcyRIIIA, the capture method was used with an immobilized anti-tetraHis antibody on a CM5 chip. Mouse anti-His antibody was diluted in sodium acetate solution, pH 5.0, to a concentration of 10 μg/ml and immobilized in all four flow cells using a 5 minute injection at a flow rate of 10 μl/min using standard amine chemistry. The recombinant human FcyRIIIA-His protein was captured on the surface of the chip by introducing a solution of FcyRIIIA-His at a concentration of 1 μg/ml for 2 min at a flow rate of 5 μl/min.

All antibodies tested were buffer exchanged with BiaCore running buffer prior to analysis. Protein concentrations in buffer exchanged samples and intermediate stock solutions were determined by measuring absorbance at 280 nm using a Nanodrop 1000 spectrophotometer.

Each sample was analyzed in a series of 8 concentrations starting at 1000 μg/ml at 2-fold dilution. Data were collected and processed using BiaCore version 4.1 control software and BIAevaluation version 4.1 software. Dose-response curves were fitted using a five-parameter logistic model (5PL). The following default weights were used for curves:

Dispersion=0.02hotresponse (RU) ^1,

Binding data was also analyzed using StatLIA version 3.2. Parallelism was assessed using the chi-square test (P<0.01) in StatLIA.

The mean response (resonance units) of AGEN1884w-FortiCHO, AGEN1884w-PowerCHO, AGEN2041w, and the reference anti-CTLA-4 IgG1 antibody to human FcyRIIA and human FcyRIIIA is shown in FIG. 9A and 9B, respectively.

6.2.7. The effect of antibodies against CTLA-4 on a cell line with an Fcy reporter IIIA receptor.

The ability of AGEN1884w to simultaneously capture CTLA-4 and signal through activation of Fcy receptors was evaluated using a cell line expressing the Fcy reporter receptor IIIA (FcyRIIIA) (Promega) together with target cells (assay scheme shown in Figure 10A). In this assay, Jurkat cells expressing human CTLA-4 were used as target cells.

Recombinant Jurkat cells stably expressing FcyRIIIA, either V158 variant (high affinity) or F158 variant (low affinity) and an NFAT response element regulating firefly luciferase expression were used as effector cells. The analysis was carried out according to the manufacturer's instructions. Briefly, 25 µl of target cells (25,000 cells) expressing CTLA4 were mixed with 25 µl of serially diluted antibodies, starting at 4 µg/ml, in 96-well flat-bottomed white plates. Then, 150,000 effector cells in 25 μl were added to the well and the plates were incubated at 37°C overnight. Binding of an antibody/antigen complex, where the antigen is located on the surface of target cells, to FcyRIIIA signals the promoter/reporter construct in effector cells and results in transcription of the luciferase gene. The next day, the plates were removed from the thermostat and left to cool for 30 minutes. 75 μl of Bio-Glo reagent (Promega) at room temperature was added to each well, luminescence was measured on a multifunctional

- 62 039322 EnVison Multimode Nanal Plate Analyzer (Perkin Elmer) and the Relative Light Units (RLU) were recorded. The following antibodies were tested: AGEN1884w-105 or AGEN 1884w (AGEN1884W-105 in Figures 10B, 10C, 10F and 10G and AGEN1884w in Figures 10D and 10E); AGEN1884w-N297A (FIGS. 10F and 10G); AGEN2041w (FIGS. 10D and 10E); reference IgG1 anti-CTLA-4 antibody (FIGS. 10B and 10C); and isotype control IgG1 antibody (FIGS. 10B-10G).

When bound to cells expressing CTLA-4, the IgG1 antibody AGEN1884w-105 activated the FcyRIIIA V158 variant (FIG. 10B) and the FcyRIIIA F158 variant (FIG. 10C). As expected, the IgG ₂ antibody AGEN2041w did not signal through FcyRIIIA (FIGS. 10D and 10E). To confirm the specificity of this reporter assay, the Fc receptor (FcR)-silent variant of AGEN1884w (AGEN1884w-N297A) was tested in the same assay, and this mutant did not signal through any FcyRIIIA (FIGS. 10F and 10G).

