Anti-CD96 antibodies and methods of use thereof

Abstract

To provide antibodies that specifically bind to CD96 (e.g., human CD96) and antagonize CD96 function.SOLUTION: The above problem is solved by an isolated antibody that specifically binds to human CD96, the antibody comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs), CDRH1, CDRH2, and CDRH3, and a light chain variable region (VL) comprising the CDRs, CDRL1, CDRL2, and CDRL3.SELECTED DRAWING: Figure 1AB

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 894,334, filed August 30, 2019, and U.S. Provisional Application No. 62 / 931,476, filed November 6, 2019, each of which is incorporated by reference herein in its entirety.

[0002] The present disclosure relates to antibodies that specifically bind to CD96 (eg, human CD96) and methods of using same. [Background technology]

[0003] CD96 (Cluster of Differentiation 96), also known as TACTILE (T cell activation, increased late expression), is a type I transmembrane protein in the immunoglobulin (Ig) superfamily. It has a single Ig domain, a type I transmembrane domain, a single intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM), and a single YXXM phosphorylation motif, and is expressed on the surface of T cells and natural killer (NK) cells.

[0004] CD96 is thought to play a role in the regulation of immune cells (e.g., NK cells and T cells) and tumor metastasis. In particular, blockade of CD96 function has been shown to suppress primary tumor growth in several mouse tumor models in a CD8+ T cell-dependent manner.

[0005] Given the role of human CD96 in regulating immune responses, therapeutic agents designed to block CD96-ligand interactions hold great promise for the treatment of diseases involving immunosuppression. Summary of the Invention

[0006] The present disclosure provides antibodies that specifically bind to CD96 (e.g., human CD96) and modulate CD96 function, e.g., CD96-mediated immunosuppression. Also provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for producing these antibodies, and methods of treating subjects using these antibodies. The antibodies disclosed herein are particularly useful for increasing immune cell activation and are therefore useful for treating cancer in a subject or treating or preventing an infectious disease in a subject.

[0007] Thus, in one aspect, the disclosure provides an isolated antibody that specifically binds to human CD96, wherein the antibody comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) CDRH1, CDRH2, and CDRH3, and a light chain variable region (VL) comprising CDRs of CDRL1, CDRL2, and CDRL3; (a) CDRH1 comprises the amino acid sequence of X1YX2X3X4 (SEQ ID NO: 135); X1 is Q or S; X2 is A or S; X3 is M or I; X4 is H or S; (b) CDRH2 is X1IX2X3X4X5X6X7X8X9YX 10 QKFQG (SEQ ID NO: 137), X1 is W or G; X2 is N or I; X3 is A, E, V, or P; X4 is V, G, W, or I; X5 is S, Y, T, N, or F; X6 is G or W, X7 is D, Y, N, or T; X8 is T or A, X9 is K or N; X 10 is S or A, (c) CDRH3 comprises the amino acid sequence of NWGX1SYGX2DV (SEQ ID NO: 180), GYDSRPLDV (SEQ ID NO: 19), or GYDSRPLDY (SEQ ID NO: 20); X1 is M or L; X2 is M or L; (d) CDRL1 comprises the amino acid sequence of RASQSIX1X2YLN (SEQ ID NO: 139) or GGNNIGSKIVH (SEQ ID NO: 26); X1 is S, T, or L; X2 is S, P, or W; (e) CDRL2 comprises the amino acid sequence of X1X2SSLQS (SEQ ID NO: 141) or DDRDRPS (SEQ ID NO: 32); X1 is S or A; X2 is A, S, or E, and / or (f) CDRL3 comprises the amino acid sequence of QQX1YSTPALX2 (SEQ ID NO: 143) or QVWDINVHHVI (SEQ ID NO: 35); X1 is S or A; X2 is T or S; Optionally, the amino acid immediately N-terminal to CDRH1 is N, T, S, D, or A.

[0008] In certain embodiments, (a) CDRH1 comprises the amino acid sequence of X1YX2MH (SEQ ID NO: 136); X1 is Q or S; X2 is A or S; (b) CDRH2 comprises the amino acid sequence of WINX1X2X3X4X5TKYSQKFQG (SEQ ID NO: 138); X1 is A, V, or E; X2 is V, W, or G; X3 is S, Y, T, or N; X4 is G or W, X5 is D, N, Y, or T; (c) CDRH3 comprises the amino acid sequence of NWGX1SYGX2DV (SEQ ID NO: 180); X1 is M or L; X2 is M or L; (d) CDRL1 comprises the amino acid sequence of RASQSIX1X2YLN (SEQ ID NO: 139); X1 is S, T, or L; X2 is S, P, or W; (e) CDRL2 comprises the amino acid sequence of X1X2SSLQS (SEQ ID NO: 141); X1 is S or A, X2 is A, S, or E, and / or (f) CDRL3 comprises the amino acid sequence of QQSYSTPALT (SEQ ID NO: 33) or QQAYSTPALS (SEQ ID NO: 34).

[0009] In certain embodiments, (a) CDRH1 comprises the amino acid sequence of SEQ ID NO: 4; (b) CDRH2 comprises the amino acid sequence of SEQ ID NO: 17; (c) CDRH3 comprises the amino acid sequence of SEQ ID NO: 19 or 20; (d) CDRL1 comprises the amino acid sequence of SEQ ID NO: 26; (e) CDRL2 comprises the amino acid sequence of SEQ ID NO: 32; and / or (f) CDRL3 comprises the amino acid sequence of SEQ ID NO: 35.

[0010] In certain embodiments, CDRH1, CDRH2, and CDRH3 comprise the amino acid sequences of SEQ ID NOs: 1, 5, and 18; 2, 6, and 18; 2, 8, and 18; 2, 9, and 18; 2, 10, and 18; 1, 7, and 18; 2, 11, and 18; 1, 12, and 18; 1, 13, and 18; 1, 14, and 18; 3, 15, and 18; 1, 16, and 18; 1, 5, and 140; 1, 5, and 142; 1, 5, and 179; 4, 17, and 19; or 4, 17, and 20, respectively.

[0011] In certain embodiments, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences of SEQ ID NOs: 21, 28, and 33; 21, 29, and 33; 21, 30, and 33; 21, 31, and 33; 22, 29, and 33; 24, 29, and 33; 23, 29, and 33; 25, 28, and 34; or 26, 32, and 35, respectively.

[0012] In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are, respectively, SEQ ID NOs: 1, 5, 18, 21, 28, and 33; 1, 5, 18, 21, 29, and 33; 1, 5, 18, 22, 29, and 33; 1, 5, 18, 23, 29, and 33; 1, 5, 18, 24, 29, and 33; 1, 5, 18, 25, 28, and 34; 1, 5, 140, 21, 28, and 33; 1, 5, 142, 21, 28, and 33; 1, 5, 179, 21, 28, and 33; 1, 7, 18, 21, 29, and 3 3; 1, 12, 18, 21, 28, and 33; 1, 13, 18, 21, 28, and 33; 1, 14, 18, 21, 28, and 33; 1, 16, 18, 21, 28, and 33; 2, 6, 18, 21, 29, and 33; 2, 8, 18, 21, 29, and 33; 2, 9, 18, 21, 30, and 33; 2, 10, 18, 21, 29, and 33; 2, 11, 18, 21, 31, and 33; 3, 15, 18, 21, 28, and 33; 4, 17, 19, 26, 32, and 35; or 4, 17, 20, 26, 32, and 35.

[0013] In certain embodiments, the antibody comprises a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence of SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61. In certain embodiments, the X of any one of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 is glutamine. In certain embodiments, the X of any one of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 is pyroglutamate.

[0014] In certain embodiments, the antibody comprises a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In certain embodiments, the amino acid sequence of the VL consists of the amino acid sequence of SEQ ID NO: 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75.

[0015] In another aspect, the disclosure provides an isolated antibody that specifically binds to human CD96, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61, and / or a VL comprising the amino acid sequence of SEQ ID NO: 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 36 and 62; 37 and 62; 37 and 63; 37 and 66; 37 and 67; 37 and 68; 37 and 69; 38 and 63; 39 and 63; 40 and 63; 41 and 63; 42 and 63; 43 and 64; 44 and 64; 45 and 63; 46 and 63; 47 and 65; 48 and 62; 49 and 62; 50 and 62; 51 and 62; 52 and 62; 53 and 62; 54 and 62; 55 and 62; 56 and 62; 57 and 62; 58 and 62; 59 and 62; 60 and 70; 60 and 71; 60 and 72; 60 and 73; 60 and 74; 60 and 75; or 61 and 70. In certain embodiments, the amino acid sequences of VH and VL consist of the amino acid sequences of SEQ ID NOs: 36 and 62; 37 and 62; 37 and 63; 37 and 66; 37 and 67; 37 and 68; 37 and 69; 38 and 63; 39 and 63; 40 and 63; 41 and 63; 42 and 63; 43 and 64; 44 and 64; 45 and 63; 46 and 63; 47 and 65; 48 and 62; 49 and 62; 50 and 62; 51 and 62; 52 and 62; 53 and 62; 54 and 62; 55 and 62; 56 and 62; 57 and 62; 58 and 62; 59 and 62; 60 and 70; 60 and 71; 60 and 72; 60 and 73; 60 and 74; 60 and 75; or 61 and 70.In certain embodiments, the X of any one of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 is glutamine. In certain embodiments, the X of any one of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 is pyroglutamate.

[0016] In certain embodiments, the antibody specifically binds to the amino acid sequence of SEQ ID NO: 130 or 131. In certain embodiments, the antibody binds to the amino acid sequence of SEQ ID NO: 134.

[0017] In certain embodiments, the antibody is internalized upon binding to cells expressing human CD96.

[0018] In another aspect, the disclosure provides an isolated antibody that specifically binds to the amino acid sequence of SEQ ID NO: 130 or 131. In certain embodiments, the isolated antibody of claim 18 or 19, wherein the antibody binds to the amino acid sequence of SEQ ID NO: 134.

[0019] In another aspect, the disclosure provides an isolated antibody that specifically binds to human CD96, wherein the antibody is internalized upon binding to a cell expressing human CD96.

[0020] In certain embodiments, the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody comprises an IgG1 heavy chain constant region. In certain embodiments, the amino acid sequence of the IgG1 heavy chain constant region comprises an N297A mutation numbered according to the EU numbering system. In certain embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 124 or 176. In certain embodiments, the amino acid sequence of the IgG1 heavy chain constant region comprises S239D, A330L, and I332E mutations numbered according to the EU numbering system. In certain embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 125 or 177. In certain embodiments, the amino acid sequence of the IgG1 heavy chain constant region comprises S267E and L328F mutations numbered according to the EU numbering system. In certain embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 126 or 178. In certain embodiments, the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, and the variant heavy chain constant region binds to an FcγR with higher affinity than the wild-type heavy chain constant region binds to the FcγR. In certain embodiments, the FcγR is FcγRIIB.

[0021] In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, or 169. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence of SEQ ID NO: 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, or 169. In certain embodiments, the Xi of any one of SEQ ID NOs: 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, and 169 is glutamine. In certain embodiments, the X of any one of SEQ ID NOs: 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, and 169 is pyroglutamate.

[0022] In certain embodiments, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 122 or 123. In certain embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115. In certain embodiments, the amino acid sequence of the light chain consists of the amino acid sequence of SEQ ID NO: 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115.

[0023] In another aspect, the disclosure provides an isolated antibody that specifically binds to human CD96, wherein the antibody is selected from the group consisting of SEQ ID NOs: 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153 , 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, or 169, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115. In certain embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, or 169, and / or the amino acid sequence of the light chain consists of the amino acid sequence of SEQ ID NO: 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115.In certain embodiments, the heavy and light chains are, respectively, SEQ ID NOs: 76 and 102; 79 and 103; 78 and 103; 82 and 103; 84 and 104; 83 and 104; 86 and 103; 85 and 103; 81 and 103; 80 and 103; 87 and 105; 77 and 102; 88 and 102; 77 and 106; 77 and 107; 77 and 108; 77 and 103; 89 and 102;90 and 102;91 and 102;92 and 102;93 and 102;77 and 109;94 and 102;95 and 102;96 and 102;97 and 102;98 and 102;99 and 102;100 and 110;100 and 111;100 and 112;100 and 113;100 and 114;100 and 115;101 and 110;144 and 102;147 and 103;146 and 103;150 and 103;152 and 104;151 and 104;154 and 103;153 and 103;149 and 103;148 and 103;155 and 105;145 and 102;156 and 102;145 and 106;145 and 107;145 and 108;145 and 103;157 and 102;158 and 102 168 and 110; 168 and 111; 168 and 112; 168 and 113; 168 and 114; 168 and 115; or 169 and 110.In certain embodiments, the amino acid sequences of the heavy and light chains are set forth in SEQ ID NOs: 76 and 102; 79 and 103; 78 and 103; 82 and 103; 84 and 104; 83 and 104; 86 and 103; 85 and 103; 81 and 103; 80 and 103; 87 and 105; 77 and 102; 88 and 102; 77 and 106; 77 and 107; 77 and 108; 77 and 109; and 103;89 and 102;90 and 102;91 and 102;92 and 102;93 and 102;77 and 109;94 and 102;95 and 102;96 and 102;97 and 102;98 and 102;99 and 102;100 and 110;100 and 111;100 and 112;100 and 113;100 and 114;100 and 115;101 and 110;144 and 102;147 and 103;146 and 103;150 and 103;152 and 104;151 and 104;154 and 103;153 and 103;149 and 103;148 and 103;155 and 105;145 and 102;156 and 102;145 and 106;145 and 107;145 and 108;145 and 103;157 and 102;158 and 10 2; 159 and 102; 160 and 102; 161 and 102; 145 and 109; 162 and 102; 163 and 102; 164 and 102; 165 and 102; 166 and 102; 167 and 102; 168 and 110; 168 and 111; 168 and 112; 168 and 113; 168 and 114; 168 and 115; or 169 and 110. In certain embodiments, X in any one of SEQ ID NOs: 76-101 or 144-169 is glutamine. In certain embodiments, X in any one of SEQ ID NOs: 76-101 or 144-169 is pyroglutamate.

[0024] In another aspect, the present disclosure provides an isolated antibody that specifically binds to human CD96, wherein the antibody binds to the same epitope on human CD96 as the antibodies disclosed herein.

[0025] In another aspect, the disclosure provides an isolated antibody that specifically binds to human CD96, wherein the antibody competes for binding to human CD96 with an antibody disclosed herein.

[0026] In certain embodiments, the antibody is a human antibody. In certain embodiments, the antibody is a multispecific antibody. In certain embodiments, the antibody is conjugated to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label. In certain embodiments, the antibody is conjugated to a second antibody.

[0027] In another aspect, the present disclosure provides isolated polynucleotides encoding the VH and / or VL of the antibodies disclosed herein. In another aspect, the present disclosure provides vectors comprising the polynucleotides. In another aspect, the present disclosure provides recombinant host cells comprising the polynucleotides or vectors. In another aspect, the present disclosure provides a method of producing an antibody that specifically binds to human CD96, the method comprising culturing host cells under suitable conditions such that the polynucleotides are expressed and the antibody is produced.

[0028] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody, polynucleotide, vector, or host cell disclosed herein and a pharmaceutically acceptable carrier or excipient.

[0029] In another aspect, the disclosure provides a method of increasing an immune response in a subject, the method comprising administering to the subject an effective amount of an antibody, polynucleotide, vector, host cell, or pharmaceutical composition disclosed herein.

[0030] In another aspect, the disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody, polynucleotide, vector, host cell, or pharmaceutical composition disclosed herein.

[0031] In another aspect, the disclosure provides a method of treating an infectious disease in a subject, the method comprising administering to the subject an antibody, polynucleotide, vector, host cell, or pharmaceutical composition disclosed herein.

[0032] In certain embodiments of the aforementioned methods, the antibody, polynucleotide, vector, host cell, or pharmaceutical composition is administered systemically, intravenously, subcutaneously, intratumorally, or delivered to a tumor-draining lymph node.

[0033] In certain embodiments of the foregoing methods, the method further comprises administering an additional therapeutic agent to the subject. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent. In certain embodiments, the additional therapeutic agent is a checkpoint targeting agent. In certain embodiments, the checkpoint targeting agent is selected from the group consisting of an antagonistic anti-PD-1 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, an antagonistic anti-CTLA-4 antibody, an antagonistic anti-TIM-3 antibody, an antagonistic anti-LAG-3 antibody, an antagonistic anti-VISTA antibody, an antagonistic anti-TIGIT antibody, an antagonistic anti-CEACAM1 antibody, an antagonistic anti-CD96 antibody, an agonist anti-GITR antibody, and an agonist anti-OX40 antibody. In certain embodiments, the additional therapeutic agent is an anti-PD-1 antibody, and optionally the anti-PD-1 antibody is pembrolizumab or nivolumab. In certain embodiments, the additional therapeutic agent is an inhibitor of indoleamine-2,3-dioxygenase (IDO). In certain embodiments, the inhibitor is selected from the group consisting of epacadostat, F001287, indoximod, and NLG919. In certain embodiments, the additional therapeutic agent is a vaccine. In certain embodiments, the vaccine comprises a heat shock protein peptide complex (HSPPC) comprising a heat shock protein complexed with an antigenic peptide. In certain embodiments, the heat shock protein is hsc70 and is complexed with a tumor-associated antigenic peptide. In certain embodiments, the heat shock protein is gp96 protein and is complexed with a tumor-associated antigenic peptide, and optionally the HSPPC is derived from a tumor obtained from the subject. [Brief explanation of the drawings]

[0034] [Figure 1AB]1A-1B are graphs showing binding of anti-CD96 antibodies BA072 or BA101, or an IgG1 isotype control antibody, to Jurkat cells engineered to express high levels of CD96 cell surface human isoform 2. In each case, the level of Jurkat cell binding of BA072 (FIG. 1A) or BA101 (FIG. 1B), as assessed by median fluorescence intensity (MFI), relative to Jurkat cell binding of the IgG1 isotype control antibody, is plotted against the concentration of each antibody incubated with the cells. [Figure 2AB] 2A-2B are graphs showing binding of anti-CD96 antibodies BA072 or BA101, or an IgG1 isotype control antibody, to CHO cells engineered to express high levels of human CD96 cell surface isoform 1. In each case, the level of binding of BA072 (FIG. 2A) or BA101 (FIG. 2B), as assessed by median fluorescence intensity (MFI), relative to CHO cell binding of the IgG1 isotype control antibody, is plotted against the concentration of each antibody incubated with the cells. [Figure 3AB] 3A-3B are graphs showing binding of anti-CD96 antibodies BA072 or BA101, or an IgG1 isotype control antibody, to CHO cells engineered to express high levels of human CD96 cell surface isoform 2. In each case, the level of binding of BA072 (FIG. 3A) or BA101 (FIG. 3B), as assessed by median fluorescence intensity (MFI), relative to CHO cell binding of the IgG1 isotype control antibody, is plotted against the concentration of each antibody incubated with the cells. [Figure 4AB] 4A-4B are graphs showing binding of anti-CD96 antibodies BA072 or BA101, or an IgG1 isotype control antibody, to CHO cells engineered to express high levels of cynomolgus CD96 cell surface isoform 2. In each case, the level of binding of BA072 (FIG. 4A) or BA101 (FIG. 4B), as assessed by median fluorescence intensity (MFI), relative to CHO cell binding of the IgG1 isotype control antibody, is plotted against the concentration of each antibody incubated with the cells. [Figure 5AB] 5A-5B are graphs showing binding of anti-CD96 antibodies BA072 or BA101, or an IgG1 isotype control antibody, to activated primary human T cells expressing cell surface CD96. In each case, the level of binding of BA072 (FIG. 5A) or BA101 (FIG. 5B), as assessed by median fluorescence intensity (MFI), relative to activated primary human T cell binding of the IgG1 isotype control antibody, is plotted against the concentration of each antibody incubated with the cells. [Figure 6A-C]

[0023] Figure 6A is a series of graphs showing binding of anti-CD96 antibodies BA072, BA083, or BA084, or an IgG1 isotype control antibody, to activated primary human T cells expressing cell surface CD96. In each case, the level of binding of BA072 (Figure 6A), BA083 (Figure 6B), or BA084 (Figure 6C), as assessed by median fluorescence intensity (MFI), relative to activated primary human T cell binding of the IgG1 isotype control antibody, is plotted against the concentration of each antibody incubated with the cells. [Figure 7A-C]

