Anti-CD3 Antibodies, Bispecific Antigen-Binding Molecules that Bind CD3 and CD20, and Uses Thereof

Abstract

The present invention provides antibodies that bind to CD3 and methods of using the same. According to certain embodiments, the antibodies of the invention bind human CD3 with high affinity and induce human T cell proliferation. The invention includes antibodies that bind CD3 and induce T cell-mediated killing of tumor cells. According to certain embodiments, the present invention provides bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds human CD3, and a second antigen-binding molecule that specifically binds human CD20. In certain embodiments, the bispecific antigen-binding molecules of the present invention are capable of inhibiting the growth of B-cell tumors expressing CD20. The antibodies and bispecific antigen-binding molecules of the invention are useful for the treatment of diseases and disorders in which an upregulated or induced targeted immune response is desired and/or therapeutically beneficial. For example, the antibodies of the invention are useful for the treatment of various cancers as well as other CD20-related diseases and disorders.

What is claimed is: 
     
         1 . An isolated antibody or antigen-binding fragment thereof that binds human CD3 and induces human T cell proliferation in vitro with an EC 50  of less than about 0.33 pM. 
     
     
         2 . An isolated antibody or antigen-binding fragment thereof that binds human CD3 and induces T cell-mediated killing of tumor cells in vitro with an EC 50  of less than about 2.3 pM. 
     
     
         3 . The isolated antibody or antigen-binding fragment of  claim 2 , wherein the antibody or antigen-binding fragment thereof induces T cell-mediated killing of tumor cells in vitro with an EC 50  of less than about 1 pM. 
     
     
         4 . An isolated antibody or antigen-binding fragment thereof that binds human CD3 with a binding dissociation equilibrium constant (K D ) of less than about 1 nM as measured in a surface plasmon resonance assay at 25° C. 
     
     
         5 . The isolated antibody or antigen-binding fragment of  claim 4 , wherein the antibody or antigen-binding fragment thereof binds human CD3 with a K D  of less than about 500 pM as measured in a surface plasmon resonance assay at 25° C. 
     
     
         6 . The isolated antibody or antigen-binding fragment of  claim 5 , wherein the antibody or antigen-binding fragment thereof binds human CD3 with a K D  of less than about 100 pM as measured in a surface plasmon resonance assay at 25° C. 
     
     
         7 . An isolated antibody or antigen-binding fragment thereof that binds human CD3 with a dissociative half-life (t½) of greater than about 10 minutes as measured in a surface plasmon resonance assay at 25° C. 
     
     
         8 . The isolated antibody or antigen-binding fragment of  claim 7 , wherein the antibody or antigen-binding fragment thereof binds human CD3 with a t½ of greater than about 100 minutes as measured in a surface plasmon resonance assay at 25° C. 
     
     
         9 . The antibody or antigen-binding fragment of any one of  claims 1  to  8 , wherein the antibody or antigen-binding fragment thereof competes for binding to CD3 with a reference antibody comprising an HCVR/LCVR amino acid sequence pair as set forth in Table 1. 
     
     
         10 . The antibody or antigen-binding fragment of  claim 9 , wherein the reference antibody comprises an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10; 114/122; 514/522; 770/778; 1050/1234; and 1090/1234. 
     
     
         11 . The antibody or antigen-binding fragment of any one of  claims 1  to  8 , wherein the antibody or antigen-binding fragment thereof binds to the same epitope on CD3 as a reference antibody comprising an HCVR/LCVR amino acid sequence pair as set forth in Table 1. 
     
     
         12 . The antibody or antigen-binding fragment of  claim 11 , wherein the antibody or antigen-binding fragment thereof binds to the same epitope on CD3 as a reference antibody comprising an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10; 114/122; 514/522; 770/778; 1050/1234; and 1090/1234. 
     
     
         13 . An isolated antibody or antigen-binding fragment thereof that binds human CD3, wherein the antibody or antigen-binding fragment comprises: (a) the complementarity determining regions (CDRs) of a heavy chain variable region (HCVR) having an amino acid sequence as set forth in Table 1; and (b) the CDRs of a light chain variable region (LCVR) having an amino acid sequence as set forth in Table 1. 
     
     
         14 . The isolated antibody or antigen-binding fragment of  claim 13 , wherein the antibody or antigen-binding fragment comprises the heavy and light chain CDRs of a HCVR/LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 2/10; 114/122; 514/522; 770/778; 1050/1234; and 1090/1234. 
     
     
         15 . The isolated antibody or antigen-binding fragment of  claim 14 , wherein the antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, selected from the group consisting of: SEQ ID NOs: 4-6-8-12-14-16; 116-118-120-124-126-128; 516-518-520-524-526-528; 772-774-776-780-782-784; 1052-1054-1056-1236-1238-1240; and 1092-1094-1096-1236-1238-1240. 
     
     
         16 . An isolated antibody or antigen-binding fragment thereof that binds human CD3, wherein the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2; 114; 514; 770; 1050; and 1090; and (b) a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10; 122; 522; 778; 1234; and 1234. 
     