6.2.8. The effect of antibodies against CTLA-4 on a cell line with an Fcy reporter receptor IIA.

The next step was to assess the ability of AGEN2041w to simultaneously capture CTLA-4 and signal through FcyRIIA using a reporter cell line expressing FcyRIIA (Promega) together with target cells (Jurkat cells expressing human CTLA-4). Jurkat cells expressing FcyRIIA with a high affinity 131 H/H polymorphism and an NFAT response element regulating the expression of firefly luciferase were used as effector cells. Briefly, 25 µl of target cells (6x106 cells/ml) were mixed with 25 µl of serially diluted antibodies, starting at 0.1 µg/ml, in wells (in duplicate) of 96-well white assay plates. The following antibodies were tested: AGEN2041w, an anti-CTLA-4 IgG ₂ reference antibody, and an IgG2 isotype control antibody. Then, 25 μl of effector cells (6x106 cells/ml) were added to each well to obtain a ratio of effector to target of 1:1. The plates were incubated for 20 hours at 37°C and 5% CO2. After this incubation, Bio-Glo Luciferase Assay Reagent (Promega) was thawed at room temperature and 75 µl was added to each well. Luminescence was measured for 5-10 min using an Envision multi-plate analyzer (PerkinElmer). The background luminescence was subtracted from the readings of each sample and the reduced relative light units (RLU) were recorded.

As shown in FIG. 11, when bound to cells expressing CTLA-4, the IgG2 antibody AGEN2041w activated FcyRIIAH ¹³¹ .

6.2.9. Effect of binding to the Fcy receptor on the antagonistic activity of antibodies against CTLA-4.

In this example, the effect of FcyR binding on the antagonistic activity of anti-CTLA-4 antibodies was investigated.

First, the binding of AGEN1884w-105 and AGEN1884w-N297A to expressed CTLA-4 on the cell surface was compared in a flow cytometric analysis similar to that described in Section 6.1.2. Briefly, Jurkat cells overexpressing human (Promega) CTLA-4 were stained first with serial dilutions of AGEN1884w-105, AGEN1884w-N297A or isotype control IgG1 and then with mouse PE-conjugated anti-human κ chain antibody (Invitrogen). Samples were examined on FACSCanto II (BD Biosciences) and mean fluorescence intensity (MFI) values were analyzed using FlowJo software (FlowJo, LLC). AGEN1884w-105 and AGEN1884w-N297A showed similar binding to Jurkat cells overexpressing human CTLA-4 (Fig. 12A).

The dependence of functional activity on Fc receptor (FcR) binding in vitro was then evaluated in an assay similar to that described in Section 6.1.6. Briefly, human PBMCs were cultured for 5 days in the presence of 100 ng/ml SEA (Toxin Technologies) and 20 μg/ml AGEN1884w-105, AGEN1884w-N297A or isotype control IgG1. The titer of IL-2 was determined using electrochemiluminescence (MSD).

Despite similar binding to cell surface expressed CTLA-4 (FIG. 12A), AGEN1884w-N297A showed reduced potentiation of IL-2 secretion compared to AGEN1884w-105 in a primary human PBMC assay (FIG. 12B).

As further confirmation of the need for FcR binding for functional activity, anti-CTLA-4 antibodies were tested for their ability to induce IL-2 secretion in the presence or absence of FcR blockers.

Cryopreserved PBMCs (105 cells/well) in RPMI1640 supplemented with penicillin, streptomycin, and 10% FBS (Hyclone) were added to NUNCLON 96-well plates with a Delta surface (NUNC™). Cells were cultured with 100 ng/ml SEA (Toxin Technologies) and 10 μg/ml anti-CTLA-4 IgG1 antibody or human IgG1 isotype control, in the presence or absence of FcR blockers, for 5 days at 37°C, 5% CO ₂ and 97% humidity. The following anti-CTLA-4 antibodies were tested: IgG1 reference antibody (FIG. 13A) and AGEN1884w-105 (FIG. 13B). Anti-CD16 (Biolegend # 302013), anti-CD32 (R&D # AF1330) and anti-CD64 (R&D #AF1257) antibodies (at 10 µg/ml each) were used as FcR blockers (Fig. 13A) or anti-CD32 antibody alone

- 63 039322 (eBiosciences # 16-0329-81) (Fig. 13B). In the control groups in Fig. 13B, AGEN1884w-105 and human isotype control IgG1 were also incubated with mouse isotype control IgG1 (Biolegend # 400124) as indicated in the figure. The clarified supernatant was collected and stored at -80°C until analysis. Titers of IL-2 were recorded using electrochemiluminescence (MSD).