[0033] Figure 7A-7F are a series of graphs showing binding of anti-CD96 antibodies BA101, BA102, BA103, BA104, BA105, or BA106, or an IgG1 isotype control antibody, to activated primary human T cells expressing cell surface CD96. In each case, the level of binding of BA101 (Figure 7A), BA102 (Figure 7B), BA103 (Figure 7C), BA104 (Figure 7D), BA105 (Figure 7E), or BA106 (Figure 7F), as assessed by median fluorescence intensity (MFI), relative to activated primary human T cell binding of the IgG1 isotype control antibody, is plotted against the concentration of each antibody incubated with the cells. Figures 7D-F are shown in the following figures. [Figure 7D-F] Continuation of Figures 7A-C. [Figure 8A-H]1 is a series of graphs showing binding of affinity matured anti-CD96 antibodies BA074, BA073, BA079, BA078, BA081, BA080, BA077, BA076, BA082, or BA075, parental antibody BA072 or BA101, germline antibody BA083, or an IgG1 isotype control antibody to NY-ESO-1 transfected CD8+ T cells expressing cell surface CD96. In each case, the level of binding of BA072 (Figure 8A), BA083 (Figure 8B), BA074 (Figure 8C), BA073 (Figure 8D), BA079 (Figure 8E), BA078 (Figure 8F), BA081 (Figure 8G), BA080 (Figure 8H), BA077 (Figure 8I), BA076 (Figure 8J), BA082 (Figure 8K), BA075 (Figure 8L), or BA101 (Figure 8M), as assessed by median fluorescence intensity (MFI), compared to NY-ESO-1 transfected CD8+ T cell binding of an IgG1 isotype control antibody, is plotted against the concentration of each antibody incubated with the cells (Figures 8I-M). [Figure 8I-M] Continuation of Figures 8A-H. [Figure 9AB] 9A-9B are graphs showing binding of anti-CD96 antibodies BA072 or BA101, or an IgG1 isotype control antibody, to activated primary cynomolgus monkey T cells expressing cell-surface cynomolgus monkey CD96. In each case, the level of binding of BA072 (FIG. 9A) or BA101 (FIG. 9B), as assessed by median fluorescence intensity (MFI), relative to activated primary cynomolgus monkey T cell binding of the IgG1 isotype control antibody is plotted against the concentration of each antibody incubated with the cells. [Figure 10AB] 10A and 10B are graphs showing blockade of PVR-Fc binding by anti-CD96 antibodies BA072 (FIG. 10A) or BA101 (FIG. 10B) to CHO cells engineered to express high levels of human CD96 cell surface isoform 2. In each case, the level of PVR-Fc binding, assessed by median fluorescence intensity (MFI), relative to blockade by an IgG1 isotype control antibody is plotted as % maximal response against the concentration of each antibody incubated with the cells. [Figure 11AB] 11A and 11B are graphs showing blockade of PVR-His binding by anti-CD96 antibodies BA072 (FIG. 11A) or BA101 (FIG. 11B) to CHO cells engineered to express high levels of human CD96 cell surface isoform 2. In each case, the level of PVR-His binding, assessed by median fluorescence intensity (MFI), relative to blockade by an IgG1 isotype control antibody is plotted as % maximal response against the concentration of each antibody incubated with the cells. [Figure 12A-C] 12A-12C are a series of graphs showing blockade of PVR-Fc binding by anti-CD96 antibodies BA072 (FIG. 12A), BA083 (FIG. 12B), or BA084 (FIG. 12C) to CHO cells engineered to express high levels of human CD96 cell surface isoform 2. In each case, the level of PVR-Fc binding, assessed by median fluorescence intensity (MFI), relative to blockade by an IgG1 isotype control antibody is plotted as % maximal response against the concentration of each antibody incubated with the cells. [Figures 13A-F]

[0033] Figure 13G-L are a series of graphs showing blockade of human PVR-Fc binding to CHO cells engineered to express high levels of human CD96 cell surface isoform 2 by anti-CD96 antibodies BA072 (Figure 13A), BA083 (Figure 13B), BA085 (Figure 13C), BA086 (Figure 13D), BA087 (Figure 13E), BA089 (Figure 13F), BA090 (Figure 13G), BA088 (Figure 13H), BA091 (Figure 13I), BA092 (Figure 13J), BA093 (Figure 13K), or BA094 (Figure 13L). In each case, the level of PVR-Fc binding, assessed by median fluorescence intensity (MFI), compared to blockade by an IgG1 isotype control antibody, is plotted as % maximal response against the concentration of each antibody incubated with the cells. Figures 13G-L are shown in the following figures. [Figure 13G-L] Continuation of Figures 13A-F. [Figure 14A-F]

[0033] Figure 14A-L are a series of graphs showing blockade of human PVR-Fc binding to CHO cells engineered to express high levels of human CD96 cell surface isoform 1 by anti-CD96 antibodies BA073 (Figure 14A), BA074 (Figure 14B), BA078 (Figure 14C), BA079 (Figure 14D), BA080 (Figure 14E), BA081 (Figure 14F), BA076 (Figure 14G), BA077 (Figure 14H), BA082 (Figure 14I), BA075 (Figure 14J), BA083 (Figure 14K), or BA072 (Figure 14L). In each case, the level of PVR-Fc binding, assessed by median fluorescence intensity (MFI), compared to blockade by an IgG1 isotype control antibody, is plotted as % maximal response against the concentration of each antibody incubated with the cells. Figures 14G-L are shown in the following figures. [Figure 14G-L] Continuation of Figures 14A-F. [Figures 15A-F]

[0033] Figure 15A-L are a series of graphs showing blockade of human PVR-Fc binding to CHO cells engineered to express high levels of human CD96 cell surface isoform 2 by anti-CD96 antibodies BA073 (Figure 15A), BA074 (Figure 15B), BA078 (Figure 15C), BA079 (Figure 15D), BA080 (Figure 15E), BA081 (Figure 15F), BA076 (Figure 15G), BA077 (Figure 15H), BA082 (Figure 15I), BA075 (Figure 15J), BA083 (Figure 15K), or BA072 (Figure 15L). In each case, the level of PVR-Fc binding, assessed by median fluorescence intensity (MFI), compared to blockade by an IgG1 isotype control antibody, is plotted as % maximal response against the concentration of each antibody incubated with the cells. Figures 15G-L are shown in the following figures. [Figure 15G-L] This is a continuation of 15A-F. [Figures 16A-F]16A-16F are a series of graphs showing blockade of PVR-Fc binding to CHO cells engineered to express high levels of CD96 cell surface human isoform 2 by anti-CD96 antibodies BA101 (FIG. 16A), BA102 (FIG. 16B), BA103 (FIG. 16C), BA104 (FIG. 16D), BA105 (FIG. 16E), or BA106 (FIG. 16F). In each case, the level of PVR-Fc binding, as assessed by median fluorescence intensity (MFI), relative to blockade by an IgG1 isotype control antibody, is plotted as % maximal response against the concentration of each antibody incubated with the cells. [Figure 17AB] 17A and 17B are graphs showing blockade of PVR-Fc binding by anti-CD96 antibodies BA101 (FIG. 17A) or BA107 (FIG. 17B) to CHO cells engineered to express high levels of CD96 cell surface human isoform 2. In each case, the level of PVR-Fc binding, assessed by median fluorescence intensity (MFI), relative to blockade by an IgG1 isotype control antibody is plotted as % maximal response against the concentration of each antibody incubated with the cells. [Figures 18A-C] 18A-18C are a series of graphs showing blockade of PVR-Fc binding by anti-CD96 antibodies BA072 (FIG. 18A), BA083 (FIG. 18B), or BA084 (FIG. 18C) to CHO cells engineered to express high levels of cynomolgus monkey CD96 cell surface isoform 2. In each case, the level of PVR-Fc binding, assessed by median fluorescence intensity (MFI), relative to blockade by an IgG1 isotype control antibody is plotted as % maximal response against the concentration of each antibody incubated with the cells. [Figures 19A-F]

[0033] Figure 19A-L are a series of graphs showing blockade of human PVR-Fc binding to CHO cells engineered to express high levels of cell surface isoform 2 of cynomolgus monkey CD96 by anti-CD96 antibodies BA072 (Figure 19A), BA083 (Figure 19B), BA085 (Figure 19C), BA086 (Figure 19D), BA088 (Figure 19E), BA087 (Figure 19F), BA089 (Figure 19G), BA090 (Figure 19H), BA091 (Figure 19I), BA092 (Figure 19J), BA093 (Figure 19K), or BA094 (Figure 19L). In each case, the level of PVR-Fc binding, assessed by median fluorescence intensity (MFI), compared to blockade by an IgG1 isotype control antibody, is plotted as % maximal response against the concentration of each antibody incubated with the cells. Figures 19G-L are shown in the following figures. [Figure 19G-L] Continuation of Figures 19A-F. [Figure 20A-F]

[0023] Figure 20A-L are a series of graphs showing blockade of human PVR-Fc binding to CHO cells engineered to express high levels of cell surface isoform 1 of cynomolgus monkey CD96 by anti-CD96 antibodies BA073 (Figure 20A), BA074 (Figure 20B), BA078 (Figure 20C), BA079 (Figure 20D), BA080 (Figure 20E), BA081 (Figure 20F), BA076 (Figure 20G), BA077 (Figure 20H), BA082 (Figure 20I), BA075 (Figure 20J), BA083 (Figure 20K), or BA072 (Figure 20L). In each case, the level of PVR-Fc binding, assessed by median fluorescence intensity (MFI), compared to blockade by an IgG1 isotype control antibody, is plotted as % maximal response against the concentration of each antibody incubated with the cells. Figures 20G-L are shown in the following figures. [Figure 20G-L] Continuation of Figures 20A-F. [Figure 21A-F]

[0023] Figure 21G-L are a series of graphs showing blockade of human PVR-Fc binding to CHO cells engineered to express high levels of cell surface isoform 2 of cynomolgus monkey CD96 by anti-CD96 antibodies BA073 (Figure 21A), BA074 (Figure 21B), BA078 (Figure 21C), BA079 (Figure 21D), BA080 (Figure 21E), BA081 (Figure 21F), BA076 (Figure 21G), BA077 (Figure 21H), BA082 (Figure 21I), BA075 (Figure 21J), BA083 (Figure 21K), or BA072 (Figure 21L). In each case, the level of PVR-Fc binding, assessed by median fluorescence intensity (MFI), compared to blockade by an IgG1 isotype control antibody, is plotted as % maximal response against the concentration of each antibody incubated with the cells. Figures 21G-L are shown in the following figures. [Figure 21G-L] Continuation of Figures 21A-F. [Figure 22A-C] Figure 22A is a graph showing conjugate formation of CHO cells engineered to express high levels of human CD96 or PVR isoform 2 in the presence of anti-CD96 antibodies BA072 (Figure 22A) or BA101 (Figure 22B), or an IgG1 isotype control antibody. In each case, the percentage of conjugates formed, compared to the IgG1 isotype control, is plotted against the concentration of each antibody incubated with the cells. Figure 22C is a scatter plot showing conjugate formation in quadrant Q2 in the presence of an isotype control and no blocking antibody. [Figure 23] 1 is a graph showing conjugate formation of CHO cells engineered to express high levels of human CD96 or PVR isoform 2 in the presence of anti-CD96 antibodies BA072, BA083, BA084, or an IgG1 isotype control antibody. In each case, the percentage of conjugates formed, compared to the IgG1 isotype control, is plotted against the concentration of each antibody incubated with the cells. [Figure 24]1 is a graph showing conjugate formation of CHO cells engineered to express high levels of human CD96 or PVR isoform 2 in the presence of anti-CD96 antibodies BA101, BA102, BA103, BA104, BA105, or BA106. In each case, the percentage of conjugates formed, compared to an IgG1 isotype control, is plotted against the concentration of each antibody incubated with the cells. [Figure 25A-H]

[0023] Figure 25 is a series of graphs showing that the anti-CD96 antibodies BA072 and BA101, when administered with or without an anti-PD-1 antibody to two different donors, promote IL-2 secretion by SEA-stimulated PBMCs in a dose-dependent manner. Figures 25A-D represent the first experiment with the first donor, and Figures 25E-H represent the second experiment with the second donor. [Figure 26A-F] Figures 26A and 26B represent one experiment without (Figure 26A) and with (Figure 26B) anti-PD-1 antibody. Figures 26C and 26D represent a second experiment with a different donor without (Figure 26C) and with (Figure 26D) anti-PD-1 antibody. Figures 26E and 26F represent a third experiment with a different donor without (Figure 26E) and with (Figure 26F) anti-PD-1 antibody. [Figure 27A-F]

[0039] Figures 27A and 27B are a series of graphs showing the ability of affinity-matured BA074, BA079, BA077, BA081, BA082, and BA075 antibodies, as well as the parental BA072 antibody, to promote IL-2 secretion by SEA-stimulated PBMCs. Figures 27A and 27B represent one experiment without (Figure 27A) and with (Figure 27B) anti-PD-1 antibody. Figures 27C and 27D represent a second experiment with a different donor without (Figure 27C) and with (Figure 27D) anti-PD-1 antibody. Figures 27E and 27F represent a third experiment with a different donor without (Figure 27E) and with (Figure 27F) anti-PD-1 antibody. [Figure 28AB]28A and 28B are graphs showing increased NFAT-luciferase (FIG. 28A) and NFκB-luciferase (FIG. 28B) signaling on CD96-expressing Jurkat reporter cells in the presence of BA072 and PVR and anti-CD3-expressing CHO cells. The delta relative light units (RLU) between BA072 and the isotype control are plotted against antibody concentration. [Figure 28C] 1 is a series of histograms showing cell surface expression of CD96, CD226, PVR, and CD3. [Figure 29AB] 29A and 29B are graphs showing increased NFAT-luciferase signaling on CD96-expressing Jurkat reporter cells with (FIG. 29A) and without (FIG. 29B) CD226 surface expression in the presence of BA072 and PVR and anti-CD3-expressing CHO cells. The delta relative light units (RLU) between BA072 and the isotype control are plotted against antibody concentration. [Figure 30A-C]

[0033] Figure 30A is a series of graphs showing the promotion of antibody-dependent cell-mediated cytotoxicity (ADCC) of CD96-expressing cells in the presence of primary NK cells, as measured by induction of caspase 3 / 7 activation by BA072 IgG1 (Figure 30A), Fc-enhanced BA072 (BA109) (Figure 30B), or an Fc-silent variant of BA072 (BA108) (Figure 30C), in each case compared to the isotype control. % induced caspase 3 / 7 activation is plotted against time (h). [Figure 31A-C]

[0039] Figure 31B is a series of graphs showing FcγRIIIA-mediated NFAT signaling from FcγRIIIA-expressing Jurkat reporter cells in the presence of anti-CD96 BA072 Fc variant, Fc-enhanced BA072 variant (BA109) (Figure 31B), Fc-silent variant of BA072 (BA108) (Figure 31C), or BA072 IgG1 (Figure 31A) bound to CD96-expressing target cells (4:1 E:T ratio). Relative light units (RLU) are plotted against antibody concentration. [Figure 32AB]32A and 32B are graphs showing the extent of IL-2 secretion induced by BA072 (FIG. 32A) and BA108 (an Fc-silent variant of BA072; FIG. 32B) in a T cell:APC co-culture assay using PBMCs from two human donors. [Figure 33A-D]

[0033] Figure 33A is a series of graphs showing percent internalization of CD96 using CD96-expressing Jurkat cells in the presence of BA072 (Figure 33A), BA101 (Figure 33B), reference antibody RefA (Figure 33C), or PVR-Fc (Figure 33D). [Fig. 34A-D]

[0033] Figure 34A is a series of graphs showing percent internalization of CD96 using CD96-expressing Jurkat cells in the presence of parental antibody BA072 (Figure 34A) or BA101 (Figure 34D), or germline variants BA083 (Figure 34B) or BA084 (Figure 34C). [Figure 35AB] 35A and 35B are graphs showing internalization of CD96 by CD96-expressing primary T cells in the presence of BA072 in donor 1 (FIG. 35A) and donor 2 (FIG. 35B). [Figure 36A] 36A-36B are sensorgrams showing binding of BA072 Fab, BA101 Fab, and Reference A Fab to Fc-tagged full-length human CD96 (FIG. 36A) or Fc-tagged domain 1 human CD96 (FIG. 36B). [Figure 36B] The above shows 36B. [Figure 37A]

[0037] Figures 37A and 37B are a series of sensorgrams showing binding of BA072 Fab, BA101 Fab, and Reference A Fab to Fc-tagged full-length human CD96 (Figures 37A and 37B) or Fc-tagged domain 1 human CD96 (Figures 37C and 37D). Figures 37A and 37C represent experiments in which the initial association was with BA072. Figures 37B and 37D represent experiments in which the initial association was with BA101. Figures 37B-D are shown in separate figures. [Figure 37B] The above Figure 37B is shown. [Figure 37C] The above Figure 37C is shown. [Figure 37D] Figure 37D above is shown. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present disclosure provides antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and antagonize CD96 function, e.g., CD96-mediated immunosuppression. Also provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for producing these antibodies, and methods of treating subjects using these antibodies. The antibodies disclosed herein are particularly useful for increasing immune cell activation and are therefore useful for treating cancer in a subject or treating or preventing an infectious disease in a subject. All examples of "isolated antibodies" described herein are additionally contemplated as antibodies that may, but need not, be isolated. All examples of "isolated polynucleotides" described herein are additionally contemplated as polynucleotides that may, but need not be isolated. All examples of "antibodies" described herein are additionally contemplated as antibodies that may, but need not be isolated. All examples of "polynucleotides" described herein are additionally contemplated as polynucleotides that may, but need not be isolated.

[0036] definition As used herein, the terms "about" and "approximately," when used to modify a numerical value or numerical range, indicate that deviations of 5% to 10% above (e.g., up to 5% to 10% above) and 5% to 10% below (e.g., up to 5% to 10% below) the value or range remain within the intended meaning of the recited value or range.

[0037] As used herein, the term "CD96" refers to cluster of differentiation 96, also known as TACTILE (T cell activation, increased late expression), which in humans is encoded by the CD96 gene. As used herein, the term "human CD96" refers to the CD96 protein encoded by the wild-type human CD96 gene (e.g., GenBank™ Accession No. NM_005816.5), fragments, or variants thereof. Exemplary extracellular portions of human CD96 are provided herein as SEQ ID NOs: 127, 128, 129, 130, and 131. Exemplary extracellular portions of cynomolgus monkey CD96 are provided herein as SEQ ID NOs: 132, 133, and 134.

[0038] As used herein, the terms "CD155," "poliovirus receptor," and "PVR" are used interchangeably and refer to the CD155 protein encoded by the CD155 gene (e.g., GenBank™ Accession No. NM_006505.5), fragments, or variants thereof.

[0039] As used herein, the term "antibody" includes full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH regions, and / or VL regions. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In certain embodiments, the antibody described herein refers to a polyclonal antibody population. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, the antibody described herein is an IgG antibody, or a class (e.g., human IgG1 or IgG4) or subclass thereof. In certain embodiments, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody.

[0040] As used herein, the terms "VH region" and "VL region" refer to a single antibody heavy chain variable region and light chain variable region, respectively, comprising FR (framework regions) 1, 2, 3, and 4, and CDR (complementarity-determining regions) 1, 2, and 3 (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is incorporated herein by reference in its entirety).

[0041] As used herein, the term "CDR" or "complementarity-determining region" refers to the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions are described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), all of which are incorporated herein by reference in their entirety. The definitions include overlapping or subsets of amino acid residues when compared to each other. In certain embodiments, the term "CDR" refers to a CDR as defined by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).In certain embodiments, the term "CDR" refers to a CDR as defined by Kabat et al., J. Biol. MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991). In certain embodiments, the heavy chain CDRs and light chain CDRs of an antibody are defined using different conventions. In certain embodiments, the heavy chain and / or light chain CDRs are defined by performing a structural analysis of the antibody and identifying residues in the variable region(s) that are predicted to contact the epitope region of the target molecule (e.g., human and / or cynomolgus CD96). CDRH1, CDRH2, and CDRH3 represent the heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 represent the light chain CDRs.

[0042] As used herein, the term "framework (FR) amino acid residues" refers to those amino acids in the framework region of an immunoglobulin chain. The term "framework region" or "FR region" as used herein includes amino acid residues that are part of the variable region but are not part of the CDRs (e.g., using the Kabat or MacCallum definitions of CDRs).

[0043] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a light or heavy chain, typically the amino-terminal 110-120 or 110-125 amino acids in a mature heavy chain and approximately 90-115 amino acids in a mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody to its particular antigen. The variability in sequence is concentrated in regions called complementarity-determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). While not wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of an antibody with an antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0044] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0045] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0046] As used herein, the terms "constant region" or "constant domain" are interchangeable and are common in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen but may exhibit various effector functions, such as interaction with Fc receptors (e.g., Fc gamma receptors).

[0047] As used herein, the term "heavy chain," when used in reference to an antibody, can refer to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0048] As used herein, the term "light chain," when used in reference to an antibody, can refer to any of the different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain.

[0049] As used herein, the term "EU numbering system" refers to the EU numbering convention for antibody constant regions as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

[0050] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ), and the equilibrium binding constant (K A) can be measured and / or expressed in a number of ways known in the art, including, but not limited to, K D is k off / k on is calculated from the ratio of A is k on / k off It is calculated from the ratio of k on refers to the binding rate constant of an antibody to an antigen, and k off k refers to the dissociation rate constant of an antibody to an antigen, for example. on and k off can be determined by techniques known to those skilled in the art, such as BIAcore® or KinExA. As used herein, "low affinity" refers to a larger K D Refers to...

[0051] As used herein, the terms "specifically bind," "specifically recognize," "immunospecifically bind," and "immunospecifically recognize" are similar terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immune complex) as such binding is understood by one of skill in the art. For example, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides with lower affinity, as determined, for example, by immunoassays, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, Idaho), or other assays known in the art. In certain embodiments, a molecule that specifically binds to an antigen has a K that is lower than the K that is observed when the molecule nonspecifically binds to another antigen. A K that is at least 2 logs (e.g., 10 times), 2.5 logs, 3 logs, or 4 logs or more A binds to the antigen.

[0052] In another specific embodiment, a molecule that specifically binds to an antigen does not cross-react with other proteins under similar binding conditions. In another specific embodiment, a molecule that specifically binds to CD96 does not cross-react with other non-CD96 proteins. In certain embodiments, provided herein are antibodies that bind to CD96 (e.g., human CD96) with higher affinity than another unrelated antigen. In certain embodiments, provided herein are antibodies that bind to CD96 (e.g., human CD96) with 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more higher affinity than another unrelated antigen, as measured, for example, by radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In certain embodiments, the extent of binding of an anti-CD96 antibody described herein to an unrelated non-CD96 protein is less than 10%, 15%, or 20% of the binding of the antibody to the CD96 protein, as measured, for example, by radioimmunoassay.