     
         17 . The isolated antibody or antigen-binding fragment of  claim 16 , wherein the antibody or antigen-binding fragment comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 2/10; 114/122; 514/522; 770/778; 1050/1234; and 1090/1234. 
     
     
         18 . A bispecific antibody comprising a first antigen-binding domain that binds human CD3 and a second antigen-binding domain that binds a target antigen, wherein the first antigen-binding domain is derived from the antibody or antigen-binding fragment of any one of  claims 1  to  17 . 
     
     
         19 . A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of  claims 1  to  17 , or the bispecific antibody of  claim 18 , and a pharmaceutically acceptable carrier or diluent. 
     
     
         20 . A method for upmodulating an immune response in a subject, the method comprising administering to the subject the pharmaceutical composition of  claim 19 . 
     
     
         21 . A method for treating a tumor in a subject, the method comprising administering the pharmaceutical composition of  claim 19  to a subject in need thereof. 
     
     
         22 . A bispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds human CD3, and a second antigen-binding domain that specifically binds human CD20. 
     
     
         23 . The bispecific antigen-binding molecule of  claim 22 , wherein the antigen-binding molecule binds human cells expressing human CD3 and cynomolgus monkey cells expressing cynomolgus CD3. 
     
     
         24 . The bispecific antigen-binding molecule of  claim 22 , wherein the antigen-binding molecule induces proliferation of human and cynomolgus peripheral blood mononuclear cells (PBMCs) in vitro. 
     
     
         25 . The bispecific antigen-binding molecule of  claim 22 , wherein the antigen-binding molecule induces IFN-gamma release and CD25 up-regulation in human whole blood. 
     
     
         26 . The bispecific antigen-binding molecule of  claim 22 , wherein the antigen-binding molecule induces T-cell mediated cytotoxicity of human B-cells. 
     
     
         27 . The bispecific antigen-binding molecule of  claim 26 , wherein the antigen-binding molecule induces T-cell mediated cytotoxicity of human B-cells that are resistant to anti-CD20-mediated cytotoxicity. 
     
     
         28 . The bispecific antigen-binding molecule of any one of  claims 22  to  27 , wherein a single administration of the bispecific antigen-binding molecule to a subject at a dose of about 0.01 mg/kg causes a reduction in the number of B cells in the subject below detectable levels by about day 1 after administration of the bispecific antigen-binding molecule to the subject. 
     
     
         29 . The bispecific antigen-binding molecule of  claim 28 , wherein the number of B-cells remains below detectable levels until at least about 14 days after administration of a single dose of about 0.01 mg/kg of the bispecific antigen-binding molecule to the subject. 
     
     
         30 . The bispecific antigen-binding molecule of  claim 28  or  29 , wherein the number of B-cells per microliter of blood drawn from the subject at about day 1 through about day 28 after administration of a single dose of about 0.01 mg/kg of the antigen-binding molecule to the subject is less than 25% the number of B-cells per microliter of blood drawn from the subject prior to administration of the bispecific antigen-binding molecule. 
     
     
         31 . The bispecific antigen-binding molecule of  claim 28  or  29 , wherein the number of B-cells per microliter of blood drawn from the subject at about day 1 through about day 56 after administration of a single dose of about 0.01 mg/kg of the antigen-binding molecule to the subject is less than 50% the number of B-cells per microliter of blood drawn from the subject prior to administration of the bispecific antigen-binding molecule. 
     
     
         32 . The bispecific antigen-binding molecule of any one of  claims 28  to  31 , wherein the number of T cells per microliter of blood drawn from the subject at about day 14 through about day 56 after administration of the antigen-binding molecule to the subject is equal to or greater than the number of T cells per microliter of blood drawn from the subject prior to administration of the bispecific antigen-binding molecule. 
     
     
         33 . The bispecific antigen-binding molecule of any one of  claims 22  to  32 , wherein the first antigen-binding domain that specifically binds human CD3 comprises the heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2 and A1-HCDR3) from a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1250, 1266, 1282, 1298, 1314 and 1329, and the light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) from a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1258, 1274, 1290, 1306, 1322 and 1333. 
     
     
         34 . The bispecific antigen-binding molecule of any one of  claims 22  to  32 , wherein the second antigen-binding domain that specifically binds human CD20 comprises the heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region (HCVR) comprising SEQ ID NO:1242, and the light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) from a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1258, 1274, 1290, 1306, 1322 and 1333. 
     
     
         35 . The bispecific antigen-binding molecule of any one of  claims 22  to  32 , wherein the first antigen-binding domain that specifically binds human CD3 comprises three heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2 and A1-HCDR3) and three light chain complementarity determining regions (A1-LCDR1, A1-LCDR2 and A1-LCDR3), wherein A1-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1252, 1268, 1284, 1300, 1316 and 1330; wherein A1-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:1254, 1270, 1286, 1302, 1318 and 1331; wherein A1-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1256, 1272, 1288, 1304, 1320 and 1332, wherein A1-LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1260, 1276, 1292, 1308, 1324 and 1334, wherein A1-LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1262, 1278, 1294, 1310, 1326 and 1335, and wherein A1-LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1264, 1280, 1296, 1312, 1328 and 1336. 
     