FcR blockade with anti-CD16, CD32 and CD64 antibodies (FIG. 13A) or with anti-CD32 antibody (FIG. 13B) reduced the ability of anti-CTLA-4 antibodies to induce IL-2 secretion in this assay with primary human PBMCs.

Subsequently, the functional activity of many anti-CTLA-4 antibodies with mutant Fc regions was examined using PBMCs. Briefly, cryopreserved human PBMCs (Research Blood Components) were seeded at a density of 105 cells/well in RPMI1640 supplemented with Normocin™ (Invivogen) and 10% heat-inactivated FBS (Gibco, Invitrogen Corporation) in NUNCLON 96-well plates with a Delta surface (NUNC™) . Cells were cultured for 5 days at 37°C, 5% CO ₂ and 97% humidity in the presence of 142 ng/ml SEA (Toxin Technologies) and 10 μg/ml AGEN1884w, AGEN1884wN297A, AGEN1884w-S267E/L328F, AGEN1884w-S239D/A330L /I332E, AGEN2041w, isotype control IgG1 antibody or isotype control IgG2 antibody. The clarified supernatant was collected and stored at -80°C until analysis. IL-2 concentrations were measured by ELISA.

AGEN1884W with the S239D/A330L/I332E triple mutation in the Fc region, which enhances binding to FcyRIIIA, more potently stimulated IL-2 secretion than AGEN1884w with the wild-type IgG1 Fc region (FIG. 14).

6.2.10. Fc-mediated potential of effector cells.

In this example, the AGEN1884w antibody was tested for its ability to mediate NK cell cytotoxicity. Briefly, Jurkat cells genetically engineered to constitutively express CTLA-4 on the cell surface (target cells) were cultured at a density of 1×10 ⁴ cells/well in 96 well round bottom plates. Elevated concentrations of AGEN1884w, anti-CTLA-4 IgG1 reference antibody or isotype control antibody (all at 0 to 10 μg/ml) and 5x10 ⁴ NK92-FcyRIIIA cells (effector cells) were added to the target cells. After incubation for 6 hours at 37° C. and 5% CO2, target cell lysis was assessed by quantitative photometric determination of lactate dehydrogenase (LDH) released into the supernatant using a cytotoxicity (LDH) kit (Roche) according to the manufacturer's instructions. Data was recorded using SoftMax® Pro Microplate Data Acquisition & Analysis software. Antibodies at all concentrations were tested in triplicate.

As shown in FIG. 15, the AGEN1884w antibody efficiently mediated the lysis of target cells expressing CTLA-4 in the presence of effector cells.

6.2.11. Mapping of anti-CTLA-4 antibody epitopes.

The interaction of the Fab fragment of AGEN1884w (AGEN1884w-Fab) with the extracellular domain of human CTLA-4 was investigated using hydrogen-deuterium exchange (HDX) mass spectrometry. The extracellular domain of CTLA-4, alone or in combination with AGEN1884w-Fab, in phosphate buffered saline at pH 7.4 was diluted with a tenfold volume of deuterium oxide labeling buffer and incubated for various time periods (0, 60, 300, 1800 and 7200 s) at room temperature. The exchange of hydrogen for deuterium was stopped by adding one volume of buffer containing 4M guanidine hydrochloride, 0.85M TCEP (tris(2-carboxyethyl)phosphine), with a final pH of 2.5. The samples were then subjected to pepsin/protease type XIII digestion on a column and LC-MS analysis. Mass spectra were recorded only in the MS mode. To calculate deuterium incorporation, the mass spectra for a given peptide were combined over the peak of the extracted ion chromatogram and the weighted average m/z was calculated. The increase in weight from the weight of the native peptide (0 min) to the weighted average weight corresponds to the level of deuterium incorporation. Curves of accumulation of deuterium during the exchange for all peptides were built for subsequent analysis and compared using the HDExaminer program.