[0053] As used herein, "epitope" is a term used in the art and refers to a region of an antigen to which an antibody can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (a linear or continuous epitope), or it can be, for example, comprised of two or more discontinuous regions of a polypeptide or multiple polypeptides (a conformational, non-linear, discontinuous, or discontinuous epitope). In certain embodiments, the epitope to which an antibody binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, mass spectrometry-linked hydrogen / deuterium exchange (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligo-peptide scanning assays (e.g., peptides constrained using CLIPS (Chemical Linkage of Peptides onto Scaffolds) to map discontinuous or conformational epitopes), and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303, each of which is incorporated herein by reference in its entirety).Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303. Antibody:antigen crystals may be studied using well-known X-ray diffraction techniques, including X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al.,; US2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed. Carter CW; Roverski P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323), each of which is incorporated herein by reference in its entirety. BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323) mutagenesis mapping studies may be performed using any method known to those skilled in the art.For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, for example, Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085, each of which is incorporated herein by reference in its entirety. CLIPS (Chemical Linkage of Peptides onto Scaffolds) is a technology that displays one or more peptides in a structurally constrained configuration that behaves as a functional mimic of a complex protein domain. See, for example, U.S. Publication Nos. US2008 / 0139407 A1 and US2007 / 099240 A1, and U.S. Patent No. 7,972,993, each of which is incorporated herein by reference in its entirety. In certain embodiments, the epitope of an antibody is determined using alanine scanning mutagenesis studies. In certain embodiments, the epitope of the antibody is determined using hydrogen / deuterium exchange coupled with mass spectrometry. In certain embodiments, the epitope of the antibody is determined using Pepscan Therapeutics' CLIPS epitope mapping technology. In certain embodiments, the epitope of the antibody is determined by protein mutagenesis, for example, by generating a switch mutant of the antigen with a portion of its ortholog from another species, and then testing the switch mutant for loss of antibody binding (e.g., by FACS-based cell binding assay as described herein).

[0054] As used herein, the terms "T cell receptor" and "TCR" are used interchangeably and refer to full-length heterodimeric αβ or γδ TCRs, antigen-binding fragments of full-length TCRs, and molecules comprising TCR CDRs or variable regions. Examples of TCRs include, but are not limited to, full-length TCRs, antigen-binding fragments of full-length TCRs, soluble TCRs lacking the transmembrane and cytoplasmic regions, single-chain TCRs comprising variable regions of TCRs joined by flexible linkers, engineered disulfide-linked TCR chains, monospecific TCRs, multispecific TCRs (including bispecific TCRs), TCR fusions, human TCRs, humanized TCRs, chimeric TCRs, recombinantly produced TCRs, and synthetic TCRs. The term encompasses wild-type TCRs and genetically engineered TCRs (e.g., chimeric TCRs comprising a chimeric TCR chain comprising a first portion from a TCR of a first species and a second portion from a TCR of a second species).

[0055] As used herein, "major histocompatibility complex" and "MHC" are used interchangeably and refer to MHC class I molecules and / or MHC class II molecules.

[0056] As used herein, a "peptide-MHC complex" refers to an MHC molecule (MHC class I or MHC class II) having a peptide bound within the art-recognized peptide-binding pocket of the MHC.

[0057] As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or prophylactic measures as described herein. Methods of "treatment" employ administration of an antibody to a subject having or prone to having a disease or disorder to prevent, cure, delay, reduce the severity of, or ameliorate a disease or disorder or one or more symptoms of a recurrent disease or disorder, or to extend the subject's lifespan beyond that expected in the absence of such treatment.

[0058] As used herein, the term "effective amount" in the context of administering a therapy to a subject refers to the amount of therapy that achieves the desired prophylactic or therapeutic effect.

[0059] As used herein, the term "internalization" or "internalized" refers to the uptake of an antibody into an intracellular compartment of a cell upon binding of the antibody to an antigen expressed on the surface of the cell.

[0060] As used herein, the term "subject" includes any human or non-human animal. In one embodiment, the subject is a human or non-human mammal. In one embodiment, the subject is a human.

[0061] The determination of "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be achieved using a mathematical algorithm.A specific, non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin S&Altschul SF (1990) PNAS 87:2264-2268, modified in Karlin S&Altschul SF (1993) PNAS 90:5873-5877, each of which is incorporated herein by reference in its entirety.Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215:403, which is incorporated herein by reference in its entirety.BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, for example, with word length=12 for score=100, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set to obtain amino acid sequences homologous to the protein molecules described herein, for example, score=50, word length=3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul SF et al., (1997) NuC Acids Res 25:3389-3402, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When using BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety.Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0062] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0063] Anti-CD96 antibody In one aspect, antibodies are provided that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and antagonize CD96 function. The amino acid sequences of exemplary antibodies are shown in Table 1 herein. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10]

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

Table 1-38

Table 1-39

Table 1-40

Table 1-41

Table 1-42

Table 1-43

Table 1-44

[0064] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96), wherein the antibody comprises a VH that comprises one, two, or all three of the CDRs of the VH shown in Table 1 herein. In certain embodiments, the antibody comprises a CDRH1 of the VH shown in Table 1. In certain embodiments, the antibody comprises a CDRH2 of the VH shown in Table 1. In certain embodiments, the antibody comprises a CDRH3 of the VH shown in Table 1.

[0065] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96), wherein the antibody comprises a VL that comprises one, two, or all three of the CDRs of a VL disclosed in Table 1 herein. In certain embodiments, the antibody comprises a CDRL1 of a VL shown in Table 1. In certain embodiments, the antibody comprises a CDRL2 of a VL shown in Table 1. In certain embodiments, the antibody comprises a CDRL3 of a VL shown in Table 1.

[0066] In certain embodiments, the CDRs of an antibody can be determined according to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest (1991), each of which is incorporated herein by reference in its entirety. In certain embodiments, the light chain CDRs of an antibody are determined according to Kabat, and the heavy chain CDRs of an antibody are determined according to MacCallum (supra). In certain embodiments, the heavy chain CDRs and / or light chain CDRs are defined by performing a structural analysis of the antibody and identifying residues in the variable region(s) that are predicted to contact the epitope region of the target molecule (e.g., human and / or cynomolgus CD96).

[0067] In certain embodiments, the CDRs of an antibody can be determined according to the Chothia numbering scheme, which refers to the position of the immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Patent No. 7,709,226, all of which are incorporated herein by reference in their entireties). Chothia C&Lesk AM,(1987),J Mol Biol 196:901-917;Al-Lazikani B et al.,(1997)J Mol Biol 273:927-948;Chothia C et al.,(1992)J Mol Biol 227:799-817;Tramontano A et al.,(1990)J Mol Biol 215(1):175-82; typically, using the Kabat numbering convention, the CDRH1 loop of Chothia is located at amino acids 26-32, 33, or 34 of the heavy chain, the CDRH2 loop of Chothia is located at amino acids 52-56 of the heavy chain, and the CDRH3 loop of Chothia is located at amino acids 95-102 of the heavy chain, while the CDRL1 loop of Chothia is located at amino acids 24-34 of the light chain, the CDRL2 loop of Chothia is located at amino acids 50-56 of the light chain, and the CDRL3 loop of Chothia is located at amino acids 89-97 of the light chain. When numbered using the Kabat numbering convention, the end of the CDRH1 loop in Chothia varies from H32 to H34 depending on the length of the loop (this is because the Kabat numbering scheme results in insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34).

[0068] In certain embodiments, the CDRs of an antibody can be determined according to MacCallum RM et al., (1996) J Mol Biol 262:732-745, which is incorporated herein by reference in its entirety. 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), which is incorporated herein by reference in its entirety.

[0069] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises the VH CDR of Chothia of the VH disclosed in Table 1 herein. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises the VL CDR of Chothia of the VL disclosed in Table 1 herein. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises the VH CDR of Chothia and the VL CDR of Chothia of an antibody disclosed in Table 1 herein. In certain embodiments, an antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) comprises one or more CDRs, wherein the Chothia and Kabat CDRs have the same amino acid sequence. In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to CD96 (eg, human CD96 or cynomolgus CD96) and comprise a combination of the Kabat and Chothia CDRs.

[0070] In certain embodiments, the CDRs of an antibody can be determined according to the IMGT numbering system as described in Lefranc MP, (1999) The Immunologist 7:132-136; Lefranc MP et al., (1999) Nucleic Acids Res 27:209-212, each of which is incorporated herein by reference in its entirety, and Lefranc MP et al., (2009) Nucleic Acids Res 37:D1006-D1012.

[0071] In certain embodiments, the present disclosure provides antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and comprise the CDRs of the antibodies disclosed in Table 1 herein, e.g., as determined by the IMGT numbering system as described in Lefranc MP (1999) supra and Lefranc MP et al., (1999) supra.

[0072] In certain embodiments, the CDRs of an antibody can be determined according to the AbM numbering scheme, which represents a compromise between the Kabat CDRs and Chothia structural loops and refers to the AbM hypervariable regions used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), the entire contents of which are incorporated herein by reference. In certain embodiments, the present disclosure provides antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and comprise the CDRs of the antibodies disclosed in Table 1 herein, as determined by the AbM numbering scheme.

[0073] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96), wherein the antibody comprises a VH comprising the amino acid sequence of the CDRH1, CDRH2, and CDRH3 regions of the VH set forth in SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61; and a VH comprising the amino acid sequence of the CDRH1, CDRH2, and CDRH3 regions of the VH set forth in SEQ ID NOs: 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 1 70, 71, 72, 73, 74, or 75, wherein each CDR is defined according to the MacCallum definition, the Kabat definition, the Chothia definition, the IMGT numbering system, the AbM definition, structural analysis, or a combination thereof, wherein the structural analysis identifies residues in the variable region(s) that are predicted to contact an epitope region of CD96 (e.g., human CD96 or cynomolgus CD96). In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and comprises a combination of CDRs defined by the Kabat definition and CDRs defined by antibody structural analysis, wherein the structural analysis identifies residues in the variable region(s) that are predicted to contact an epitope region of CD96 (e.g., human CD96 or cynomolgus CD96).

[0074] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96), wherein the antibody: (a) a CDRH1 comprising the amino acid sequence of X1YX2X3X4 (SEQ ID NO: 135), X1 is Q or S; X2 is A or S; X3 is M or I; CHRH1, wherein X4 is H or S; (b)X1IX2X3X4X5X6X7X8X9YX 10a CDRH2 comprising the amino acid sequence of QKFQG (SEQ ID NO: 137), X1 is W or G; X2 is N or I; X3 is A, E, V, or P; X4 is V, G, W, or I; X5 is S, Y, T, N, or F; X6 is G or W, X7 is D, Y, N, or T; X8 is T or A; X9 is K or N; X 10 is S or A, (c) a CDRH3 comprising the amino acid sequence of NWGX1SYGX2DV (SEQ ID NO: 180), GYDSRPLDV (SEQ ID NO: 19), or GYDSRPLDY (SEQ ID NO: 20), X1 is M or L; CDRH3, wherein X2 is M or L; (d) a CDRL1 comprising the amino acid sequence of RASQSIX1X2YLN (SEQ ID NO: 139) or GGNNIGSKIVH (SEQ ID NO: 26), X1 is S, T, or L; CDRL1, wherein X2 is S, P, or W; (e) a CDRL2 comprising the amino acid sequence of X1X2SSLQS (SEQ ID NO: 141) or DDRDRPS (SEQ ID NO: 32), X1 is S or A, CDRL2, in which X2 is A, S, or E, and / or (f) a CDRL3 comprising the amino acid sequence of QQX1YSTPALX2 (SEQ ID NO: 143) or QVWDINVHHVI (SEQ ID NO: 35), X1 is S or A, X2 is T or S. In certain embodiments of the antibodies disclosed herein, the amino acid immediately N-terminal to CDRH1 (eg, as described in Table 1 herein) is N, T, S, D, or A.

[0075] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96), wherein the antibody: (a) a CDRH1 comprising the amino acid sequence of X1YX2X3X4 (SEQ ID NO: 135), X1 is Q or S; X2 is A or S; X3 is M or I; CDRH1, wherein X4 is H or S; (b)X1IX2X3X4X5X6X7X8X9YX 10 a CDRH2 comprising the amino acid sequence of QKFQG (SEQ ID NO: 137), X1 is W or G; X2 is N or I; X3 is A, E, V, or P; X4 is V, G, W, or I; X5 is S, Y, T, N, or F; X6 is G or W, X7 is D, Y, N, or T; X8 is T or A; X9 is K or N; X 10 is S or A, (c) a CDRH3 comprising the amino acid sequence of NWGX1SYGX2DV (SEQ ID NO: 180), GYDSRPLDV (SEQ ID NO: 19), or GYDSRPLDY (SEQ ID NO: 20), X1 is M or L; CDRH3, wherein X2 is M or L; (d) a CDRL1 comprising the amino acid sequence of RASQSIX1X2YLN (SEQ ID NO: 139) or GGNNIGSKIVH (SEQ ID NO: 26), X1 is S, T, or L; CDRL1, wherein X2 is S, P, or W; (e) a CDRL2 comprising the amino acid sequence of X1X2SSLQS (SEQ ID NO: 141) or DDRDRPS (SEQ ID NO: 32), X1 is S or A, X2 is A, S, or E; and (f) CDRL3 comprises the amino acid sequence of QQX1YSTPALX2 (SEQ ID NO: 143) or QVWDINVHHVI (SEQ ID NO: 35); X1 is S or A, An isolated antibody, wherein X2 is T or S.

[0076] In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibodies comprise VHs comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 1, 5, and 18; 2, 6, and 18; 2, 8, and 18; 2, 9, and 18; 2, 10, and 18; 1, 7, and 18; 2, 11, and 18; 1, 12, and 18; 1, 13, and 18; 1, 14, and 18; 3, 15, and 18; 1, 16, and 18; 1, 5, and 140; 1, 5, and 142; 1, 5, and 179; 4, 17, and 19; or 4, 17, and 20, respectively.

[0077] In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus monkey CD96), wherein the antibodies comprise a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 21, 28, and 33; 21, 29, and 33; 21, 30, and 33; 21, 31, and 33; 22, 29, and 33; 24, 29, and 33; 23, 29, and 33; 25, 28, and 34; or 26, 32, and 35, respectively.

[0078] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96), wherein the antibody comprises a VH comprising CDRH1, CDRH2, and CDRH3 regions, and a VL comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions are set forth in SEQ ID NOs: 1, 5, 18, 21, 28, and 33; 1, 5, 18, 21, 29, and 33; 1, 5, 18, 22, 29, and 33; 1, 5, 18, 23, 29, and 33; 1, 5, 18, 24, 29, and 33; 1, 5, 18, 25, 28, and 34; 1, 5, 140, 21, 28, and 35; and 33; 1, 5, 142, 21, 28, and 33; 1, 5, 179, 21, 28, and 33; 1, 7, 18, 21, 29, and 33; 1, 12, 18, 21, 28, and 33; 1, 13, 18, 21, 28, and 33; 1, 14, 18, 21, 28, and 33; 1, 16, 18, 21, 28, and 33; 2, 6, 18, 21, 29, and and 33; 2, 8, 18, 21, 29, and 33; 2, 9, 18, 21, 30, and 33; 2, 10, 18, 21, 29, and 33; 2, 11, 18, 21, 31 and 33; 3, 15, 18, 21, 28, and 33; 4, 17, 19, 26, 32, and 35; or 4, 17, 20, 26, 32, and 35.

[0079] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), comprising a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96), comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence set forth in SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61. In certain embodiments, X in any one of SEQ ID NOs: 36-61 is glutamine. In certain embodiments, X of any one of SEQ ID NOs: 36-61 is pyroglutamate.

[0080] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), comprising a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In certain embodiments, the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75.

[0081] In certain embodiments, the present disclosure provides a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61; and a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), comprising a VH comprising the amino acid sequence of SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61, and a VL comprising the amino acid sequence of SEQ ID NO: 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence set forth in SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61, and the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In certain embodiments, X in any one of SEQ ID NOs: 36-61 is glutamine. In certain embodiments, X in any one of SEQ ID NOs: 36-61 is pyroglutamate.

[0082] In certain embodiments, the present disclosure provides the nucleic acids of SEQ ID NOs: 36 and 62; 37 and 62; 37 and 63; 37 and 66; 37 and 67; 37 and 68; 37 and 69; 38 and 63; 39 and 63; 40 and 63; 41 and 63; 42 and 63; 43 and 64; 44 and 64; 45 and 63; 46 and 63; 47 and 65; 48 and 62; 49 and 62; 50 and 62; 51 and 62; 52 and 62; 53 and 62; 54 and 62; 55 and 62; 56 and 62; 57 and 62; 58 and 62; 59 and 62; 60 and 70; 60 and 71; 60 and 72; 60 and 73; 60 and 74; 60 and 75; or 61 and 70. In certain embodiments, the VH and VL amino acid sequences consist of the VH and VL amino acid sequences set forth in SEQ ID NOs: 36 and 62; 37 and 62; 37 and 63; 37 and 66; 37 and 67; 37 and 68; 37 and 69; 38 and 63; 39 and 63; 40 and 63; 41 and 63; 42 and 63; 43 and 64; 44 and 64; 45 and 63; 46 and 63; 47 and 65; 48 and 62; 49 and 62; 50 and 62; 51 and 62; 52 and 62; 53 and 62; 54 and 62; 55 and 62; 56 and 62; 57 and 62; 58 and 62; 59 and 62; 60 and 70; 60 and 71; 60 and 72; 60 and 73; 60 and 74; 60 and 75; or 61 and 70. In certain embodiments, X in any one of SEQ ID NOs: 36-61 is glutamine. In certain embodiments, X in any one of SEQ ID NOs: 36-61 is pyroglutamate.

[0083] In certain embodiments, the present disclosure provides SEQ ID NOs: 36 and 62; 37 and 62; 37 and 63; 37 and 66; 37 and 67; 37 and 68; 37 and 69; 38 and 63; 39 and 63; 40 and 63; 41 and 63; 42 and 63; 43 and 64; 44 and 64; 45 and 63; 46 and 63; 47 and 65; 48 and 62; 49 and 62; 50 and 62; 51 and 62; 52 and 63; and 62; 53 and 62; 54 and 62; 55 and 62; 56 and 62; 57 and 62; 58 and 62; 59 and 62; 60 and 70; 60 and 71; 60 and 72; 60 and 73; 60 and 74; 60 and 75; or 61 and 70.

[0084] In certain embodiments, the present disclosure relates to antibodies described herein, for example, those set forth in SEQ ID NOs: 36 and 62; 37 and 62; 37 and 63; 37 and 66; 37 and 67; 37 and 68; 37 and 69; 38 and 63; 39 and 63; 40 and 63; 41 and 63; 42 and 63; 43 and 64; 44 and 64; 45 and 63; 46 and 63; 47 and 65; 48 and 62; 49 and 62; 50 and 62; 51 and 62; 52 and 63; and 62; 53 and 62; 54 and 62; 55 and 62; 56 and 62; 57 and 62; 58 and 62; 59 and 62; 60 and 70; 60 and 71; 60 and 72; 60 and 73; 60 and 74; 60 and 75; or 61 and 70. In certain embodiments, the epitope of the antibody can be determined by, for example, NMR spectroscopy, surface plasmon resonance (BIAcore®), or other methods. (登録商標)), X-ray diffraction crystallography studies, ELISA assays, mass spectrometry-linked hydrogen / deuterium exchange (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligo-peptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303, all of which are incorporated herein by reference in their entireties).Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303. Antibody:antigen crystals may be studied using well-known X-ray diffraction techniques, including X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al., U.S. Patent Application No. 2004 / 0014194), BUSTER (Bricogne G (1993) Acta Refinement may be performed using computer software such as Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter CW; Roverski P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323, all of which are incorporated herein by reference in their entireties. Meth Enzymol (1985) volumes 114&115, eds Wyckoff HW et al.; US Patent Application No. 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323. Mutagenesis mapping studies may be performed using any method known to those skilled in the art.For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, for example, Champe M et al., (1995) supra and Cunningham BC & Wells JA (1989) supra. In certain embodiments, the epitope of an antibody is determined using alanine scanning mutagenesis studies. In addition, antibodies that recognize and bind to the same or overlapping epitopes of CD96 (e.g., human CD96 or cynomolgus monkey CD96) can be identified using routine techniques, such as immunoassays, by demonstrating the ability of one antibody to block the binding of another antibody to a target antigen, i.e., by competitive binding assays. Competitive binding assays can also be used to determine whether two antibodies have similar binding specificities for an epitope. Competitive binding can be determined in an assay in which an immunoglobulin inhibits the specific binding of a reference antibody under test to a common antigen, such as CD96 (e.g., human CD96 or cynomolgus monkey CD96).There are many types of competitive binding assays, such as solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competitive assays (see Stahli C et al., (1983) Methods Enzymol 9:242-253); solid-phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137:3614-9); solid-phase direct label assay, solid-phase direct label sandwich assay (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using I-125 label (see Morel GA et al., (1988) Mol Immunol 25(1):7-15); solid-phase direct biotin-avidin EIA (see Cheung RC et al., (1986) J Immunol 137:3614-9); al., (1990) Virology 176:546-52); and direct labeling RIA (Moldenhauer G et al., (1990) Scand J Immunol 32:77-82) are known, all of which are incorporated herein by reference in their entirety. Typically, such assays involve the use of purified antigen (e.g., CD96, such as human CD96 or cynomolgus CD96) bound to a solid surface or cells bearing either of these unlabeled test immunoglobulins and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, when a competing antibody is present in excess, it will inhibit the specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Competitive binding assays can be designed in a number of different formats, using either labeled antigen or labeled antibody. In a common version of this assay, the antigen is immobilized on a 96-well plate.The ability of unlabeled antibodies to block the binding of labeled antibodies to antigen is then measured using radioactive or enzyme labels. For further details, see, for example, Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al., (1984) J Immunol Methods 68:269-274; Kuroki M at al., (1990) Cancer Res 50:4872-4879; Kuroki M et al., (1992) Immunol Invest 21:523-538; Kuroki M et al., (1992) Hybridoma 11:391-407; and Antibodies: Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al., (1984) J Immunol Methods 68:269-274; Kuroki M et al. al., (1990) Cancer Res 50:4872-4879; Kuroki M et al., (1992) Immunol Invest 21:523-538; Kuroki M et al., (1992) Hybridoma 11:391-407 A Laboratory Manual, Ed Harlow E & Lane D editors (supra), pp. 386-389, all of which are incorporated herein by reference in their entirety.