Most of the CTLA-4 peptides showed identical or similar levels of deuterium with and without Fab against human CTLA-4. Several peptide segments, however, were found to have significantly reduced deuterium incorporation after Fab binding. All residues in this paragraph are numbered according to SEQ ID NO: 77. Two regions, Q80VT ⁸² (SEQ ID NO: 78) and Y ¹³⁵ PPPYYLGINGTQI ¹⁴⁹ (SEQ ID NO: 79), experienced strong deuterium protection when human CTLA-4 was associated with Fab. The strongest decrease in deuterium uptake was observed in Y ¹⁴⁰ L ¹⁴¹ , thus appearing to be the main feature of the AGEN1884w epitope on CTLA-4. Sequence review of human and cynomolgus monkey CTLA-4, which AGEN1884w strongly binds, showed almost complete sequence identity in the two regions described above, except for the replacement of leucine with methionine at position 141 (FIG. 17A). In contrast, AGEN1884w does not bind to any significant extent to either mouse or rat CTLA-4 (data not shown), which differ from human CTLA-4 in Y ¹⁴⁰ LGI ¹⁴³ (SEQ ID NO: 80) in three out of four

- 64 039322 positions (Fig. 17A). Additional selectivity data show that AGEN1884w binds with high specificity to human and cynomolgus monkey 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 in sequence Y ¹⁴⁰ LGI ¹⁴³ (SEQ ID NO: 80) (FIG. 17B), further confirming the importance of this epitope for AGEN1884w binding.

6.2.12. T-dependent humoral response (TDAR) study in cynomolgus monkeys.

An eight-week TDAR study in cynomolgus monkeys was performed to test the ability of AGEN1884w and AGEN2041w to potentiate a T cell-dependent humoral immune response to hepatitis B vaccine (ENERIX-G®) (HBsAg). The HBsAg vaccine was administered in three subcutaneous injections, at 10 μg per injection (total of 30 μg), in the hind limb on Day 1 (primary vaccination) and Day 29 (booster vaccination). AGEN1884w (N=6) or AGEN2041w (N=6) was given twice by intravenous bolus injection (10 mg/kg), on Day 1 and Day 29, along with vaccine antigens. Using the same administration schedule, one control group of animals (N=3) received the HBsAg vaccine in combination with 3 mg/kg anti-CTLA-4 IgG1 reference antibody, and the other control group of animals (N=6) received the HBsAg with the control product: 20 mM trishydrochloride, 100 mM NaCl, 1% mannitol, 0.10 mM DTPA, 0.01% polysorbate 80, pH 7.0, but no anti-CTLA-4 antibody. During the eight-week study, anti-HBsAg (IgG) antibody assays were performed in serum samples collected at specific time points during the study (Day -20, -7, Day +15, +29, +43, +59, and +69). Serum titers were measured by colorimetric ELISA.

Animals treated with AGEN1884w, AGEN2041w, anti-CTLA-4 IgG1 reference antibody, or control product had normal secondary immune responses with measurable antibody titers after the second dose of vaccine that peaked and then declined (FIGS. 18A and 18B). However, AGEN1884w, AGEN2041w and the reference anti-CTLA-4 IgG1 antibody were shown to enhance the anti-HBsAg IgG response compared to the control product given without anti-CTLA-4 antibody.

6.3. Example 3 Anti-CTLA-4 antibody assay.

In this example, the following 19 anti-CTLA-4 antibodies were tested for ligand binding and blocking: AGEN1884, AGEN1885, AGEN1886, AGEN1887, AGEN1888, AGEN1889, AGEN1890, AGEN1891, AGEN1892, AGEN1893, AGEN1894, AGEN1895, AGEN1896, AGEN1898, AGEN1897, AGEN1897 , AGEN1901 and AGEN1902. The sequences of the heavy chain variable region and the light chain variable region of these antibodies were disclosed in table. 6. In FIG. 19A and 19B show the sequence alignment of heavy chain variable regions and light chain variable regions, respectively.