[0085] In certain embodiments, the antibody inhibits binding of human CD96 to human CD155 (also known as poliovirus receptor (PVR)). In certain embodiments, binding of human CD96 to human CD155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the antibody relative to binding of human CD96 to human CD155 in the absence of the antibody.

[0086] In certain embodiments, the antibody inhibits binding of a soluble fragment of human CD96 to a soluble fragment of human CD 155. In certain embodiments, binding of the soluble fragment of human CD96 to the soluble fragment of human CD 155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the antibody relative to binding of the soluble fragment of human CD96 to the soluble fragment of human CD 155 in the absence of the antibody.

[0087] In certain embodiments, the antibody inhibits binding of CD96-expressing cells to a soluble fragment of human CD 155. In certain embodiments, binding of CD96-expressing cells to a soluble fragment of human CD 155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the antibody relative to binding of CD96-expressing cells to a soluble fragment of human CD 155 in the absence of the antibody.

[0088] In certain embodiments, the antibody inhibits binding of CD96-expressing cells to cells expressing human CD 155. In certain embodiments, binding of CD96-expressing cells to CD155-expressing cells is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the antibody relative to binding of CD96-expressing cells to CD155-expressing cells in the absence of the antibody.

[0089] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, or 169. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 76. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 77. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 78. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 79. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 80. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 81. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 82. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 83. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 84. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 85. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 86. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 87. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 88. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 89. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 90. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 91. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 92.In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 93. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 94. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 95. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 96. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 97. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 98. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 99. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 100. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 101. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 144. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 145. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 147. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 148. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 149. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 150. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 151. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 152. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 153. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 154. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 155. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 156. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 157.In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 158. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 159. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 160. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 161. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 162. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 163. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 164. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 165. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 166. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 167. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 168. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 169.

[0090] In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 76. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 77. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 78. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 79. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 80. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 81. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 82. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 83. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 84. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 85. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 86. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 87. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 88. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 89. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 90. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 91. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 92. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 93. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 94. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 95. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 96. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 97.In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 98. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 99. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 100. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 101. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 144. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 145. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 146. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 147. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 148. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 149. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 150. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 151. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 152. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 153. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 154. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 155. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 156. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 157. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 158. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 159. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 160.In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 161. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 162. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 163. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 164. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 165. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 166. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 167. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 168. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 169. In certain embodiments, X in any one of SEQ ID NOs: 76-101 or 144-169 is glutamine. In certain embodiments, X of any one of SEQ ID NOs: 76-101 or 144-169 is pyroglutamate.

[0091] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115. In certain embodiments, the amino acid sequence of the light chain consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115. In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus monkey CD96), comprising a heavy chain and a light chain, wherein the heavy chain and light chain are set forth in SEQ ID NOs: 76 and 102; 79 and 103; 78 and 103; 82 and 103; 84 and 104; 83 and 104; 86 and 103; 85 and 103; 81 and 103; 80 and 103; 87 and 105; 77 and 102;88 and 102;77 and 106;77 and 107;77 and 108;77 and 103;89 and 102;90 and 102;91 and 102;92 and 102;93 and 102;77 and 109;94 and 102;95 and 102;96 and 102;97 and 102;98 and 102;99 and 102;100 and 110;100 and 111;100 and 112;100 and 113;100 and 114;100 and 115;101 and 110;144 and 102;147 and 103;146 and 103;150 and 103;152 and 104;151 and 104;154 and 103;153 and 103;149 and 103;148 and 103;155 and 105;145 and 102;156 and 102;145 and 106;145 and 107;145 and 108;145 and 103;157 and 102; 158 and 102; 159 and 102; 160 and 102; 161 and 102; 145 and 109; 162 and 102; 163 and 102; 164 and 102; 165 and 102; 166 and 102; 167 and 102; 168 and 110; 168 and 111; 168 and 112; 168 and 113; 168 and 114; 168 and 115; or 169 and 110.In certain embodiments, the amino acid sequences of the heavy and light chains are set forth in SEQ ID NOs: 76 and 102; 79 and 103; 78 and 103; 82 and 103; 84 and 104; 83 and 104; 86 and 103; 85 and 103; 81 and 103; 80 and 103; 87 and 105; 77 and 102; 88 and 102; 77 and 106; 77 and 107; 77 and 108; 77 and 109; and 103;89 and 102;90 and 102;91 and 102;92 and 102;93 and 102;77 and 109;94 and 102;95 and 102;96 and 102;97 and 102;98 and 102;99 and 102;100 and 110;100 and 111;100 and 112;100 and 113;100 and 114;100 and 115;101 and 110;144 and 102;147 and 103;146 and 103;150 and 103;152 and 104;151 and 104;154 and 103;153 and 103;149 and 103;148 and 103;155 and 105;145 and 102;156 and 102;145 and 106;145 and 107;145 and 108;145 and 103;157 and 102;158 and 10 2; 159 and 102; 160 and 102; 161 and 102; 145 and 109; 162 and 102; 163 and 102; 164 and 102; 165 and 102; 166 and 102; 167 and 102; 168 and 110; 168 and 111; 168 and 112; 168 and 113; 168 and 114; 168 and 115; or 169 and 110. In certain embodiments, X in any one of SEQ ID NOs: 76-101 or 144-169 is glutamine. In certain embodiments, X in any one of SEQ ID NOs: 76-101 or 144-169 is pyroglutamate.

[0092] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein 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: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 79 and a light chain comprising the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 78 and a light chain comprising the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 82 and a light chain comprising the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 104. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 83 and a light chain comprising the amino acid sequence of SEQ ID NO: 104. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 85 and a light chain comprising the amino acid sequence of SEQ ID NO: 103.In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 81 and a light chain comprising the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence of SEQ ID NO: 105. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 77 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 77 and a light chain comprising the amino acid sequence of SEQ ID NO: 106. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 77 and a light chain comprising the amino acid sequence of SEQ ID NO: 107. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 77 and a light chain comprising the amino acid sequence of SEQ ID NO: 108.In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 77 and a light chain comprising the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 89 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 90 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein 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: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 77 and a light chain comprising the amino acid sequence of SEQ ID NO: 109. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein 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: 102.In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 95 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 96 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein 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: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 98 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 99 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 110. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 111. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 112.In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 113. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 114. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 115. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 110. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 144 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 147 and a light chain comprising the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 146 and a light chain comprising the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 150 and a light chain comprising the amino acid sequence of SEQ ID NO: 103.In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 152, and a light chain comprising the amino acid sequence of SEQ ID NO: 104. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 151, and a light chain comprising the amino acid sequence of SEQ ID NO: 104. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 154, and a light chain comprising the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 153 and a light chain comprising the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 149, and a light chain comprising the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 148, and a light chain comprising the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 155, and a light chain comprising the amino acid sequence of SEQ ID NO: 105. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 145 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 156 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 145 and a light chain comprising the amino acid sequence of SEQ ID NO: 106. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 145 and a light chain comprising the amino acid sequence of SEQ ID NO: 107. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 145 and a light chain comprising the amino acid sequence of SEQ ID NO: 108.In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 145 and a light chain comprising the amino acid sequence of SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 157 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 158 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 159 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 160 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 161 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 145 and a light chain comprising the amino acid sequence of SEQ ID NO: 109. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 162 and a light chain comprising the amino acid sequence of SEQ ID NO: 102.In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 163, and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 164, and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 165, and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 166, and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 167, and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 168, and a light chain comprising the amino acid sequence of SEQ ID NO: 110. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain comprising the amino acid sequence of SEQ ID NO: 111. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain comprising the amino acid sequence of SEQ ID NO: 112.In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain comprising the amino acid sequence of SEQ ID NO: 113. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain comprising the amino acid sequence of SEQ ID NO: 114. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain comprising the amino acid sequence of SEQ ID NO: 115. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 169 and a light chain comprising the amino acid sequence of SEQ ID NO: 110.

[0093] In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 76 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 79 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 78 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 82 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 84 and 104, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 83 and 104, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 86 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 85 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 81 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 80 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 87 and 105, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 77 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 88 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 77 and 106, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 77 and 107, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 77 and 108, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 77 and 103, respectively.In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 89 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 90 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 91 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 92 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 93 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 77 and 109, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 94 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 95 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 96 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 97 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 98 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 99 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 100 and 110, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 100 and 111, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 100 and 112, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 100 and 113, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 100 and 114, respectively.In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 100 and 115, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 101 and 110, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 144 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 147 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 146 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 150 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 152 and 104, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 151 and 104, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 154 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 153 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 149 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 148 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 155 and 105, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 145 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 156 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 145 and 106, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 145 and 107, respectively.In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 145 and 108, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 145 and 103, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 157 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 158 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 159 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 160 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 161 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 145 and 109, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 162 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 163 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 164 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 165 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 166 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 167 and 102, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 168 and 110, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 168 and 111, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 168 and 112, respectively.In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 168 and 113, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 168 and 114, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 168 and 115, respectively. In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences of SEQ ID NOs: 169 and 110, respectively.

[0094] Any antibody format can be used for the antibodies disclosed herein. In certain embodiments, the antibody is a single-chain antibody or a single-chain Fv (scFv). In certain embodiments, the antibody is an scFv fused with an Fc region (scFv-Fc). In certain embodiments, the antibody is a Fab fragment. In certain embodiments, the antibody is a F(ab')2 fragment.

[0095] In certain embodiments, the antibodies disclosed herein are multispecific antibodies (e.g., bispecific antibodies) that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen.

[0096] In certain embodiments, the antibodies disclosed herein are conjugated to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label. In certain embodiments, a cytotoxic agent is capable of inducing death or destruction of cells in contact therewith. In certain embodiments, a cytostatic agent is capable of preventing or substantially reducing the proliferation and / or inhibiting the activity or function of cells in contact therewith. In certain embodiments, the cytotoxic agent or cytostatic agent is a chemotherapeutic agent. In certain embodiments, the radionuclide is an isotope 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58Co, 59 Fe, 67 Cu, 90 Y, 99 Tc, 111 In, 117 Lu, 121 I, 124 I, 125 I, 131 I, 198 Au, 211 At, 213 Bi, 225 Ac and 186 Re. In certain embodiments, the detectable label comprises a fluorescent moiety or a click chemistry handle.

[0097] Any immunoglobulin (Ig) constant region can be used in the antibodies disclosed herein. In certain embodiments, the Ig region is a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b).

[0098] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 121 or 175. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 122 or 123.

[0099] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1)) and / or hinge region of an antibody described herein, numbered according to the EU numbering system, to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0100] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), e.g., as described in U.S. Patent No. 5,677,425, the entire contents of which are incorporated herein by reference. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered to, for example, facilitate assembly of the light and heavy chains or to alter (e.g., increase or decrease) the stability of the antibody.

[0101] In certain embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant domain, or an FcRn-binding fragment thereof (preferably, an Fc or hinge-Fc domain fragment), to alter (e.g., decrease or increase) the half-life of the antibody in vivo. For example, for examples of mutations that alter (e.g., decrease or increase) the half-life of an antibody in vivo, see International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, all of which are incorporated by reference in their entirety. In certain embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant domain, or an FcRn-binding fragment thereof (preferably, an Fc or hinge-Fc domain fragment), to decrease the half-life of the antibody in vivo. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant domain, or an FcRn-binding fragment thereof (preferably, an Fc or hinge-Fc domain fragment), to increase the half-life of the antibody in vivo. In certain embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1), numbered according to the EU numbering system. In certain embodiments, the IgG1 constant region of an antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU numbering system. See U.S. Patent No. 7,658,921, which is incorporated herein by reference in its entirety.Mutant IgGs of this type, termed "YTE mutants," have been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24, incorporated herein by reference in its entirety). In certain embodiments, the antibody comprises an IgG constant domain comprising one, two, three, or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU numbering system.

[0102] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1)) and / or hinge region of an antibody described herein, numbered according to the EU numbering system, to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that decrease or increase the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptors of antibodies that can be made to alter the affinity of the antibody for the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, all of which are incorporated herein by reference in their entireties.

[0103] In certain embodiments, the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, and the variant heavy chain constant region binds to FcγRIIB with a higher affinity than the wild-type heavy chain constant region binds to FcγRIIB. In certain embodiments, the variant heavy chain constant region is a variant human heavy chain constant region, such as a variant human IgG1, variant human IgG2, or variant human IgG4 heavy chain constant region. In certain embodiments, the variant human IgG heavy chain constant region comprises one or more of the following amino acid mutations according to the EU numbering system: G236D, P238D, S239D, S267E, L328F, and L328E. In certain embodiments, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of: S267E and L328F; P238D and L328E; P238D and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G, and A330R; P238D, E233D, G237D, H268D, P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, and L328F; and V264E, S267E, and L328F, according to the EU numbering system. In certain embodiments, FcγRIIB is expressed on cells selected from the group consisting of macrophages, monocytes, B cells, dendritic cells, endothelial cells, and activated T cells.

[0104] In further embodiments, one, two, or more amino acid substitutions are introduced into the IgG constant domain Fc region to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396, numbered according to the EU numbering system, can be replaced with different amino acid residues so that the antibody has altered affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, each of which is incorporated herein by reference in its entirety. In certain embodiments, deletion or inactivation (by point mutation or other means) of the constant region domain may reduce Fc receptor binding of circulating antibodies, thereby increasing tumor localization. See, e.g., U.S. Patent Nos. 5,585,097 and 8,591,886 for a description of mutations that delete or inactivate constant domains, thereby increasing tumor localization. In certain embodiments, one or more amino acid substitutions can be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on the Fc region, which may reduce Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604, incorporated herein by reference in its entirety).In various embodiments, one or more of the following mutations in the constant regions of the antibodies described herein, numbered according to the EU numbering system, may be made: an N297A substitution, an N297Q substitution, an L234A substitution, an L234F substitution, an L235A substitution, an L235F substitution, an L235V substitution, an L237A substitution, an S239D substitution, an E233P substitution, an L234V substitution, an L235A substitution, a deletion of C236, a P238A substitution, an S239D substitution, an F243L substitution, a D265A substitution, an S267E substitution, an L328F substitution, an R292P substitution, a Y300L substitution, an A327Q substitution, a P329A substitution, an A332L substitution, an I332E substitution, or a P396L substitution.

[0105] In certain embodiments, a mutation selected from the group consisting of D265A, P329A, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of the antibody described herein.In certain embodiments, a mutation selected from the group consisting of L235A, L237A, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of the antibody described herein.In certain embodiments, a mutation selected from the group consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of the antibody described herein.In certain embodiments, a mutation selected from the group consisting of S239D, I332E, optionally A330L, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of the antibody described herein. In certain embodiments, a mutation selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein. In certain embodiments, a mutation selected from the group consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein.

[0106] In certain embodiments, the antibodies described herein comprise an IgG1 constant domain with an N297Q or N297A amino acid substitution, numbered according to the EU numbering system. In one embodiment, the antibodies described herein comprise an IgG1 constant domain with a mutation selected from the group consisting of D265A, P329A, and combinations thereof, numbered according to the EU numbering system. In another embodiment, the antibodies described herein comprise an IgG1 constant domain with a mutation selected from the group consisting of L234A, L235A, and combinations thereof, numbered according to the EU numbering system. In another embodiment, the antibodies described herein comprise an IgG1 constant domain with a mutation selected from the group consisting of L234F, L235F, N297A, and combinations thereof, numbered according to the EU numbering system. In certain embodiments, the amino acid residues in the constant regions of the antibodies described herein at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, numbered according to the EU numbering system, are not L, L, and D, respectively. This approach is described in detail in International Publication No. WO 14 / 108483, which is incorporated herein by reference in its entirety. In certain embodiments, the amino acids corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, numbered according to the EU numbering system, are F, E, and A, or A, A, and A, respectively.

[0107] In certain embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322, numbered according to the EU numbering system, in the constant region of an antibody described herein can be replaced with a different amino acid residue, such that the antibody has altered C1q binding and / or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 (Idusogie et al.), incorporated herein by reference in its entirety. In certain embodiments, one or more amino acid residues within amino acid positions 231-238 in the N-terminal region of the CH2 domain of an antibody described herein are altered, thereby altering the antibody's ability to fix complement. This approach is further described in International Publication No. WO 94 / 29351, incorporated herein by reference in its entirety. In certain embodiments, the Fc region of an antibody described herein has been modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to contain a nucleotide sequence at any of the following positions, numbered according to the EU numbering system: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 352, 354, 355, 356, 358, 365, 367, 368, 369, 270, The affinity of the antibody for the Fc receptor is increased by mutating (e.g., introducing amino acid substitutions) one or more amino acids at positions 01, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This approach is further described in International Publication No. WO 00 / 42072, which is incorporated herein by reference in its entirety.

[0108] In certain embodiments, the antibodies described herein comprise a modified IgG1 constant domain, where the modification increases the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, 0.1, 1, or 10 μg / mL of the antibody can induce cell death of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of CD96-expressing cells within 1, 2, or 3 hours, as assessed by methods described herein and / or known to those skilled in the art. In certain embodiments, the modified IgG1 constant domain comprises S239D and I332E substitutions, numbered according to the EU numbering system. In certain embodiments, the modified IgG1 constant domain comprises S239D, A330L, and I332E substitutions, numbered according to the EU numbering system. In certain embodiments, the modified constant domain of IgG1 comprises L235V, F243L, R292P, Y300L, and P396L substitutions, numbered according to the EU numbering system. In certain embodiments, the antibody can induce cell death in effector T cells and Tregs, and the percentage of Tregs undergoing cell death is at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold higher than the percentage of effector T cells undergoing cell death.

[0109] In certain embodiments, the antibodies described herein comprise the constant region of an IgG4 antibody, wherein the serine at amino acid residue 228 of the heavy chain, numbered according to the EU numbering system, is substituted with a proline. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:26.

[0110] In certain embodiments, any of the constant region mutations or modifications described herein may be introduced into one or both of the heavy chain constant regions of an antibody described herein that has two heavy chain constant regions.

[0111] In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and function as antagonists (e.g., decrease or increase CD96 activity).

[0112] In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and reduce or inhibit CD96 (e.g., human CD96 or cynomolgus CD96) activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% when assessed relative to CD96 (e.g., human CD96 or cynomolgus CD96) activity, including any antibody not included or an unrelated antibody (e.g., an antibody that does not specifically bind CD96 (e.g., human CD96 or cynomolgus CD96)). In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and reduce or inhibit CD96 (e.g., human CD96 or cynomolgus CD96) activity by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more when assessed by methods described herein and / or methods known to those of skill in the art relative to CD96 (e.g., human CD96 or cynomolgus CD96) activity, including any antibody not included or an unrelated antibody (e.g., an antibody that does not specifically bind CD96 (e.g., human CD96)). Non-limiting examples of CD96 (e.g., human CD96 or cynomolgus CD96) activities can include CD96 (e.g., human CD96 or cynomolgus CD96) signaling, CD96 (e.g., human CD96 or cynomolgus CD96) binding to its ligand (e.g., CD155) or a fragment and / or fusion protein thereof, activation of T cells (e.g., T cells expressing human CD96), activation of natural killer (NK) cells, reduction or inhibition of Tregs, increased cytokine (e.g., IL-2) production, and increased activity of CD155 (e.g., human CD155).In certain embodiments, increased CD96 (eg, human CD96 or cynomolgus CD96) activity is assessed as described in the Examples.

[0113] In certain embodiments, the present disclosure provides isolated antibodies that specifically bind CD96 (e.g., human CD96 or cynomolgus CD96) and reduce or inhibit CD96 (e.g., human or cynomolgus CD96) binding to its ligand (e.g., CD155) or a fragment and / or fusion protein thereof by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% when assessed by methods described herein or known to those of skill in the art for CD96 (e.g., human CD96 or cynomolgus CD96) binding to its ligand, including any antibody free of or unrelated antibody (e.g., an antibody that does not specifically bind CD96 (e.g., human or cynomolgus CD96)). In certain embodiments, the present disclosure provides antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and its ligand (e.g., CD155 (e.g., human or cynomolgus CD96)) as assessed by methods described herein or known to one of skill in the art for CD96 (e.g., human CD96) binding to its ligand, including any antibody or unrelated antibody (e.g., an antibody that does not specifically bind to CD96 (e.g., human or cynomolgus CD96)).

[0023] Provided are isolated antibodies that reduce or inhibit CD96 (e.g., human or cynomolgus CD96) binding to CD96 (e.g., human or cynomolgus CD155) or fragments and / or fusion proteins thereof) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

[0114] In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and activate T cells (e.g., T cells expressing human CD96). In certain embodiments, the T cells are memory T cells. In certain embodiments, the T cells are primary CD3-expressing T cells. In certain embodiments, the T cells are CD96-expressing Jurkat cells. In certain embodiments, the antibodies disclosed herein increase nuclear factor of activated T cells (NFAT) activity by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% when assessed by a method described herein or a method known to one of skill in the art, without any antibody or an unrelated antibody (e.g., an antibody that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)). In certain embodiments, the antibodies disclosed herein increase NFAT activity by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more relative to NFAT activity without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)), as assessed by methods described herein or known to those of skill in the art. In certain embodiments, the antibodies increase NFAT activity in the presence of a ligand of CD96 (e.g., CD155) or a fragment and / or fusion protein thereof, and / or cells (e.g., monocytes or dendritic cells) expressing a ligand of CD96.

[0115] In certain embodiments, the present disclosure provides isolated antibodies that specifically bind CD96 (e.g., human CD96 or cynomolgus CD96) and increase cytokine production (e.g., IL-2) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% when assessed by methods described herein or known to those of skill in the art relative to cytokine production without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind CD96 (e.g., human CD96 or cynomolgus CD96)). In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and increase cytokine production (e.g., IL-2) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more when assessed by a method described herein or a method known to one of skill in the art relative to cytokine production without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind CD96 (e.g., human CD96 or cynomolgus CD96)). In certain embodiments, the antibody increases cytokine production (e.g., IL-2) in the presence of a ligand of CD96 (e.g., CD155) or a fragment and / or fusion protein thereof, and / or cells (e.g., monocytes or dendritic cells) expressing a ligand of CD96. In certain embodiments, the antibody increases IL-2 production relative to IL-2 production without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)).

[0116] In certain embodiments, the present disclosure provides a method for the production of IFNγ and / or IL-2 comprising administering to a subject a therapeutic target for IFNγ and / or IL-2, as assessed by a method described herein or a method known to one of skill in the art, that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and that is administered alone or in combination with an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), without any antibody or an unrelated antibody (e.g., an antibody that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)). and / or IL-2 production in human peripheral blood mononuclear cells (PBMCs) in response to Staphylococcus enterotoxin A (SEA) stimulation by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, either in combination with a phosphodiesterase inhibitor (mAb) or a phosphodiesterase inhibitor (mAb).

[0117] In certain embodiments, human peripheral blood mononuclear cells (PBMCs) stimulated with Staphylococcus enterotoxin A (SEA) in the presence of an antibody described herein that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) do not bind to any antibody or to an unrelated antibody (e.g., CD96 (e.g., human CD96 or cynomolgus monkey CD96) when assessed by a method described herein or a method known to one of skill in the art). The antibody (antibody that does not specifically bind to monkey CD96) increased IFNγ and / or IL-2 production by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold relative to IFNγ and / or IL-2 production from PBMCs stimulated with SEA alone.

[0118] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and increases or promotes memory recall of memory T cells. In certain embodiments, the memory T cells are CD8 effector memory T cells. In certain embodiments, the memory T cells are CD4 effector memory T cells. In certain embodiments, the antibody increases the number of memory T cells that proliferate when memory T cells are contacted with their cognate antigen(s) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, as assessed by methods described herein or known to those of skill in the art, relative to the number of memory T cells that proliferate when memory T cells are contacted with their cognate antigen(s) in the absence of any antibody or in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)). In certain embodiments, the antibody increases cytokine (e.g., IFNγ, TNFα) production by memory T cells upon contact with their cognate antigen by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, as assessed by methods described herein or known to those of skill in the art relative to cytokine production by memory T cells upon contact with their cognate antigen in the absence of any antibody or in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)).

[0119] In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and activate NK cells. In certain embodiments, the NK cells are isolated. In certain embodiments, the NK cells are in a mixed culture of PBMCs. In certain embodiments, the antibodies disclosed herein increase the expression level of CD107a on NK cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by the activity methods described herein or methods known to those of skill in the art, relative to the expression level of CD107a on NK cells, including any antibody or an unrelated antibody (e.g., an antibody that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)). In certain embodiments, the antibodies disclosed herein increase the expression level of CD107 on NK cells by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more, as assessed by methods described herein or known to one of skill in the art, relative to the expression level of CD107 on NK cells without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)).In certain embodiments, the antibodies disclosed herein increase cytokine production (e.g., IFNγ and / or TNFα) from NK cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods described herein or known to one of skill in the art relative to cytokine production (e.g., IFNγ and / or TNFα) from NK cells not including any antibody or an unrelated antibody (e.g., an antibody that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)). In certain embodiments, the antibodies disclosed herein increase cytokine production (e.g., IFNγ and / or TNFα) from NK cells by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, as assessed by methods described herein or known to one of skill in the art, relative to cytokine production (e.g., IFNγ and / or TNFα) from NK cells not containing any antibody or containing an unrelated antibody (e.g., an antibody that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)).

[0120] Pharmaceutical Composition Compositions comprising the antibodies described herein, having the desired degree of purity, in a physiologically acceptable carrier, excipient, or stabilizer are provided herein (see, e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0121] In certain embodiments, a pharmaceutical composition comprises an anti-CD96 antibody described herein (e.g., human CD96 or cynomolgus CD96) in a pharmaceutically acceptable carrier, and optionally one or more additional prophylactic or therapeutic agents. In certain embodiments, a pharmaceutical composition comprises an effective amount of an antibody described herein in a pharmaceutically acceptable carrier, and optionally one or more additional prophylactic or therapeutic agents. In certain embodiments, the antibody is the only active ingredient contained in the pharmaceutical composition. The pharmaceutical compositions described herein may be useful for increasing or promoting CD96 (e.g., human CD96 or cynomolgus CD96) activity and treating conditions such as cancer or infectious diseases. In one embodiment, the present invention relates to a pharmaceutical composition of the present invention comprising an anti-CD96 antibody of the present invention for use as a medicament. In another embodiment, the present invention relates to a pharmaceutical composition of the present invention for use in a method for treating cancer or infectious diseases.

[0122] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonicity agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at bacteriostatic or fungistatic concentrations may be added to parenteral preparations packaged in multi-dose containers, including phenol or cresol, mercuric, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonicity agents include sodium chloride and dextrose. Buffering agents include phosphate and citrate. Antioxidants include sodium disulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN (登録商標) 80). Sequestering or chelating agents include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0123] Pharmaceutical compositions may be formulated for any route of administration to a subject. Specific examples of administration routes include intranasal, oral, pulmonary, transdermal, intradermal, and parenteral. Parenteral administration, characterized by subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, as solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. Furthermore, if desired, the administered pharmaceutical composition may also contain small amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0124] Preparations for parenteral administration of antibodies include sterile solutions ready for injection, sterile dry soluble products such as lyophilized powders ready to be mixed with a solvent immediately before use, including tablets for hypodermic injection, sterile suspensions ready for injection, sterile dry insoluble products ready to be mixed with a vehicle immediately before use, and sterile emulsions. Solutions may be either aqueous or non-aqueous.

[0125] If administered intravenously, suitable carriers include saline or phosphate buffered saline (PBS), solutions containing thickening agents and solubilizing agents such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0126] Topical mixtures containing antibodies are prepared as described for local and systemic administration. The resulting mixtures may be solutions, suspensions, emulsions, etc., and may be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigants, sprays, suppositories, bandages, skin patches, or any other formulation suitable for topical administration.

[0127] The anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies disclosed herein can be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for steroid delivery useful in the treatment of inflammatory diseases, particularly asthma, and are incorporated herein by reference in their entireties). These formulations for administration to the respiratory tract may be in the form of an aerosol or solution for a nebulizer, or as a fine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation, in one embodiment, have a diameter of less than 50 microns, in one embodiment, less than 10 microns.

[0128] The anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies disclosed herein can be formulated for local or topical application, such as topical application to the skin and mucous membranes, such as the eyes, and for ocular, intracisternal, or intraspinal application, in the form of gels, creams, and lotions. Topical administration is also contemplated for transdermal delivery, as well as administration to the eyes or mucous membranes, or inhalation therapy. Nasal solutions of the antibodies can also be administered alone or in combination with other pharmaceutically acceptable excipients.

[0129] Transdermal patches, including iontophoresis and electrophoresis devices, are well known to those skilled in the art and can be used to administer antibodies. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of which are incorporated herein by reference in their entireties.

[0130] In certain embodiments, pharmaceutical compositions containing antibodies described herein are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. They may also be reconstituted and formulated as solids or gels. Lyophilized powders are prepared by dissolving the antibodies described herein, or pharmaceutically acceptable derivatives thereof, in a suitable solvent. In certain embodiments, the lyophilized powder is sterile. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, such as citrate, sodium, or potassium phosphate, in one embodiment, at approximately neutral pH, or other such buffers known to those of skill in the art. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those of skill in the art, provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial contains a single or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature. Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound selected. Such amounts can be determined empirically.

[0131] The anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies disclosed herein, and other compositions provided herein, can also be formulated to target specific tissues, receptors, or other areas of the body of the subject being treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in the present compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874, all of which are incorporated herein by reference in their entireties. In certain embodiments, the antibodies described herein target tumors.

[0132] Compositions to be used for in vivo administration can be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0133] How to use In another aspect, the present disclosure provides methods of treating a subject using the anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies disclosed herein. Any disease or disorder in a subject that would benefit from reduced CD96 (e.g., human CD96 or cynomolgus CD96) function can be treated using the anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies disclosed herein. In certain embodiments, the disease or disorder is resistant to a checkpoint targeting agent (e.g., an antagonistic anti-CTLA-4 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, or an antagonistic anti-PD-1 antibody). In certain embodiments, the disease or disorder recurs after treatment with a checkpoint targeting agent (e.g., an antagonistic anti-CTLA-4 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, or an antagonistic anti-PD-1 antibody).

[0134] The anti-CD96 (e.g., human CD96) antibodies disclosed herein are particularly useful for inhibiting the immune system's tolerance to tumors and, therefore, can be used as immunotherapy for subjects with cancer. For example, in certain embodiments, the present disclosure provides antibodies to T cells (e.g., CD8 + Cytotoxic T cells, CD4 +

[0013] In certain embodiments, the present disclosure provides a method for increasing T cell (helper T cell, NKT cell, effector T cell, or memory T cell) activation, the method comprising administering to a subject an effective amount of an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody or pharmaceutical composition thereof disclosed herein. In certain embodiments, the present disclosure provides a method for treating cancer in a subject, the method comprising administering to a subject an effective amount of an antibody or pharmaceutical composition as disclosed herein.

[0135] Cancers that may be treated with the anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies or pharmaceutical compositions disclosed herein include, but are not limited to, solid tumors, hematological cancers (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and metastatic lesions. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignant tumors, e.g., sarcomas and carcinomas, e.g., adenocarcinomas of various organ systems, such as those affecting the lung, breast, ovary, lymphatic, gastrointestinal tract (e.g., colon), anus, genital and genitourinary tract (e.g., kidney, urothelium, bladder cells, prostate), pharynx, CNS (e.g., brain, neural or glial cells), head and neck, skin (e.g., melanoma), and pancreas, as well as adenocarcinomas, including malignant tumors such as colon cancer, kidney cancer, renal cell carcinoma, liver cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), small intestine cancer, and esophageal cancer. The cancer may be early-stage, mid-stage, late-stage, or metastatic. In certain embodiments, the cancer is resistant to a checkpoint targeting agent (e.g., an antagonistic anti-CTLA-4 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, or an antagonistic anti-PD-1 antibody). In certain embodiments, the cancer recurs after treatment with a checkpoint targeting agent (e.g., an antagonistic anti-CTLA-4 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, or an antagonistic anti-PD-1 antibody).

[0136] In one embodiment, the cancer is lung cancer (e.g., lung adenocarcinoma or non-small cell lung cancer (NSCLC) (e.g., NSCLC with squamous and / or non-squamous histology, or NSCLC adenocarcinoma)), melanoma (e.g., advanced melanoma), kidney cancer (e.g., renal cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), myeloma (e.g., multiple myeloma), prostate cancer, breast cancer (e.g., estrogen receptor, progesterone receptor, or Her2 / neu cysts), or In certain embodiments, the cancer is selected from breast cancer that does not express one, two, or all of these genes (e.g., triple-negative breast cancer), ovarian cancer, colorectal cancer, pancreatic cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), anal cancer, gastroesophageal cancer (e.g., esophageal squamous cell carcinoma), mesothelioma, nasopharyngeal cancer, thyroid cancer, cervical cancer, epithelial carcinoma, peritoneal cancer, or lymphoproliferative disease (e.g., post-transplant lymphoproliferative disease). In certain embodiments, the cancer is cervical cancer.

[0137] In one embodiment, the cancer is a blood cancer, such as leukemia, lymphoma, or myeloma. In one embodiment, the cancer is a leukemia, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myeloblastic leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelogenous leukemia (CMML), chronic myelogenous leukemia (CMML), chronic lymphocytic leukemia (CLL), or hairy cell leukemia. In one embodiment, the cancer is a lymphoma, e.g., B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell-like (ABC) diffuse large B-cell lymphoma, germinal center B-cell (GCB) diffuse large B-cell lymphoma, mantle cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, relapsed non-Hodgkin's lymphoma, refractory non-Hodgkin's lymphoma, relapsed follicular non-Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, or extranodal marginal zone lymphoma. In one embodiment, the cancer is a myeloma, e.g., multiple myeloma.

[0138] In another embodiment, the cancer is selected from carcinoma (eg, advanced or metastatic cancer), melanoma, or lung cancer, eg, non-small cell lung cancer.

[0139] In one embodiment, the cancer is lung cancer, for example, lung adenocarcinoma, non-small cell lung cancer, or small cell lung cancer.

[0140] In one embodiment, the cancer is melanoma, e.g., advanced melanoma. In one embodiment, the cancer is advanced or unresectable melanoma that is unresponsive to other therapies. In another embodiment, the cancer is melanoma with a BRAF mutation (e.g., a BRAF V600 mutation). In yet another embodiment, the anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody or pharmaceutical composition disclosed herein is administered after treatment with an anti-CTLA-4 antibody (e.g., ipilimumab) with or without a BRAF inhibitor (e.g., vemurafenib or dabrafenib).

[0141] In another embodiment, the cancer is hepatocellular carcinoma, e.g., advanced hepatocellular carcinoma, with or without associated viral infection, e.g., chronic viral hepatitis.

[0142] In another embodiment, the cancer is prostate cancer, e.g., advanced prostate cancer.

[0143] In one embodiment, the cancer is a myeloma, for example, multiple myeloma.

[0144] In yet another embodiment, the cancer is a kidney cancer, for example, a renal cell carcinoma (RCC) (e.g., metastatic RCC, clear cell renal cell carcinoma (CCRCC), or papillary renal cell carcinoma).

[0145] In yet another embodiment, the cancer is selected from lung cancer, melanoma, kidney cancer, breast cancer, colon cancer, leukemia, or a metastatic lesion of cancer.

[0146] In certain embodiments, the present disclosure provides a method for preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody or pharmaceutical composition thereof as disclosed herein. In one embodiment, a method for preventing and / or treating an infection (e.g., a viral infection, a bacterial infection, a fungal infection, a protozoan infection, or a parasitic infection) is provided herein. The infection prevented and / or treated according to the method may be caused by an infectious agent identified herein. In certain embodiments, an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody or composition thereof described herein is the only active agent administered to the subject. In certain embodiments, an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody or composition thereof described herein is used in combination with an anti-infective intervention (e.g., an antiviral, antibacterial, antifungal, or antihermetic) for the treatment of an infectious disease. Thus, in one embodiment, the invention relates to an antibody and / or pharmaceutical composition of the invention for use in a method for preventing and / or treating an infectious disease, optionally wherein the antibody or pharmaceutical composition is the only active agent administered to a subject, or wherein the antibody or pharmaceutical composition is used in combination with an anti-infective intervention.

[0147] Infectious diseases that can be treated and / or prevented by the anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies or pharmaceutical compositions disclosed herein are caused by infectious agents including, but not limited to, bacteria, parasites, fungi, protozoa, and viruses. In certain embodiments, infectious diseases treated and / or prevented by the anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies or pharmaceutical compositions disclosed herein are caused by viruses. Viral diseases or infections that may be prevented and / or treated in accordance with 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 type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, papovavirus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackievirus, mumps virus, measles virus, rubella virus, poliovirus, smallpox, Epstein-Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), and agents of viral diseases such as viral meningitis, encephalitis, dengue, or smallpox.

[0148] Bacterial infections that may be prevented and / or treated include infections caused by Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris, Staphylococcus viridans, and Pseudomonas aeruginosa. Bacterial diseases caused by bacteria (e.g., Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris, Staphylococcus viridans, and Pseudomonas aeruginosa) that can be prevented and / or treated in accordance with the methods described herein include, but are not limited to, Mycobacteria rickettsia, Mycoplasma, Neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease), Bacillus antracis (anthrax), tetanus, Streptococcus, Staphylococcus, mycobacterium, whooping cough, cholera, plague, diphtheria, chlamydia, S. aureus, and Legionnaires' disease.

[0149] Protozoal diseases or 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 schistosoma, or malaria. Parasitic diseases or infections caused by parasites that can be prevented and / or treated according to the methods described herein include, but are not limited to, chlamydia and rickettsia.

[0150] Fungal diseases or infections that may be prevented and / or treated according to the methods described herein include, but are not limited to, Candida infection, zygomycosis, Candida mastitis, progressive disseminated trichosporonosis with occult trichosporonemia, disseminated candidiasis, pulmonary paracoccidioidomycosis, pulmonary aspergillosis, Pneumocystis carinii pneumonia, cryptococcal meningitis, coccidioidal meningoencephalitis and cerebral vasculitis, Aspergillus niger infection, Fusarium keratitis, sinus mycosis, Aspergillus fumigatus endocarditis, tibial chondrodysplasia, Candida glabrata vaginitis, oral candidiasis, X-linked chronic granulomatous disease, tinea pedis, cutaneous candidiasis, fungal placentitis, disseminated trichosporonosis, allergic bronchopulmonary aspergillosis, fungal keratitis, Cryptococcus These include those caused by C. neoformans infection, fungal peritonitis, Curvularia geniculata infection, staphylococcal endophthalmitis, sporotrichosis, and dermatophytosis.

[0151] In certain embodiments, the method further comprises administering an additional therapeutic agent to the subject. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, or a checkpoint targeting agent. In certain embodiments, the chemotherapeutic agent is a hypomethylating agent (e.g., azacytidine). In certain embodiments, the chemotherapeutic agent is a DNA damage-inducing agent (e.g., gemcitabine). In certain embodiments, the checkpoint targeting agent is selected from the group consisting of an antagonistic anti-CTLA-4 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, an antagonistic anti-PD-1 antibody, an antagonistic anti-TIM-3 antibody, an antagonistic anti-LAG-3 antibody, an antagonistic anti-VISTA antibody, an antagonistic anti-CD96 antibody, an antagonistic anti-CEACAM1 antibody, an agonist anti-CD137 antibody, an agonist anti-GITR antibody, and an agonist anti-OX40 antibody. In certain embodiments, the checkpoint targeting agent is selected from the group consisting of an antagonistic anti-CTLA-4 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, and an antagonistic anti-PD-1 antibody, and the anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody or pharmaceutical composition disclosed herein exhibits synergistic effects with the checkpoint targeting agent.

[0152] In one embodiment, the present invention relates to an antibody and / or pharmaceutical composition of the present invention for use in a method of the present invention, the method further comprising administering an additional therapeutic agent to a subject. In one embodiment, the present invention relates to (a) an antibody and / or pharmaceutical composition of the present invention, and (b) an additional therapeutic agent for use as a medicament. In one embodiment, the present invention relates to (a) an antibody and / or pharmaceutical composition of the present invention, and (b) an additional therapeutic agent for use in a method for treating cancer. In a further embodiment, the present invention relates to a pharmaceutical composition, kit, or kit-of-parts comprising (a) an antibody and / or pharmaceutical composition of the present invention, and (b) an additional therapeutic agent. In one embodiment, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, or a checkpoint targeting agent.

[0153] In certain embodiments, an anti-PD-1 antibody is used in the methods disclosed herein. In certain embodiments, the anti-PD-1 antibody is nivolumab, also known as BMS-936558 or MDX1106, developed by Bristol-Myers Squibb. In certain embodiments, the anti-PD-1 antibody is pembrolizumab, also known as lambrolizumab or MK-3475, developed by Merck & Co. In certain embodiments, the anti-PD-1 antibody is pidilizumab, also known as CT-011, developed by CureTech. In certain embodiments, the anti-PD-1 antibody is MEDI0680, also known as AMP-514, developed by Mediimmune. In certain embodiments, the anti-PD-1 antibody is PDR001, developed by Novartis Pharmaceuticals. In certain embodiments, the anti-PD-1 antibody is REGN2810, developed by Regeneron Pharmaceuticals. In certain embodiments, the anti-PD-1 antibody is PF-06801591 developed by Pfizer. In certain embodiments, the anti-PD-1 antibody is BGB-A317 developed by BeiGene. In certain embodiments, the anti-PD-1 antibody is TSR-042 developed by AnaptysBio and Tesaro. In certain embodiments, the anti-PD-1 antibody is SHR-1210 developed by Hengrui.

[0154] Further non-limiting examples of anti-PD-1 antibodies that may be used in the therapeutic methods disclosed herein are described in the following patents and patent applications: U.S. Patent No. 6,808,710, U.S. Patent No. 7,332,582, U.S. Patent No. 7,488,802, U.S. Patent No. 8,008,449, U.S. Patent No. 8,114,845, U.S. Patent No. 8,168,757, U.S. Patent No. 8,354,509, U.S. Patent No. Nos. 8,686,119, 8,735,553, 8,747,847, 8,779,105, 8,927,697, 8,993,731, 9,102,727, 9,205,148, U.S. Publication No. US2013 / 0202623A1, U.S. Publication No. US2013 / 0291136A1, U.S. Publication No. U S2014 / 0044738A1, U.S. Publication No. US2014 / 0356363A1, U.S. Publication No. US2016 / 0075783A1, and PCT Publication Nos. WO2013 / 033091A1, WO2015 / 036394A1, WO2014 / 179664A2, WO2014 / 209804A1, and WO2014 / 2061 07A1, PCT Publication No. WO2015 / 058573A1, PCT Publication No. WO2015 / 085847A1, PCT Publication No. WO2015 / 200119A1, PCT Publication No. WO2016 / 015685A1, and PCT Publication No. WO2016 / 020856A1, all of which are incorporated herein by reference in their entirety for all purposes.