6.3.1. Anti-CTLA-4 antibody ligand binding and blocking assay.

The affinity of the 19 anti-CTLA-4 antibodies described above was examined by surface plasmon resonance in an assay similar to that described in Section 6.1.1. The CTLA-4 antigen tested was recombinant human CTLA-4-Fc (R&D Systems, #7268-CT). Anti-CTLA-4 antibodies (6 μg/ml in running buffer) were captured on the surface of the CM5 sensor chip. These 19 antibodies bound to recombinant human CTLA-4 with an affinity of nM order (data not shown).

Ligand blocking activity of 19 anti-CTLA-4 antibodies was examined using suspension chip technology in an assay similar to that described in Section 6.1.5. Ligand blocking was tested in the presence of various concentrations of anti-CTLA-4 antibodies (from 3000 to 12 ng/ml, prior to the addition of spheres). As shown in FIG. 20A-20E, all 19 anti-CTLA-4 antibodies blocked CTLA-4 binding to CD80 and CD86.

The scope of the invention should not be limited to the specific embodiments described herein. In fact, various modifications of the invention in addition to those described will be apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are to be included within the scope of the appended claims.

All references (e.g., publications or patents or patent applications) cited herein are incorporated by reference in their entirety and in all respects to the same extent as if it were expressly and individually stated that each individual reference (e.g., publication or patent or patent application) is incorporated by reference in its entirety in all respects. Other embodiments are contained in the following claims.

Claims (34)