[0155] In certain embodiments, an anti-PD-L1 antibody is used in the methods disclosed herein. In certain embodiments, the anti-PD-L1 antibody is atezolizumab, developed by Genentech. In certain embodiments, the anti-PD-L1 antibody is durvalumab, developed by AstraZeneca, Celgene, and Mediimmune. In certain embodiments, the anti-PD-L1 antibody is avelumab, also known as MSB0010718C, developed by Merck Serono and Pfizer. In certain embodiments, the anti-PD-L1 antibody is MDX-1105, developed by Bristol-Myers Squibb. In certain embodiments, the anti-PD-L1 antibody is AMP-224, developed by Amplimmune and GSK.

[0156] Non-limiting examples of anti-PD-L1 antibodies that may be used in the methods of treatment disclosed herein are described in the following patents and patent applications: U.S. Patent No. 7,943,743, U.S. Patent No. 8,168,179, U.S. Patent No. 8,217,149, U.S. Patent No. 8,552,154, U.S. Patent No. 8,779,108, U.S. Patent No. 8,981,063, U.S. Patent No. 9,175,082, U.S. Publication No. US2010 / 0203056A1 ... US Publication No. US2003 / 0232323A1, US Publication No. US2013 / 0323249A1, US Publication No. US2014 / 0341917A1, US Publication No. US2014 / 0044738A1, US Publication No. US2015 / 0203580A1, US Publication No. US2015 / 0225483A1, US Publication No. US2015 / 0346208A1, US Publication No. US2015 / 0355184A1, and PCT Publication No. WO and PCT Publication Nos. WO2014 / 100079A1, PCT Publication No. WO2014 / 022758A1, PCT Publication No. WO2014 / 055897A2, PCT Publication No. WO2015 / 061668A1, PCT Publication No. WO2015 / 109124A1, PCT Publication No. WO2015 / 195163A1, PCT Publication No. WO2016 / 000619A1, and PCT Publication No. WO2016 / 030350A1, all of which are incorporated herein by reference in their entirety for all purposes.

[0157] In certain embodiments, an anti-CTLA-4 antibody is used in the methods disclosed herein. In certain embodiments, the anti-CTLA-4 antibody is ipilimumab, developed by Bristol-Myers Squibb.

[0158] In certain embodiments, an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody disclosed herein is administered to a subject in combination with a compound that targets immunomodulatory enzyme(s), such as IDO (indoleamine-(2,3)-dioxygenase) and / or TDO (tryptophan 2,3-dioxygenase). Accordingly, in one embodiment, the additional therapeutic agent is a compound that targets immunomodulatory enzyme(s), such as an inhibitor of indoleamine-(2,3)-dioxygenase (IDO). In certain embodiments, such a compound is selected from the group consisting of epacadostat (Incyte Corp; see, e.g., WO2010 / 005958, incorporated herein by reference in its entirety), F001287 (Flexus Biosciences / Bristol-Myers Squibb), indoximod (NewLink Genetics), and NLG919 (NewLink Genetics). In one embodiment, the compound is epacadostat. In another embodiment, the compound is F001287. In another embodiment, the compound is indoximod. In another embodiment, the compound is NLG919. In certain embodiments, an anti-CD96 (e.g., human CD96) antibody disclosed herein is administered to a subject in combination with an IDO inhibitor for treating cancer. The IDO inhibitor described herein for use in treating cancer is present in a solid dosage form of a pharmaceutical composition, such as a tablet, pill, or capsule, wherein the pharmaceutical composition comprises an IDO inhibitor and a pharmaceutically acceptable excipient. Thus, the antibody described herein and the IDO inhibitor described herein can be administered separately, sequentially, or simultaneously as separate dosage forms. In one embodiment, the antibody is administered parenterally and the IDO inhibitor is administered orally. In certain embodiments, the inhibitor is selected from the group consisting of epacadostat (Incyte Corporation), F001287 (Flexus Biosciences / Bristol-Myers Squibb), indoximod (NewLink Genetics), and NLG919 (NewLink Genetics).Epacadostat is described in PCT Publication No. WO2010 / 005958, which is incorporated herein by reference in its entirety for all purposes. In one embodiment, the inhibitor is epacadostat. In another embodiment, the inhibitor is F001287. In another embodiment, the inhibitor is indoximod. In another embodiment, the inhibitor is NLG919.

[0159] In certain embodiments, an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody disclosed herein is administered to a subject in combination with a vaccine. The vaccine may be, for example, a peptide vaccine, a DNA vaccine, or an RNA vaccine. In certain embodiments, the vaccine is a heat shock protein-based tumor vaccine or a heat shock protein-based pathogen vaccine. In certain embodiments, an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody disclosed herein is administered to a subject in combination with a heat shock protein-based tumor vaccine. Heat shock proteins (HSPs) are a family of highly conserved proteins found uniformly across all species. Their expression can be strongly induced to much higher levels as a result of heat shock or other forms of stress, including exposure to toxins, oxidative stress, or glucose deprivation. Five families have been classified according to molecular weight: HSP-110, -90, -70, -60, and -28. HSPs deliver immunogenic peptides through cross-presentation pathways in antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs), leading to T cell activation. HSPs function as chaperone carriers for tumor-associated antigenic peptides, forming complexes capable of inducing tumor-specific immunity. Upon release from dying tumor cells, HSP-antigen complexes are uptaken by antigen-presenting cells (APCs), where the antigens are processed into peptides that bind to MHC class I and class II molecules, leading to the activation of anti-tumor CD8+ and CD4+ T cells. The immunity elicited by HSP complexes derived from tumor preparations is specifically directed against the unique antigenic peptide repertoire expressed by each subject's cancer. Thus, in one embodiment, the present invention relates to (a) antibodies and / or pharmaceutical compositions of the present invention, and (b) vaccines for use as pharmaceuticals, e.g., in methods for treating cancer. In one embodiment, the present invention relates to pharmaceutical compositions, kits, or kits-of-parts comprising (a) antibodies and / or pharmaceutical compositions of the present invention, and (b) vaccines. In one embodiment, the vaccine is a heat shock protein-based tumor vaccine.In one embodiment, the vaccine is a heat shock protein-based pathogen vaccine. In certain embodiments, the vaccine is as described in WO2016 / 183486, the entire contents of which are incorporated herein by reference.

[0160] Heat shock protein peptide complexes (HSPPCs) are protein-peptide complexes consisting of heat shock proteins non-covalently complexed with antigenic peptides. HSPPCs elicit both innate and adaptive immune responses. In certain embodiments, the antigenic peptide(s) present antigenicity to the cancer being treated. HSPPCs are efficiently sized by APCs via membrane receptors (primarily CD91) or by binding to Toll-like receptors. HSPPC internalization leads to functional maturation of APCs with chemokine and cytokine production, leading to the activation of natural killer cells (NK), monocytes, and Th1- and Th2-mediated immune responses. In certain embodiments, HSPPCs used in the methods disclosed herein comprise one or more heat shock proteins from the hsp60, hsp70, or hsp90 families of stress proteins complexed with antigenic peptides. In certain embodiments, the HSPPC comprises hsc70, hsp70, hsp90, hsp110, grp170, gp96, calreticulin, or a combination of two or more thereof.

[0161] In certain embodiments, the heat shock protein peptide complex (HSPPC) comprises a recombinant heat shock protein (e.g., hsp70 or hsc70) or its peptide-binding domain complexed with a recombinant antigenic peptide. The recombinant heat shock protein can be produced by recombinant DNA technology using the human hsc70 sequence, for example, as described in Dworniczak and Mirault, Nucleic Acids Res. 15:5181-5197 (1987) and GenBank Accession Nos. P11142 and / or Y00371, each of which is incorporated herein by reference in its entirety. In certain embodiments, the Hsp70 sequence is as described in Hunt and Morimoto, Proc. Natl. Acad. Sci. USA 82(19), 6455-6459 (1985) and GenBank Accession Nos. P0DMV8 and / or M11717, each of which is incorporated herein by reference in its entirety. Antigenic peptides can also be prepared by recombinant DNA methods known in the art.

[0162] In certain embodiments, the antigenic peptide comprises a modified amino acid. In certain embodiments, the modified amino acid comprises a post-translational modification. In certain embodiments, the modified amino acid comprises a mimic of a post-translational modification. In certain embodiments, the modified amino acid is Tyr, Ser, Thr, Arg, Lys, or His phosphorylated on the side chain hydroxyl or amine. In certain embodiments, the modified amino acid is a mimic of Tyr, Ser, Thr, Arg, Lys, or His amino acid phosphorylated on the side chain hydroxyl or amine.

[0163] In certain embodiments, an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody disclosed herein is administered to a subject in combination with a heat shock protein peptide complex (HSPPC), e.g., heat shock protein peptide complex 96 (HSPPC-96), to treat cancer. HSPPC-96 comprises a 96 kDa heat shock protein (Hsp), gp96, complexed with an antigenic peptide. HSPPC-96 is a cancer immunotherapy produced from a subject's tumor and contains the antigenic "fingerprint" of the cancer. In certain embodiments, this fingerprint contains unique antigens present only in specific cancer cells of a particular subject, and injection of the vaccine is intended to stimulate the subject's immune system to recognize and attack any cells with the specific cancer fingerprint. Thus, in one embodiment, the invention relates to an antibody and / or pharmaceutical composition of the invention in combination with a heat shock protein peptide complex (HSPPC) for use as a medicament and / or in a method for treating cancer.

[0164] In certain embodiments, the HSPPC, e.g., HSPPC-96, is produced from the subject's tumor tissue. In certain embodiments, the HSPPC (e.g., HSPPC-96) is produced from a tumor of the type of cancer or metastasis thereof being treated. In another specific embodiment, the HSPPC (e.g., HSPPC-96) is autologous to the subject being treated. In certain embodiments, the tumor tissue is non-necrotic tumor tissue. In certain embodiments, at least 1 gram (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 grams) of non-necrotic tumor tissue is used to generate the vaccine regimen. In certain embodiments, after surgical resection, the non-necrotic tumor tissue is frozen prior to use in vaccine preparation. In certain embodiments, the HSPPC, e.g., HSPPC-96, is isolated from the tumor tissue by purification techniques, filtered, and prepared for an injectable vaccine. In certain embodiments, a subject is administered 6-12 doses of HSPPC, e.g., HSPPC-96. In such embodiments, the HSPPC, e.g., HSPPC-96, doses may be administered weekly for the first 4 doses, followed by 2-8 additional doses every other week.

[0165] Further examples of HSPPCs that may be used in accordance with the methods described herein are described in the following patents and patent applications: U.S. Patent Nos. 6,391,306, 6,383,492, 6,403,095, 6,410,026, 6,436,404, 6,447,780, 6,447,781, and 6,610,659, all of which are incorporated herein by reference in their entirety.

[0166] In certain embodiments, an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody disclosed herein is administered to a subject in combination with an adjuvant. Depending on the treatment situation, various adjuvants can be used. Non-limiting examples of suitable adjuvants include, but are not limited to, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), Montanide ISA (incomplete Seppic adjuvant), Ribi adjuvant system (RAS), Titer Max, muramyl peptides, Syntex adjuvant formulation (SAF), alum (aluminum hydroxide and / or aluminum phosphate), aluminum salt adjuvants, Gerbu (登録商標) Adjuvants include nitrocellulose-absorbed antigens, encapsulated or entrapped antigens, 3-de-O-acylated monophosphoryl lipid A (3D-MPL), immunostimulatory oligonucleotides, toll-like receptor (TLR) ligands, mannan-binding lectin (MBL) ligands, STING agonists, immune stimulatory complexes such as saponins, Quil A, QS-21, QS-7, ISCOMATRIX, etc. Other adjuvants include CpG oligonucleotides and double-stranded RNA molecules such as poly(A) and poly(U). Combinations of the above adjuvants can also be used. See, for example, U.S. Patent Nos. 6,645,495, 7,029,678, and 7,858,589, all of which are incorporated herein by reference in their entirety. In one embodiment, the adjuvant used herein is QS-21 STIMULON.

[0167] In certain embodiments, an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody disclosed herein is administered to a subject in combination with an additional therapeutic agent comprising a TCR. In certain embodiments, the additional therapeutic agent is a soluble TCR. In certain embodiments, the additional therapeutic agent is a cell expressing a TCR. Thus, in one embodiment, the invention relates to an antibody and / or pharmaceutical composition of the invention in combination with an additional agent comprising a TCR for use as a medicament and / or for use in a method for the treatment of cancer.

[0168] In certain embodiments, an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody disclosed herein is administered to a subject in combination with a cell expressing a chimeric antigen receptor (CAR). In certain embodiments, the cell is a T cell.

[0169] In certain embodiments, the anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody disclosed herein is administered to a subject in combination with a TCR mimetic antibody. In certain embodiments, the TCR mimetic antibody is an antibody that specifically binds to a peptide-MHC complex. For non-limiting examples of TCR mimetic antibodies, see, for example, U.S. Patent No. 9,074,000 and U.S. Publication Nos. US2009 / 0304679A1 and US2014 / 0134191A1, all of which are incorporated herein by reference in their entirety.

[0170] In certain embodiments, an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody disclosed herein is administered to a subject in combination with a bispecific T cell engager (BiTE) (e.g., as described in WO2005 / 061547A2, the entirety of which is incorporated herein by reference) and / or a dual affinity retargeting antibody (DART) (e.g., as described in WO2012 / 162067A2, the entirety of which is incorporated herein by reference). In certain embodiments, the BiTE and / or DART specifically binds to a tumor-associated antigen (e.g., a polypeptide overexpressed in tumors, a polypeptide derived from an oncovirus, a polypeptide comprising a tumor-specific post-translational modification, or a polypeptide specifically mutated in tumors) and a molecule on an effector cell (e.g., CD3 or CD16). In certain embodiments, the tumor-associated antigen is EGFR (e.g., human EGFR), and optionally, the BiTE and / or DART comprises the VH and VL sequences of cetuximab. In certain embodiments, the tumor-associated antigen is Her2 (e.g., human Her2), and optionally, the BiTE and / or DART comprises the VH and VL sequences of trastuzumab. In certain embodiments, the tumor-associated antigen is CD20 (e.g., human CD20).

[0171] The anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody and additional therapeutic agent (e.g., a chemotherapeutic agent, a radiotherapeutic agent, a checkpoint targeting agent, an IDO inhibitor, a vaccine, an adjuvant, a soluble TCR, a TCR-expressing cell, a chimeric antigen receptor-expressing cell, and / or a TCR mimetic antibody) may be administered separately, sequentially, or simultaneously as separate dosage forms. In one embodiment, the anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody is administered parenterally and the IDO inhibitor is administered orally.

[0172] The antibodies or pharmaceutical compositions described herein can be delivered to a subject by various routes. These include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, intraarterial, and subcutaneous routes. Pulmonary administration can also be used, for example, by using an inhaler or nebulizer and formulating with an aerosolizing agent for use as a spray. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered subcutaneously or intravenously. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered intraarterially. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered intratumorally. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered to tumor-draining lymph nodes.

[0173] The amount of the antibody or composition that will be effective in the treatment and / or prevention of the condition will depend on the nature of the disease, and can be determined by standard clinical techniques.

[0174] The exact dosage to be used in the composition also depends on the route of administration and the severity of the infection or disease caused by it, and should be determined according to the judgment of the practitioner and the circumstances of each patient.For example, the effective dosage may also vary depending on the means of administration, target site, the patient's physiological condition (including age, weight, and health), whether the patient is human or animal, other medicines administered, or whether the treatment is preventive or therapeutic.Usually, the patient is human, but non-human mammals, including transgenic mammals, can also be treated.The therapeutic dosage is optimally titrated to optimize safety and effectiveness.

[0175] The anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies described herein can also be used to assay CD96 (e.g., human CD96 or cynomolgus CD96) protein levels in biological samples using classical immunohistological methods known to those skilled in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase, iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium ( 99 Labels include radioisotopes such as Tc), luminescent labels such as luminol, and fluorescent labels such as fluorescein and rhodamine, and biotin. Such labels can be used to label the antibodies described herein. Alternatively, the anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies described herein can be labeled and used in combination with an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody to detect CD96 (e.g., human CD96 or cynomolgus CD96) protein levels. Thus, in one embodiment, the present invention relates to the use of the antibodies of the present invention for in vitro detection of CD96 (e.g., human CD96 or cynomolgus CD96) protein in a biological sample. In a further embodiment, the invention relates to the use of an anti-CD96 antibody of the invention for assaying and / or detecting CD96 (e.g., human CD96 or cynomolgus CD96) protein levels in a biological sample in vitro, optionally wherein the anti-CD96 antibody is conjugated to a radionuclide or detectable label and / or carries a label as described herein, and / or wherein immunohistological methods are used.

[0176] Assaying for CD96 (e.g., human CD96 or cynomolgus CD96) protein expression levels is intended to include qualitatively or quantitatively measuring or estimating the level of CD96 (e.g., human CD96 or cynomolgus CD96) protein in a first biological sample, either directly (e.g., by determining or estimating absolute protein levels) or relatively (e.g., by comparing to disease-associated protein levels in a second biological sample). The CD96 (e.g., human CD96 or cynomolgus CD96) polypeptide expression level in a first biological sample can be measured or estimated and compared to a standard CD96 (e.g., human CD96 or cynomolgus CD96) protein level, where the standard is, for example, taken from a second biological sample obtained from an individual without the disorder or determined by averaging levels from a population of individuals without the disorder. As is understood in the art, once a "standard" CD96 (e.g., human CD96 or cynomolgus monkey CD96) polypeptide level is known, it can be repeatedly used as a standard for comparison. Accordingly, in a further embodiment, the present invention relates to an in vitro method for assaying and / or detecting CD96 protein levels, e.g., human CD96 protein levels, in a biological sample, comprising qualitatively or quantitatively measuring or estimating the level of CD96 protein, e.g., human CD96 protein, in the biological sample by immunohistological methods.

[0177] 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 CD96 (e.g., human CD96 or cynomolgus monkey CD96). Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans or cynomolgus monkeys) are well known in the art. Biological samples include human peripheral blood mononuclear cells (PBMCs).

[0178] The anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies described herein can be used for prognostic, diagnostic, monitoring, and screening applications, including in vitro and in vivo applications, that are well known to those skilled in the art and are based on the present description. Prognostic, diagnostic, monitoring, and screening assays and kits for in vitro determination and evaluation of immune system status and / or immune response may be utilized to predict, diagnose, and monitor patient samples, including those known to have or suspected of having an immune deficiency, or those for expected or desired immune system, antigen, or vaccine responses. Determination and evaluation of immune system status and / or immune response is also useful in determining a patient's suitability for drug clinical trials against different agents or antibodies, or for the administration of a particular chemotherapeutic agent, radiotherapeutic agent, or antibody (including combinations thereof). This type of prognostic and diagnostic monitoring and determination is already being performed utilizing antibodies against the HER2 protein in breast cancer, where assays are also used to evaluate patients for antibody therapy using Herceptin® (HercepTest™, Dako). In vivo applications include directed cell therapy, and immune system modulation and radioimaging of immune responses. Thus, in one embodiment, the invention relates to an anti-CD96 antibody and / or pharmaceutical composition of the invention for use as a diagnostic. In one embodiment, the invention relates to an anti-CD96 antibody and / or pharmaceutical composition of the invention for use in a method for predicting, diagnosing, and / or monitoring a subject having or suspected of having an immune system deficiency and / or with respect to an expected or desired immune system response, antigen response, or vaccine response.In another embodiment, the invention relates to an anti-CD96 antibody of the invention for prognosing, diagnosing, and / or monitoring a subject having or suspected of having an immune system deficiency, and / or with respect to an expected or desired immune system response, antigen response, or vaccine response, by assaying and / or detecting human CD96 protein levels in a biological sample of the subject in vitro.

[0179] In one embodiment, anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies can be used in immunohistochemistry of biopsy samples. In one embodiment, the method is an in vitro method. In another embodiment, anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies can be used to detect levels of CD96 (e.g., human CD96 or cynomolgus CD96) or levels of cells containing CD96 (e.g., human CD96 or cynomolgus CD96) on their membrane surface, which levels can then be linked to certain disease symptoms. The anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies described herein may carry a detectable or functional label and / or be conjugated to a radionuclide or detectable label. When fluorescent labels are used, specific binding members can be identified and quantified using currently available microscopy and fluorescence-activated cell sorting analysis (FACS) or a combination of both method procedures known in the art. The anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies described herein may carry or be conjugated to a fluorescent label. Exemplary 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-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies may be isotopic or 3 H, 14 C. 32 P, 35 S, 36 Cl, 51 Cr,57 Co、 58 Co、 59 Fe、 67 Cu、 90 Y、 99 Tc、 111 In、 117 Is, 121 I、 124 I、 125 I、 131 I、 198 I、 211 And、 213 Bi、 225 Ac、および 186The label may carry or be conjugated to a radioactive label or radionuclide such as Re. When a radioactive label is used, specific binding of an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody to CD96 (e.g., human CD96 or cynomolgus CD96) can be identified and quantified using currently available counting procedures known in the art. When the label is an enzyme, detection can be achieved by any of the currently available colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric techniques known in the art. This can be achieved by contacting a sample or control sample with an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody under conditions that allow the formation of a complex between the antibody and CD96 (e.g., human CD96 or cynomolgus CD96). Any complexes formed between the antibody and CD96 (e.g., human CD96 or cynomolgus monkey CD96) are detected and compared in the sample and the control. In light of the specific binding of the antibodies described herein to CD96 (e.g., human CD96 or cynomolgus monkey CD96), the antibodies can be used to specifically detect CD96 (e.g., human CD96 or cynomolgus monkey CD96) expression on the surface of cells. The antibodies described herein can also be used to purify CD96 (e.g., human CD96 or cynomolgus monkey CD96) via immunoaffinity purification. Also included herein are assay systems that can be prepared in the form of test kits, kits, or kits of parts, for example, for quantitative analysis of the degree of presence of CD96 (e.g., human CD96 or cynomolgus monkey CD96) or CD96 (e.g., human CD96 or cynomolgus monkey CD96) / CD96 (e.g., human CD96 or cynomolgus monkey CD96) ligand complexes. The system, test kit, kit, or kit-of-parts may include a labeled component, eg, a labeled antibody, and one or more additional immunochemical reagents.

[0180] Polynucleotides, vectors, and methods for producing anti-CD96 antibodies In another aspect, provided herein are polynucleotides comprising a nucleotide sequence encoding an antibody described herein, or a portion or fragment thereof (e.g., VL and / or VH, and light chain and / or heavy chain), which specifically binds to the CD96 (e.g., human CD96 or cynomolgus CD96) antigen, and vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells). Provided herein are polynucleotides comprising a nucleotide sequence encoding the heavy and / or light chain of any of the antibodies provided herein, and vectors comprising such polynucleotide sequences, e.g., expression vectors for their efficient expression in host cells, e.g., mammalian cells.

[0181] As used herein, an "isolated" polynucleotide or nucleic acid molecule is one that is separated from other nucleic acid molecules present in the nucleic acid molecule's natural source (e.g., mouse or human). Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the term "substantially free" includes preparations of polynucleotides or nucleic acid molecules having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (particularly less than about 10%) of other materials, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals. In certain embodiments, the nucleic acid molecule(s) encoding the antibodies described herein are isolated or purified.

[0182] In certain aspects, provided herein are antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) polypeptide and that comprise an amino acid sequence described herein, as well as polynucleotides comprising nucleotide sequences encoding antibodies that compete with such antibodies (e.g., in a dose-dependent manner) for binding to CD96 (e.g., human CD96 or cynomolgus CD96) polypeptide or that bind to the same epitope as such antibodies.

[0183] In certain embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding the light chain or heavy chain of an antibody described herein. The polynucleotide may comprise a nucleotide sequence encoding a light chain comprising the VL FRs and CDRs of an antibody described herein (see, e.g., Table 1), or a nucleotide sequence encoding a heavy chain comprising the VH FRs and CDRs of an antibody described herein (see, e.g., Table 1).

[0184] Also provided herein are polynucleotides encoding anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies that have been optimized, for example, by codon / RNA optimization, substitution with a heterologous signal sequence, and removal of mRNA instability elements. Methods for generating optimized nucleic acids encoding anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies or fragments thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes in the mRNA and / or excluding inhibitory regions can be carried out by appropriately applying the optimization methods described in, for example, U.S. Patent Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498 (all of which are incorporated herein by reference in their entireties). For example, potential splice sites and instability elements (e.g., A / T- or A / U-rich elements) within the RNA can be mutated without altering the amino acid encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. Modifications can take advantage of the degeneracy of the genetic code, for example, by using alternative codons for the same amino acid. In certain embodiments, conservative mutations, e.g., altering one or more codons that encode a similar amino acid with a similar chemical structure and properties and / or function as the original amino acid, may be desirable. Such methods can increase expression of an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody or fragment thereof by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more relative to expression of an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody encoded by a non-optimized polynucleotide.

[0185] In certain embodiments, optimized polynucleotide sequences encoding an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody, or fragment thereof (e.g., VL domain and / or VH domain) described herein may hybridize to an antisense (e.g., complementary) polynucleotide of a non-optimized polynucleotide sequence encoding an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody, or fragment thereof (e.g., VL domain and / or VH domain) described herein. In certain embodiments, optimized nucleotide sequences encoding an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody or fragment thereof described herein hybridize under high stringency conditions to an antisense polynucleotide of a non-optimized polynucleotide sequence encoding an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody, or fragment thereof described herein. In certain embodiments, an optimized nucleotide sequence encoding an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody, or fragment thereof, described herein hybridizes to an antisense polynucleotide of a non-optimized nucleotide sequence encoding an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody, or fragment thereof, described herein under highly stringent, moderately stringent, or less stringent hybridization conditions. Information regarding hybridization conditions is described in U.S. Patent Application Publication No. 2005 / 0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference in its entirety.

[0186] Polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotide can be determined. The nucleotide sequences encoding the antibodies described herein, such as those described in Table 1, and modified versions of these antibodies can be determined using methods known in the art, i.e., nucleotide codons known to encode specific amino acids are assembled to generate nucleic acids encoding antibodies. Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17:242-6, the entire contents of which are incorporated herein by reference), which briefly involves synthesizing overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0187] Alternatively, polynucleotides encoding the antibodies described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding the light and / or heavy chains of the antibody. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding the variable light and / or variable heavy chain regions of the antibody. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning to generate, for example, chimeric and humanized antibodies.

[0188] If a clone containing nucleic acid encoding a particular antibody is not available but the sequence of the antibody molecule is known, nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from any tissue or cell that expresses an antibody, such as a hybridoma cell selected to express an antibody described herein, or nucleic acid isolated therefrom, preferably polyA+ RNA) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence to identify a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0189] DNA encoding the anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies in the recombinant host cells.

[0190] To generate whole antibodies, the VH or VL sequence can be amplified in an scFv clone using PCR primers containing the VH or VL nucleotide sequence, a restriction enzyme site, and flanking sequences to protect the restriction enzyme site. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region, such as the human gamma 1 or human gamma 4 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region, such as the human kappa or lambda constant region. In certain embodiments, the vector for expressing the VH or VL domain contains an EF-1α promoter, a secretion signal, a cloning site for the variable region, a constant domain, and a selection marker such as neomycin. The VH and VL domains can also be cloned into a vector expressing the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line to generate stable or transient cell lines expressing full-length antibodies, such as IgG, using techniques known to those skilled in the art.

[0191] The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the murine sequences, or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0192] Also provided are polynucleotides that hybridize to polynucleotides encoding the antibodies described herein under highly stringent, moderately stringent, or less stringent hybridization conditions. In certain embodiments, the polynucleotides described herein hybridize to polynucleotides encoding the VH domains and / or VL domains provided herein under highly stringent, moderately stringent, or less stringent hybridization conditions.

[0193] Hybridization conditions are described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions may include hybridization to filter-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2x SSC / 0.1% SDS at about 50-65°C; hybridization under highly stringent conditions may include hybridization to filter-bound nucleic acid in 6x SSC at about 45°C, followed by one or more washes in 0.1x SSC / 0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is known to those skilled in the art and has been described, see, e.g., Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3, which is incorporated herein by reference in its entirety.

[0194] In certain aspects, provided herein are cells (e.g., host cells) that express (e.g., recombinantly) an antibody described herein that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), as well as related polynucleotides and expression vectors. Provided herein are vectors (e.g., expression vectors) comprising a polynucleotide that includes a nucleotide sequence encoding an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody or fragment thereof for recombinant expression in a host cell, preferably a mammalian cell (e.g., a CHO cell). Also provided herein are host cells comprising such vectors for recombinantly expressing an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody (e.g., a human or humanized antibody) described herein. In certain aspects, provided herein are methods of producing an antibody described herein, comprising expressing such an antibody from a host cell.

[0195] Recombinant expression of an antibody described herein (e.g., a full-length antibody, antibody heavy or light chain, or single-chain antibody described herein) that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) generally involves construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule described herein, the antibody heavy and / or light chain, or a fragment thereof (e.g., a heavy or light chain variable region) has been obtained, vectors for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Accordingly, described herein are methods for preparing proteins by expressing polynucleotides comprising antibody or antibody fragment (e.g., light or heavy chain) encoding nucleotide sequences. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody or antibody fragment (e.g., light or heavy chain) coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors containing a nucleotide sequence encoding the antibody molecule described herein, the heavy or light chain of the antibody, the heavy or light chain variable region of the antibody or its fragment, or the heavy or light chain CDR, operably linked to a promoter. Such vectors can contain, for example, a nucleotide sequence encoding the constant region of the antibody molecule (see, for example, International Publication Nos. WO86 / 05807 and WO89 / 01036, and U.S. Patent No. 5,122,464, the entire contents of which are incorporated herein by reference), and the variable region of the antibody can be cloned into such a vector for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

[0196] The expression vector can be introduced into cells (e.g., host cells) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce the antibodies or fragments thereof described herein. Accordingly, provided herein are host cells comprising a polynucleotide encoding an antibody or fragment thereof described herein, or a heavy or light chain thereof, or a fragment thereof, or a single-chain antibody described herein, operably linked to a promoter for expression of such sequences in the host cell. In certain embodiments, for expression of a double-chain antibody, vectors encoding both the heavy and light chains individually can be coexpressed in the host cell for expression of the entire immunoglobulin molecule, as described in more detail below. In certain embodiments, the host cell comprises a vector comprising polynucleotides encoding both the heavy and light chains of an antibody or fragment thereof described herein. In certain embodiments, the host cell comprises two different vectors: a first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody described herein, and a second vector comprising a polynucleotide encoding the light chain or light chain variable region of an antibody or fragment thereof described herein. In other embodiments, a first host cell comprises a first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody or fragment thereof described herein, and a second host cell comprises a second vector comprising a polynucleotide encoding the light chain or light chain variable region of an antibody described herein. 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-CD96 (e.g., human CD96 or cynomolgus CD96) antibody described herein. In certain embodiments, a population of host cells comprising such a first host cell and such a second host cell is provided herein.

[0197] In certain embodiments, provided herein are populations of vectors comprising a first vector comprising a polynucleotide encoding the light chain / light chain variable region of an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody described herein, and a second vector comprising a polynucleotide encoding the heavy chain / heavy chain variable region of an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody described herein.

[0198] A variety of host-expression vector systems can be utilized to express the antibody molecules described herein (see, e.g., U.S. Pat. No. 5,807,715, incorporated herein by reference in its entirety). Such host-expression systems represent vehicles in which coding sequences of interest may be produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequences, are capable of expressing the antibody molecules described herein in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems (e.g., Chlamydomonas) infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences. reinhardtii); or mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) harboring a recombinant expression construct comprising, for example, a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter; a vaccinia virus 7.5K promoter). In certain embodiments, cells expressing an antibody described herein are Chinese hamster ovary (CHO) cells, e.g., CHO cells from the CHO GS System™ (Lonza).In certain embodiments, the heavy and / or light chains of antibodies produced by CHO cells may have an N-terminal glutamine or glutamate residue replaced with pyroglutamate. In certain embodiments, cells for expressing the antibodies described herein are human cells, e.g., human cell lines. In certain embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In certain embodiments, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells) are used for expression of recombinant antibody molecules, particularly whole recombinant antibody molecules. For example, mammalian cells such as CHO cells in association with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus are effective expression systems for antibodies (Foecking MK & Hofstetter H (1986) Gene 45:101-5, and Cockett MI et al., (1990) Biotechnology 8(7):662-7, each of which is incorporated herein by reference in its entirety). In certain embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In certain embodiments, expression of a nucleotide sequence encoding an antibody described herein that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) is controlled by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0199] In bacterial systems, several expression vectors can be advantageously selected depending on the use intended for the expressed antibody molecule. For example, when large quantities of such antibodies are to be produced for the generation of pharmaceutical compositions of the antibody molecule, vectors directing the expression of high levels of fusion protein products that are easily purified may be desirable. Such vectors include, but are not limited to, E. coli expression pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2:1791-1794), pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13:3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24:5503-5509), in which the antibody coding sequence can be individually ligated into the vector in frame with the lac Z coding region to produce a fusion protein, and the like, all of which are incorporated herein by reference in their entirety. Inouye S & Inouye M (1985) Nuc Acids Res 13:3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24:5503-5509. For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins containing glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads, followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0200] In an insect system, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus is grown in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0201] Several viral-based expression systems are available for mammalian host cells. When adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription / translation control complex, such as the late promoter and tripartite leader sequence. This 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., region E1 or E3) results in a recombinant virus that is viable and capable of expressing antibody molecules in infected hosts (see, e.g., Logan & Shenk T (1984) PNAS 81(12):3655-9, incorporated herein by reference in its entirety). Specific initiation signals may also be required for efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol. 153:516-544, incorporated herein by reference in its entirety).

[0202] In addition, a host cell strain can be selected that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the initial transcription, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2OPER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 In certain embodiments, the anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies described herein are produced in mammalian cells, such as CHO cells.

[0203] In certain embodiments, the antibodies described herein have reduced or no fucose content. Such antibodies can be produced using techniques known to those skilled in the art. For example, the antibodies can be expressed in cells that are deficient or lack the ability to fucosylate. In certain instances, a cell line with a knockout of both alleles of α1,6-fucosyltransferase can be used to produce antibodies with reduced fucose content. (登録商標) The system (Lonza) is an example of such a system that can be used to produce antibodies with reduced fucose content.

[0204] For long-term, high-yield production of recombinant proteins, stable expression cells can be generated. For example, cell lines can be engineered that stably express the anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies described herein. In certain embodiments, the cells provided herein stably express the light chain / light chain variable region and heavy chain / heavy chain variable region that assemble to form the antibodies described herein.

[0205] In certain embodiments, rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA / polynucleotide, engineered cells can be grown in enriched media for 1-2 days and then switched to selective media. The selectable marker in the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which are then cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing the anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibodies or fragments thereof described herein. Such engineered cell lines can be particularly useful for screening and evaluating compositions that interact directly or indirectly with the antibody molecule.

[0206] Several 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), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12):2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3):817-23) genes in tk, hgprt, or aprt cells, respectively, all of which are incorporated herein by reference in their entirety. Additionally, antimetabolite resistance is conferred by the following genes: dhfr, which confers resistance to methotrexate (Wingler M et al., (1980) PNAS 77(6):3567-70; O'Hare K at al., (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4):2072-6); and the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3:87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32:573-596; Mulligan RC (1993) Science 260:926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62:191-217, Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5):211-5), and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3):147-56).Wigler M et al., (1980) PNAS 77(6):3567-70; O'Hare K et al., (1981) PNAS 78:1527-31) G-418 (Wu GY & Wu CH (1991) Biotherapy 3:87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32:573-596; Mulligan RC (1993) Science 260:926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62:191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5):211-5); methods generally known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clones, such methods being described, for example, in Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993), and Dracopoli NC et al., (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994), Chapters 12 and 13 of Colbere-Garapin F et al., (1981) J Mol Biol 150:1-14, all of which are incorporated herein by reference in their entireties.

[0207] The expression level of an antibody molecule can be increased by vector amplification (for a review, see Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987), which is incorporated herein by reference in its entirety). If the marker in the antibody-expressing vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Because the amplified region is associated with the antibody gene, antibody production will also increase (Crouse GF et al., (1983) Mol Cell Biol 3:257-66, which is incorporated herein by reference in its entirety).

[0208] Host cells can be transfected with two or more expression vectors described herein: a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide. The two vectors can contain the same selectable marker to enable equal expression of heavy and light chain polypeptides. Host cells can be co-transfected with different amounts of two or more expression vectors. For example, host cells can be transfected with any one of the following ratios of the first expression vector to the second expression vector: approximately 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.

[0209] Alternatively, a single vector capable of encoding and expressing both heavy and light chain polypeptides can be used. In such a situation, the light chain should be placed before the heavy chain to avoid excess, non-toxic free heavy chain (Proudfoot NJ (1986) Nature 322:562-565, and Kohler G (1980) PNAS 77:2197-2199, each of which is incorporated herein by reference in its entirety). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA. Expression vectors can be monocistronic or multicistronic. Multicistronic nucleic acid constructs can encode two, three, four, five, six, seven, eight, nine, ten, or more genes / nucleotide sequences, or in the range of 2-5, 5-10, or 10-20 genes / nucleotide sequences. For example, a bicistronic nucleic acid construct can include, in the following order: a promoter, a first gene (e.g., the heavy chain of an antibody described herein), and a second gene (e.g., the light chain of an antibody described herein). In such an expression vector, transcription of both genes can be driven by a promoter, but translation of mRNA from the first gene can occur by a cap-dependent scanning mechanism, and translation of mRNA from the second gene can occur by a cap-independent mechanism, e.g., via an IRES.

[0210] Once an antibody molecule described herein is produced by recombinant expression, it may be purified by any method known in the art for the purification of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity, particularly affinity for specific antigens following Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. Additionally, the antibodies described herein may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0211] In certain embodiments, the antibodies described herein are isolated or purified. Generally, an isolated antibody is substantially free of other antibodies having antigenic specificities different from the isolated antibody. For example, in certain embodiments, preparations of the antibodies described herein are substantially free of cellular material and / or chemical precursors. The term "substantially free of cellular material" includes preparations of antibodies in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, antibodies that are substantially free of cellular material include preparations of antibodies having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or antibody variants, e.g., different post-translationally modified forms of antibodies or other different versions of antibodies (e.g., antibody fragments). When an antibody is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When an antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in the synthesis of the protein. Thus, such preparations of antibodies have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In certain embodiments, the antibodies described herein are isolated or purified.

[0212] Antibodies or fragments thereof that specifically bind to CD96 (e.g., human CD96 or cynomolgus monkey CD96) can be produced by any method known in the art for synthesizing antibodies, for example, by chemical synthesis or by recombinant expression technology. The methods described herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described, for example, in the references cited herein and are explained fully in the literature.For example, Maniatis T at 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 at al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Ausubel FM et al. al.,Current Protocols in Molecular Biology,John Wiley&Sons(1987 and annual updates),Current Protocols in Immunology,John Wiley&Sons(1987 and annual updates)Gait(ed.)(1984)Oligonucleotide Synthesis:A Practical Approach,IRL Press;Eckstein(ed.)(1991)Oligonucleotides and Analogues:A Practical Approach,IRL Press, Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, all of which are incorporated herein by reference in their entireties.

[0213] In certain embodiments, the antibodies described herein are antibodies (e.g., recombinant antibodies) that are prepared, expressed, engineered, or isolated by any means, including, for example, synthesis of DNA sequences, through genetic engineering, or by creation. In certain embodiments, such antibodies comprise sequences (e.g., DNA sequences or amino acid sequences) that do not naturally exist within the antibody germline repertoire of an animal or mammal (e.g., a human) in vivo.

[0214] In one aspect, provided herein is a method for producing an antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), comprising culturing a cell or host cell described herein. In one embodiment, the method is performed in vitro. In certain aspects, provided herein is a method for producing an antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), comprising expressing (e.g., recombinantly expressing) the antibody using a cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody described herein). In certain embodiments, the cell is an isolated cell. In certain embodiments, an exogenous polynucleotide has been introduced into the cell. In certain embodiments, the method further comprises purifying the antibody obtained from the cell or host cell.

[0215] Methods for producing polyclonal antibodies are known in the art (see, e.g., Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York, which is incorporated herein by reference in its entirety).

[0216] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including those known in the art and taught in, for example, Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ at al., in: Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981) (each of which is incorporated herein by reference in its entirety). As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be recombinantly produced from host cells that exogenously express an antibody described herein or a fragment thereof, such as the light chain and / or heavy chain of such an antibody.

[0217] In certain embodiments, a "monoclonal antibody," as used herein, is an antibody produced by a single cell (e.g., a hybridoma or host cell producing a recombinant antibody), which specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) as determined, for example, by ELISA or other antigen-binding or competitive binding assays known in the art or in the Examples provided herein. In certain embodiments, a monoclonal antibody may be a chimeric or humanized antibody. In certain embodiments, a monoclonal antibody is a monovalent or multivalent (e.g., bivalent) antibody. In certain embodiments, a monoclonal antibody may be a monospecific or multispecific (e.g., bispecific) antibody. The monoclonal antibodies described herein can be made, for example, by the hybridoma method described in Kohler G & Milstein (1975) Nature 256:495, which is incorporated herein by reference in its entirety, or can be isolated, for example, from a phage library, using, for example, the techniques described herein. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, e.g., Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

[0218] As used herein, an antibody binds to an antigen multivalently (e.g., bivalently) when it comprises at least two (e.g., two or more) monovalent binding domains, each capable of binding to an epitope on the antigen. Each monovalent binding domain may bind to the same or a different epitope on the antigen.

[0219] Methods for producing and screening specific antibodies using hybridoma technology are conventional and well known in the art. For example, in the hybridoma method, mice or other suitable host animals, such as sheep, goats, rabbits, rats, hamsters, or macaques, are immunized to induce lymphocytes that produce or can produce antibodies that specifically bind to the protein used for immunization (e.g., CD96 (e.g., human CD96 or cynomolgus CD96)). Alternatively, lymphocytes may be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986), incorporated herein by reference in its entirety. Additionally, RIMMS (repeated immunization multiple site) technology can be used to immunize animals (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, incorporated herein by reference in its entirety).

[0220] In certain embodiments, a mouse (or other animal such as a rat, monkey, donkey, pig, sheep, hamster, or dog) can be immunized with an antigen (e.g., CD96 (e.g., human CD96 or cynomolgus CD96)), and once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested, and splenocytes are isolated. The splenocytes can then be transfected with any suitable myeloma cells, e.g., from the American Type Culture Collection (ATCC 94444), by well-known techniques. (登録商標) The cells are fused to cells from cell line SP20, available from NIH (Manassas, VA), to form hybridomas. Hybridomas are selected and cloned by limiting dilution. In certain embodiments, lymph nodes from immunized mice are harvested and fused with NS0 myeloma cells.