1. An isolated antibody that specifically binds to the human CTLA-4 protein containing a heavy chain variable region containing complementarity determining regions - 65 039322 CDRH1, CDRH2 and CDRH3, and a light chain variable region containing the complementarity-determining regions of CDRL1, CDRL2 and CDRL3, where: (a) CDRH1 contains the amino acid sequence SYX1MX ₂ (SEQ ID NO: 22), where X ₁ is S or A; and X ₂ is N or S; (b) CDRH2 contains the amino acid sequence of SEQ ID NO: 2; (c) CDRH3 contains the amino acid sequence VGLMGPFXI (SEQ ID NO: 23), where X is D or N; (d) CDRL1 contains the amino acid sequence RASQSVX ₁ X ₂ YLX ₃ (SEQ ID NO: 24), where X ₁ is S or G; X ₂ represents R, S or T; and X ₃ is G or A; (e) CDRL2 contains the amino acid sequence X1X2SX3RAT (SEQ ID NO: 25), where X ₁ is G or A; X2 is A or T; and X3 is T, S, R or N; and (f) CDRL3 contains the amino acid sequence QQYGX1SPX2T (SEQ ID NO: 26), where X ₁ is S or T; and X ₂ is W or F. 2. An isolated antibody according to claim 1, where: (a) CDRH1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 27; (b) CDRH3 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 28; (c) CDRL1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 29 and 30; (d) CDRL2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 31-35; (e) CDRL3 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 36 and 37; (f) CDRH1, CDRH2 and CDRH3 contain the amino acid sequences of CDRH1, CDRH2 and CDRH3, respectively, SEQ ID NOs: 1, 2 and 3; 27, 2 and 3; or 27, 2 and 28; and/or (g) CDRL1, CDRL2 and CDRL3 contain the amino acid sequences of CDRL1, CDRL2 and CDRL3, respectively, SEQ ID NOs: 4, 5 and 6; 29, 32 and 36; 29, 33 and 37; 30, 31 and 6; 29, 34 and 6; or 29, 35 and 37. 3. Selected antibody according to any one of claims 1 or 2, where CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 contain the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5 and 6; 1, 2, 3, 29, 32 and 36; 1, 2, 3, 29, 33 and 37; 27, 2, 3, 4, 5 and 6; 27, 2, 3, 29, 33 and 37; 1, 2, 3, 30, 31 and 6; 1, 2, 3, 29, 34 and 6; 1, 2, 3, 29, 35 and 37; 27, 2, 28, 4, 5 and 6; 27, 2, 28, 29, 32 and 36; 27, 2, 28, 29, 33 and 37; or 27, 2, 28, 29, 35 and 37 respectively. 4. An isolated antibody according to any one of claims 1-3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. 5. An isolated antibody according to any one of claims 1-4, where: (a) the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 72, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 73; (b) the heavy chain variable region contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 38-42, and the light chain variable region contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 43- 47; or (c) the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 7 and 8; 7 and 44; 7 and 45; 38 and 8; 38 and 45; 39 and 43; 39 and 45; 39 and 46; 39 and 47; 40 and 43; 40 and 8; 40 and 44; 40 and 45; 41 and 8; 41 and 44; 41 and 45; 41 and 47; 42 and 43; or 42 and 45, respectively. 6. An isolated antibody according to any one of claims 1 to 5, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 7 and 8, respectively. 7. An isolated antibody according to any one of claims 1 to 6, wherein the antibody comprises a human IgG ₁ heavy chain constant region, optionally wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 12, 76, 93, or 97. 8. An isolated antibody according to any one of claims 1 to 7, wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 12 or 93 and a light chain containing the amino acid sequence of SEQ ID NO: 13. 9. An isolated antibody according to any one of claims 1 to 8, wherein the antibody comprises a human IgG heavy chain constant region that is a variant of the native human IgG heavy chain constant region, wherein the variant of the human IgG heavy chain constant region binds to a higher human Fcy receptor. level of affinity relative to binding to the human Fcy receptor of the corresponding native human IgG heavy chain constant region, optionally wherein the human IgG heavy chain constant region variant is a human IgG1 heavy chain constant region variant, a human IgG2 variant, or a human IgG4 heavy chain constant region variant . 10. The isolated antibody of claim 9, wherein the human Fcy receptor is FcyRIIIA or FcyRIIB and the human IgG heavy chain constant region variant contains: - 66 039322 (a) one or more of the following amino acid mutations according to the EU numbering system: G236D, P238D, S239D, S267E, L328F and L328E; or (b) a set of amino acid mutations selected from the group consisting of: S267E and L328F; P238D and L328E; P238D and one or more replacements selected from the group consisting of E233D, G237D, H268D, P271G and A330R; P238D, E233D, G237D, H268D, P271G and A330R; G236D and S267E; S239D and S267E; V262E, S267E and L328F; and V264E, S267E and L328F according to the EU numbering system. 11. The isolated antibody of claim 9 or 10, wherein the human Fcy receptor is FcyRIIB and the human IgG heavy chain constant region variant also contains one or more amino acid mutations that reduce the affinity of the IgG for human FcyRIIIA, human FcyRIIA, or human FcyRI. 