[0221] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0222] Certain embodiments use myeloma cells that fuse efficiently, support stable, high-level production of antibody by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. These myeloma cell lines are mouse myeloma lines such as those derived from the NS0 cell line, or the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, CA, USA), and SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection (Rockville, MD, USA). Human myeloma and mouse-human heteromyeloma cell lines 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)), each of which is incorporated herein by reference in its entirety.

[0223] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies directed against CD96 (e.g., human CD96 or cynomolgus CD96). The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by methods known in the art, for example, by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0224] After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice (supra)). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 medium. Additionally, hybridoma cells may be grown in vivo as ascites tumors in animals.

[0225] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0226] The antibodies described herein include, for example, antibody fragments that recognize a specific CD96 (e.g., human CD96 or cynomolgus monkey CD96) and can be generated by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). The Fab fragment corresponds to one of the two identical arms of an antibody molecule and contains an intact light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains the two antigen-binding arms of an antibody molecule linked by disulfide bonds in the hinge region.

[0227] Furthermore, the antibodies described herein can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of affected tissue). The DNA encoding the VH and VL domains is recombined with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli, and the E. coli is infected with helper phage. The phages used in these methods are typically filamentous phages, including fd and M13, and the VH and VL domains are usually recombinantly fused to either phage gene III or gene VIII. Phage expressing antigen-binding fragments that bind to a specific antigen can be selected or identified using, for example, labeled antigen or antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to generate the antibodies described herein include those described in Brinkman U et al., (1995) J Immunol Methods 182:41-50, Ames RS et al., (1995) J Immunol Methods 184:177-186, Kettleborough CA et al., (1994) Eur J Immunol 24:952-958, Persic L et al., (1997) Gene 187:9-18, Burton DR & Barbas CF (1994) Advan Immunol 57:191-280, PCT Application No. PCT / GB91 / 001134, International Publication Nos. WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and WO97 / 13844, and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,488, Nos. 4, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108, all of which are incorporated herein by reference in their entirety.

[0228] After phage selection, as described in the above references, the antibody coding region from the phage is isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, which can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as described below. Techniques for recombinantly producing antibody fragments, such as Fab, Fab', and F(ab')2 fragments, can also be employed using methods known in the art, such as those disclosed in PCT Publication No. WO92 / 22324, Mullinax RL et al., (1992) BioTechniques 12(6):864-9, Sawai H et al., (1995) Am J Reprod Immunol 34:26-34, and Better M et al., (1988) Science 240:1041-1043, all of which are incorporated herein by reference in their entireties.

[0229] In certain embodiments, to generate whole antibodies, PCR primers containing the VH or VL nucleotide sequence, a restriction enzyme site, and flanking sequences to protect the restriction enzyme site can be used to amplify the VH or VL sequence from a template, e.g., an scFv clone. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a VH constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a VL constant region, e.g., a human kappa or lambda constant region. The VH and VL domains can also be cloned into a vector expressing the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line to generate stable or transient cell lines expressing full-length antibodies, e.g., IgG, using techniques known to those skilled in the art.

[0230] Chimeric antibodies are molecules in which different parts of the antibody are derived from different immunoglobulin molecules.For example, chimeric antibodies can comprise the variable region of a mouse or rat monoclonal antibody fused with the constant region of a human antibody.Methods for producing chimeric antibodies are known in the art.See, for example, Morrison SL (1985) Science 229:1202-7; Oi VT & Morrison SL (1986) BioTechniques 4:214-221; Gillies SD at al., (1989) J Immunol Methods 125:191-202, and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397 and 6,331,415, all of which are incorporated herein by reference in their entirety.

[0231] A humanized antibody can bind to a predetermined antigen and comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin (e.g., a murine immunoglobulin). In certain embodiments, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody may be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4.Humanized antibodies can be prepared by techniques including, but not limited to, CDR grafting (European Patent No. EP239400, International Publication No. WO91 / 09967, and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (European Patent Nos. EP592106 and EP519596, 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), chain shuffling (U.S. Pat. No. 5,565,332), as well as e.g. U.S. Pat. No. 6,407,213, U.S. Pat. al.,(2000)Protein Eng.13(5):353-60, Morea V et al.,(2000)Methods 20(3)267-79, Baca M et al.,(1997)J Biol Chem 272(16):10678-84, Roguska MA et al.,(1996)Protein Eng 9(10):895 904, Couto JR et. et al., (1995) Cancer Res. 55(23 Supp):5973s-5977s, Couto JR et al., (1995) Cancer Res 55(8):1717-22, Sandhu JS (1994) Gene 150(2):409-10, and Pedersen JT et al., (1994) J Mol Biol 235(3):959-73, all of which are incorporated herein by reference in their entireties. See also U.S. Application Publication No. US2005 / 0042664A1 (February 24, 2005), which is incorporated herein by reference in its entirety.

[0232] Methods for making multispecific (e.g., bispecific) antibodies have been described, see, e.g., U.S. Patent Nos. 7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992; and 8,586,713, all of which are incorporated by reference herein in their entireties.

[0233] Single domain antibodies, for example, antibodies lacking light chains, can be produced by methods well known in the art. See Riechmann L & Muyldermans S (1999) J Immunol 231:25-38, Nuttall SD et al., (2000) Curr Pharm Biotechnol 1(3):253-263, Muyldermans S, (2001) J Biotechnol 74(4):277-302, U.S. Patent No. 6,005,079, and International Publication Nos. WO94 / 04678, WO94 / 25591, and WO01 / 44301, all of which are incorporated herein by reference in their entirety. Riechmann L&Muyldermans S(1999)J Immunol 231:25-38;Nuttall SD et al.,(2000)Curr Pharm Biotechnol 1(3):253-263;Muyldermans S,(2001)J Biotechnol 74(4):277-302;

[0234] Furthermore, antibodies that specifically bind to the CD96 (e.g., human CD96 or cynomolgus CD96) antigen can then be utilized to generate anti-idiotypic antibodies that "mimic" the antigen using techniques well known to those of skill in the art. See, e.g., Greenspan NS & Bona CA (1989) FASEB J 7(5):437-444, and Nissinoff A (1991) J Immunol 147(8):2429-2438, each of which is incorporated herein by reference in its entirety.

[0235] In certain embodiments, an antibody described herein that binds to the same epitope of CD96 (e.g., human CD96 or cynomolgus CD96) as an anti-CD96 (e.g., human CD96 or cynomolgus CD96) antibody described herein is a human antibody. In certain embodiments, an antibody described herein that competitively blocks (e.g., in a dose-dependent manner) any one of the antibodies described herein from binding to CD96 (e.g., human CD96 or cynomolgus CD96) is a human antibody. Human antibodies can be produced using any method known in the art. For example, transgenic mice that are incapable of expressing functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. In particular, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. Mouse heavy and light chain immunoglobulin genes may be provided separately or simultaneously, non-functionally, with the introduction of human immunoglobulin loci by homologous recombination. HHomozygous deletion of the region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the usual manner with all or part of a selected antigen, such as an antigen (e.g., CD96 (e.g., human CD96 or cynomolgus CD96)). Monoclonal antibodies directed against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation and undergo substantial class switching and somatic mutation. Thus, such technology can be used to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93, which is incorporated herein by reference in its entirety. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735, and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, all of which are incorporated herein by reference in their entirety. 5,413,923,5,625,126,5,633,425,5,569,825,5,661,016,5,545,806,5,814,318 An example of a mouse capable of producing human antibodies is the Xenomouse (商標) (Abgenix, Inc.; U.S. Patent Nos. 6,075,181 and 6,150,184), HuAb-Mouse (商標) (Medarex, Inc. / Gen Pharm; U.S. Patent Nos. 5,545,806 and 5,569,825), Trans Chromo Mouse (商標)(Kirin), and KM Mouse (商標) (Medarex / Kirin), all of which are incorporated herein by reference in their entirety.

[0236] Human antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) can be made by a variety of methods known in the art, including the phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887, 4,716,111, and 5,885,793, and International Publication Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741, all of which are incorporated herein by reference in their entireties. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735,

[0237] In certain embodiments, human antibodies can be produced using mouse-human hybridomas. For example, Epstein-Barr virus (EBV)-transformed human peripheral blood lymphocytes can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine those that secrete human monoclonal antibodies that specifically bind to a target antigen (e.g., CD96 (e.g., human CD96 or cynomolgus CD96)). Such methods are known and described in the art; see, e.g., Shinmoto H et al., (2004) Cytotechnology 46:19-23; Nakagawa Y et al., (2005) Human Antibodies 14:27-31, each of which is incorporated herein by reference in its entirety. Shinmoto H et al.,(2004)Cytotechnology 46:19-23;Naganawa Y et al.,(2005)Human Antibodies 14:27-31

[0238] kit Also provided herein are kits containing one or more antibodies described herein, or pharmaceutical compositions or conjugates thereof. In certain embodiments, provided herein are pharmaceutical packs or kits containing one or more containers filled with one or more components of the pharmaceutical compositions described herein, such as one or more antibodies provided herein. In certain embodiments, the kits include the pharmaceutical compositions described herein and any prophylactic or therapeutic agent, such as those described herein. In certain embodiments, the kits may include, for example, T cell mitogens such as phytohemagglutinin (PHA) and / or phorbol myristate acetate (PMA), or TCR complex-stimulating antibodies such as anti-CD3 and anti-CD28 antibodies. A notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, reflecting agency approval for manufacture, use, or sale for human administration, may optionally be associated with such container(s).

[0239] Also provided herein are kits that can be used in the above methods. In one embodiment, the kit comprises an antibody, preferably a purified antibody, described herein in one or more containers. In a specific embodiment, the kit described herein comprises substantially isolated CD96 (e.g., human CD96 or cynomolgus CD96) as a control. In another specific embodiment, the kit described herein further comprises a control antibody that does not react with the CD96 (e.g., human CD96 or cynomolgus CD96) antigen. In another specific embodiment, the kit described herein comprises one or more elements for detecting binding of the antibody to the CD96 (e.g., human CD96 or cynomolgus CD96) antigen (e.g., the antibody may be conjugated to a detectable substrate, such as a fluorescent compound, an enzymatic substrate, a radioactive compound, or a luminescent compound, or a secondary antibody that recognizes the primary antibody may be conjugated to a detectable substrate). In a specific embodiment, the kit provided herein may comprise a recombinantly produced or chemically synthesized CD96 (e.g., human CD96 or cynomolgus CD96) antigen. The CD96 (e.g., human CD96 or cynomolgus CD96) antigen provided in the kit can also be bound to a solid support. In a more specific embodiment, the detection means of the above-described kit comprises a solid support to which the CD96 (e.g., human CD96 or cynomolgus CD96) antigen is bound. Such a kit can also include an unbound reporter-labeled anti-human antibody or anti-mouse / rat antibody. In this embodiment, binding of the antibody to the CD96 (e.g., human CD96 or cynomolgus CD96) antigen can be detected by binding of the reporter-labeled antibody. In one embodiment, the invention relates to the use of the kit of the invention for in vitro assays and / or detection of CD96 (e.g., human CD96 or cynomolgus CD96) in biological samples. [Example]

[0240] The examples in this section (ie, Section 6) are presented by way of illustration and not by way of limitation.

[0241] Example 1: Characterization of anti-CD96 antibodies This example describes the characterization of antibodies that specifically bind to human CD96. The amino acid sequences of exemplary antibodies are shown in Table 1.

[0242] Anti-human CD96 antibodies bind to purified human and cynomolgus monkey CD96 proteins Binding of parental and germline anti-CD96 antibodies to His-tagged isoform 2 of human CD96 carrying the C89S mutation The binding affinity of the parent antibody BA072 and the germline variants BA083 and BA084 to full-length isoform 2 of human CD96 carrying the C89S mutation with a His tag (SEQ ID NO: 129) was assessed by surface plasmon resonance.

[0243] Briefly, surface plasmon resonance experiments were performed using a Biacore T200 instrument to determine the association rate (K a ), dissociation rate (K d ), and the dissociation constant (K D ) was calculated from each experiment using a 1:1 binding model with Biacore T200 evaluation software.

[0244] Approximately 4 μg / ml of BA072, BA083, BA084, BA0833, and BA0834 diluted in running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20) were captured onto individual flow cells of a Series S Protein A Sensor Chip (GE Healthcare Ltd, catalog number 29-1275-56) holding a single flow cell as a reference. Antibodies were captured using a 15-second injection at a flow rate of 10 μl / min to reach approximately 150 resonance units (RU). Full-length isoform 2 of human CD96 with a His-tag and C89S (SEQ ID NO: 129) mutation, diluted in running buffer at concentrations of 0.41, 1.23, 3.7, 11.1, 33.3, 100, and 300 nM, was flowed over the chip surface at a flow rate of 30 μl / min for a 3-minute association phase and either a 10-minute or 15-minute dissociation phase. The sensor chip was regenerated between cycles with a 30-second injection of 10 mM glycine, pH 1.5. Sensorgrams were evaluated and fitted to a simple Langmuir 1:1 interaction model using the global data analysis option in BIAevaluation 3.1 software. Data quality was assessed by visually inspecting deviations and curve fits and by R. max The binding kinetics (K a、 K d , and K. D ) were determined from sensorgram analysis and are shown in Table 3. [Table 3]

[0245] The binding affinity of the parent antibody BA101 and germline variants BA102, BA103, BA104, BA105, BA106, and BA107 to full-length isoform 2 of human CD96 carrying the C89S mutation with a His tag (SEQ ID NO: 129) was assessed by surface plasmon resonance.

[0246] Briefly, surface plasmon resonance experiments were performed using a Biacore T200 instrument to determine the association rate (Ka), dissociation rate (Kd), and dissociation constant (K D ) was calculated from each experiment using a 1:1 binding model with Biacore T200 evaluation software.

[0247] Approximately 4 μg / ml of BA102, BA103, BA104, BA105, BA106, and BA107 diluted in running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20) were captured onto individual flow cells of a Series S Protein A Sensor Chip (GE Healthcare Ltd, catalog number 29-1275-56) holding a single flow cell as a reference. Antibodies were captured using a 15-second injection at a flow rate of 10 μl / min to reach approximately 200 resonance units (RU). Full-length isoform 2 of human CD96 with a His-tag and C89S mutation (SEQ ID NO: 129) diluted in running buffer at concentrations of 0.41, 1.23, 3.7, 11.1, 33.3, 100, and 300 nM was flowed over the chip surface at a flow rate of 30 μl / min for a 3-minute association phase and either a 10-minute (for gl6) or 15-minute dissociation phase. The sensor chip was regenerated between cycles with a 30-second injection of 10 mM glycine, pH 1.5. Sensorgrams were evaluated and fitted to a simple Langmuir 1:1 interaction model using the global data analysis option in BIAevaluation 3.1 software. Data quality was assessed by visually inspecting deviations and curve fits and by using R. max The binding kinetics (Ka, Kd, ​​and K D ) were determined from sensorgram analysis and are shown in Table 4. [Table 4]

[0248] Binding of affinity-matured anti-CD96 antibodies to His-tagged domain 1 of human CD96 with the C89S mutation or His-tagged domain 1 of cynomolgus monkey CD96 The binding affinity of the parent antibody BA072, germline antibody BA083, and affinity-matured variants BA093, BA092, BA091, BA089, BA086, BA094, BA088, BA090, BA087, and BA085 to domain 1 of human CD96 with a C89S mutation and a His tag (SEQ ID NO: 131) or domain 1 of cynomolgus CD96 with a His tag (SEQ ID NO: 134) was assessed by surface plasmon resonance. Affinity-matured variants were selected for increased affinity to cynomolgus CD96.

[0249] Briefly, surface plasmon resonance experiments were performed using a Biacore T200 instrument to determine the association rate (K a ), dissociation rate (K d ), and the dissociation constant (K D ) was calculated from each experiment using a 1:1 binding model with Biacore T200 evaluation software.

[0250] Specifically, antibodies diluted in running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20) were captured onto individual flow cells of a Series S Protein A Sensor Chip (GE Healthcare Ltd, catalog number 29-1275-56), with a single flow cell held as a reference. Antibodies were captured using a 15-second injection at a flow rate of 10 μl / min to reach a capture level optimized for body analysis (approximately 430 RU). The antibody concentration to reach the optimal capture level was determined separately for each experiment (approximately 8 μg / ml was used for each antibody). Domain 1 of human CD96 with a C89S mutation containing a His tag (SEQ ID NO: 131) or domain 1 of cynomolgus monkey CD96 with a His tag (SEQ ID NO: 134) diluted in running buffer at concentrations of 0.41, 1.23, 3.7, 11.1, 33.3, 100, and 300 nM was flowed over the chip surface at a flow rate of 30 μl / min with a 3-minute association phase and a 15-minute dissociation phase. The sensor chip was regenerated between cycles with a 30-second injection of 10 mM glycine, pH 1.5. Sensorgrams were evaluated and fitted to a simple Langmuir 1:1 interaction model using the global data analysis option in BIAevaluation 3.1 software. Data quality was assessed by visually inspecting deviations and curve fits and by using R. max The binding kinetics (K a、 K d , and K. D ) were determined from sensorgram analysis and are shown in Table 5 (human) and Table 6 (cynomolgus monkey). [Table 5] [Table 6]

[0251] Binding of affinity-matured anti-CD96 antibodies to His-tagged domain 1 of human CD96 with the C89S mutation or His-tagged domain 1 of cynomolgus monkey CD96 The binding affinity of the parent antibody BA072, germline antibody BA083, and affinity-matured variants BA073, BA074, BA078, BA079, BA080, BA081, BA076, BA077, BA082, and BA075 to domain 1 of human CD96 with a C89S mutation and a His tag (SEQ ID NO: 131) or domain 1 of cynomolgus CD96 with a His tag (SEQ ID NO: 134) was assessed by surface plasmon resonance. Affinity-matured variants were selected for their increased affinity to cynomolgus CD96.

[0252] Briefly, surface plasmon resonance experiments were performed using a Biacore T200 instrument to determine the association rate (K a ), dissociation rate (K d ), and the dissociation constant (K D ) was calculated from each experiment using a 1:1 binding model with Biacore T200 evaluation software.

[0253] Specifically, antibodies diluted in running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20) were captured onto individual flow cells of a Series S Protein A Sensor Chip (GE Healthcare Ltd, catalog number 29-1275-56), with a single flow cell held as a reference. Antibodies were captured using a 15-second injection at a flow rate of 10 μl / min to reach a capture level optimized for body analysis (approximately 250 RU). The antibody concentration to reach the optimal capture level was determined separately for each experiment (approximately 4 μg / ml was used for each antibody). Domain 1 of human CD96 with a C89S mutation containing a His tag (SEQ ID NO: 131) or domain 1 of cynomolgus monkey CD96 with a His tag (SEQ ID NO: 134) diluted in running buffer at concentrations of 0.75, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 nM was flowed over the chip surface at a flow rate of 30 μl / min with a 3-minute association phase and a 15-minute dissociation phase. The sensor chip was regenerated between cycles with a 30-second injection of 10 mM glycine, pH 1.5. Sensorgrams were evaluated and fitted to a simple Langmuir 1:1 interaction model using the global data analysis option in BIAevaluation 3.1 software. Data quality was assessed by visually inspecting deviations and curve fits and by R. max The binding kinetics (K a、 K d , and K. D ) were determined from sensorgram analysis and are shown in Table 7 (human) and Table 8 (cynomolgus monkey). [Table 7] [Table 8]

[0254] Anti-human CD96 antibodies bind to cells expressing human and cynomolgus CD96 The ability of human anti-CD96 IgG1 antibodies to bind to cells expressing human CD96 or cynomolgus CD96 was tested on various cell types. Binding of anti-CD96 antibodies to Jurkat cells expressing human CD96 isoform 2

[0255] The ability of the parent antibodies BA072 and BA101 to bind to human CD96 isoform 2 expressed on the surface of Jurkat cells was evaluated. Briefly, Jurkat cells were transfected with a vector encoding full-length human CD96 isoform 2 (SEQ ID NO: 128), and clones stably expressing high levels of CD96 were selected. The stable cell line was cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 1% puromycin (R10 medium).

[0256] For antibody binding ass...

Claims

[Claim 1] 1. An isolated antibody that specifically binds to human CD96, the antibody comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) CDRH1, CDRH2, and CDRH3, and a light chain variable region (VL) comprising CDRs CDRL1, CDRL2, and CDRL3; (a) CDRH1 is X １ YX ２ X ３ X ４ (SEQ ID NO: 135), X １ is Q or S, X ２ is A or S, X ３ is M or I, X ４ is H or S, (b) CDRH2 is X １ IX ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ YX １０ QKFQG (SEQ ID NO: 137), X １ is W or G, X ２ is N or I, X ３ is A, E, V, or P; X ４ is V, G, W, or I; X ５ is S, Y, T, N, or F; X ６ is G or W, X ７ is D, Y, N, or T; X ８ is T or A, X ９ is K or N, X １０ is S or A, (c) CDRH3 is NWGX １ SYGX ２ DV (SEQ ID NO: 180), GYDSRPLDV (SEQ ID NO: 19), or GYDSRPLDY (SEQ ID NO: 20), X １ is M or L, X ２ is M or L, (d) CDRL1 is RASQSIX １ X ２ YLN (SEQ ID NO: 139) or GGNNIGSKIVH (SEQ ID NO: 26), X １ is S, T, or L; X ２ is S, P, or W; (e) CDRL2 is X １ X ２ comprising the amino acid sequence of SSLQS (SEQ ID NO: 141) or DDRDRPS (SEQ ID NO: 32), X １ is S or A, X ２ is A, S, or E, and / or (f) CDRL3 is QQX １ YSTPALX ２ (SEQ ID NO: 143) or QVWDINVHHVI (SEQ ID NO: 35), X １ is S or A, X ２ is T or S.

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