12. The isolated antibody of claim 9, wherein the human Fcy receptor is FcyRIIIA, FcyRIIA or FcyRI and the human IgG heavy chain constant region variant contains: (a) one or more of the following amino acid mutations according to EU numbering: G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T and P396L; or (b) a set of amino acid mutations selected from the group consisting of: S239D; T256A; K290A; S298A; I332E; E333A; K334A; A339T; S239D and I332E; S239D, A330L and I332E; S298A, E333A and K334A; G236A, S239D and I332E; and F243L, R292P, Y300L, V305I and P396L according to the EU numbering system. 13. An isolated antibody according to any one of claims 1-12, wherein the antibody comprises an afucosylated Fco region. 14. An isolated antibody according to any one of claims 1-13, having one or more of the following properties: (a) the antibody is a human antibody; (b) the antibody is CTLA-4 antagonistic; (c) the antibody inhibits the binding of human CTLA-4 protein to human CD80 or to human CD86. 15. A bispecific isolated antibody that specifically binds to the human CTLA-4 protein, containing a heavy chain variable region containing complementarity-determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region containing complementarity-determining regions CDRL1, CDRL2 and CDRL3, where: (a) CDRH1 contains the amino acid sequence SYX1MX ₂ (SEQ ID NO: 22), where X ₁ is S or A; and X ₂ is N or S; (b) CDRH2 contains the amino acid sequence of SEQ ID NO: 2; (c) CDRH3 contains the amino acid sequence VGLMGPFXI (SEQ ID NO: 23), where X is D or N; (d) CDRL1 contains the amino acid sequence RASQSVX1X2YLX3 (SEQ ID NO: 24), where X1 is S or G; X2 is R, S or T; and X ₃ is G or A; (e) CDRL2 contains the amino acid sequence X1X2SX3RAT (SEQ ID NO: 25), where X1 is G or A; X ₂ is A or T; and X ₃ is T, S, R or N; and (f) CDRL3 contains the amino acid sequence QQYGX1SPX2T (SEQ ID NO: 26), where X1 is S or T; and X ₂ is W or F. 16. A pharmaceutical composition for the treatment of a CTLA-4 related disorder, comprising an antibody according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier or excipient. 17. An isolated polynucleotide encoding the heavy chain variable region of an antibody according to any one of claims 1-14. 18. An isolated polynucleotide encoding the light chain variable region of an antibody according to any one of claims 1-14. 19. An isolated polynucleotide encoding the heavy chain of an antibody according to any one of claims 1-14. 20. An isolated polynucleotide encoding an antibody light chain according to any one of claims 1-14. 21. An isolated polynucleotide encoding a heavy chain variable region and a light chain variable region of an antibody according to any one of claims 1-14. 22. An isolated polynucleotide encoding the heavy chain and light chain of an antibody according to any one of claims 1-14. 23. A recombinant host cell containing a polynucleotide according to any one of claims 17-22. 24. A method for producing an antibody that binds to human CTLA-4, wherein the method comprises culturing a host cell containing: (a) an isolated polynucleotide encoding the heavy chain variable region of an antibody according to any one of claims 1 to 14 and an isolated polynucleotide encoding a light chain variable region of an antibody according to any one of claims 1 to 14; or (b) an isolated polynucleotide encoding an antibody heavy chain according to any one of claims 1 to 9 and an isolated polynucleotide encoding an antibody light chain according to any one of claims 1 to 14, under conditions allowing expression of the polynucleotide and production of an antibody. - 67 039322 25. The use of an isolated antibody according to any one of claims 1 to 15 to increase T cell activation in response to an antigen in an individual. 26. The use of an isolated antibody according to any one of claims 1 to 15 for the treatment of cancer. 27. The use of an isolated antibody according to any one of claims 1 to 15 for the treatment of an infectious disease. 28. The use of a pharmaceutical composition according to claim 16 to increase T cell activation in response to an antigen in an individual. 29. The use of a pharmaceutical composition according to claim 16 for the treatment of cancer. 30. The use of a pharmaceutical composition according to claim 16 for the treatment of an infectious disease. 31. Use according to any one of claims 25-30, further comprising administering an additional therapeutic agent to an individual, optionally wherein the additional agent is administered systemically. 32. Use according to claim 31 wherein the subject is suffering from a solid cancer and wherein the additional therapeutic agent is an anti-PD-1 antibody, optionally wherein the anti-PD-1 antibody is pembrolizumab or nivolumab. 33. Use according to claim 31, wherein the subject suffers from squamous cell carcinoma of the head and neck, and where the additional therapeutic agent is an anti-EGFR antibody, optionally, where the anti-EGFR antibody is cetuximab, optionally, where the use further comprises administering a chemotherapeutic agent to an individual, optionally, where the chemotherapeutic agent is administered systemically, optionally, the chemotherapeutic agent is gemcitabine. 34. Use according to claim 31, wherein the individual is suffering from HER2+ breast cancer and where the additional therapeutic agent is an anti-HER2 antibody, optionally, where the anti-HER2 antibody is trastuzumab, optionally, where the use further comprises administering a chemotherapeutic agent to the individual, optionally where the chemotherapeutic agent is administered systemically, optionally, the chemotherapeutic agent is gemcitabine.