     
         36 . The bispecific antigen-binding molecule of any one of  claims 22  to  32 , wherein the second antigen-binding domain that specifically binds human CD20 comprises three heavy chain complementarity determining regions (A2-HCDR1, A2-HCDR2 and A2-HCDR3) and three light chain complementarity determining regions (A2-LCDR1, A2-LCDR2 and A2-LCDR3), wherein A2-HCDR1 comprises the amino acid sequence of SEQ ID NO:1244, wherein A2-HCDR2 comprises the amino acid sequence of SEQ ID NO:1246, wherein A2-HCDR3 comprises SEQ ID NO:1248, wherein A2-LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:1260, 1276, 1292, 1308, 1324 and 1334, wherein A2-LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1262, 1278, 1294, 1310, 1326 and 1335, and wherein A2-LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1264, 1280, 1296, 1312, 1328 and 1336. 
     
     
         37 . The bispecific antigen-binding molecule of any one of  claims 22  to  32 , wherein the first antigen-binding domain that specifically binds human CD3 comprises three heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2 and A1-HCDR3) and three light chain complementarity determining regions (A1-LCDR1, A1-LCDR2 and A1-LCDR3), and wherein the second antigen-binding domain that specifically binds human CD20 comprises three heavy chain complementarity determining regions (A2-HCDR1, A2-HCDR2 and A2-HCDR3) and three light chain complementarity determining regions (A2-LCDR1, A2-LCDR2 and A2-LCDR3);
 wherein A1-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1252, 1268, 1284, 1300, 1316 and 1330; wherein A1-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1254, 1270, 1286, 1302, 1318 and 1331; wherein A1-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1256, 1272, 1288, 1304, 1320 and 1332, wherein A1-LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1260, 1276, 1292, 1308, 1324 and 1334, wherein A1-LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1262, 1278, 1294, 1310, 1326 and 1335, and wherein A1-LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1264, 1280, 1296, 1312, 1328 and 1336; and 
 wherein A2-HCDR1 comprises the amino acid sequence of SEQ ID NO:1244, wherein A2-HCDR2 comprises the amino acid sequence of SEQ ID NO:1246, wherein A2-HCDR3 comprises SEQ ID NO:1248, wherein A2-LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1260, 1276, 1292, 1308, 1324 and 1334, wherein A2-LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1262, 1278, 1294, 1310, 1326 and 1335, and wherein A2-LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1264, 1280, 1296, 1312, 1328 and 1336. 
 
     
     
         38 . The bispecific antigen-binding molecule of any one of  claims 22  to  32 , wherein the first antigen-binding domain competes for binding to human CD3 with a reference antigen-binding protein comprising three heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2 and A1-HCDR3) and three light chain complementarity determining regions (A1-LCDR1, A1-LCDR2 and A1-LCDR3), wherein A1-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1252, 1268, 1284, 1300, 1316 and 1330; wherein A1-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1254, 1270, 1286, 1302, 1318 and 1331; wherein A1-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1256, 1272, 1288, 1304, 1320 and 1332, wherein A1-LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1260, 1276, 1292, 1308, 1324 and 1334, wherein A1-LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1262, 1278, 1294, 1310, 1326 and 1335, and wherein A1-LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1264, 1280, 1296, 1312, 1328 and 1336. 
     
     
         39 . The bispecific antigen-binding molecule of any one of  claims 22  to  32 , wherein the first antigen-binding domain competes for binding to human CD3 with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1250, 1266, 1282, 1298, 1314 and 1329, and a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1258, 1274, 1290, 1306, 1322 and 1333. 
     
     
         40 . The bispecific antigen-binding molecule of any one of  claims 22  to  32 , wherein the second antigen-binding domain competes for binding to human CD20 with a reference antigen-binding protein comprising three heavy chain complementarity determining regions (A2-HCDR1, A2-HCDR2 and A2-HCDR3) and three light chain complementarity determining regions (A2-LCDR1, A2-LCDR2 and A2-LCDR3), wherein A2-HCDR1 comprises the amino acid sequence of SEQ ID NO:1244, wherein A2-HCDR2 comprises the amino acid sequence of SEQ ID NO:1246, wherein A2-HCDR3 comprises SEQ ID NO:1248, wherein A2-LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1260, 1276, 1292, 1308, 1324 and 1334, wherein A2-LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1262, 1278, 1294, 1310, 1326 and 1335, and wherein A2-LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1264, 1280, 1296, 1312, 1328 and 1336. 
     
     
         41 . The bispecific antigen-binding molecule of any one of  claims 22  to  32 , wherein the second antigen-binding domain competes for binding to human CD20 with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:1242, and a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1258, 1274, 1290, 1306, 1322 and 1333. 
     
     
         42 . The bispecific antigen-binding molecule of any one of  claims 22  to  32 , wherein the first antigen-binding domain competes for binding to human CD3 with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1250, 1266, 1282, 1298, 1314 and 1329, and a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1258, 1274, 1290, 1306, 1322 and 1333; and wherein the second antigen-binding domain competes for binding to human CD20 with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:1242, and a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1258, 1274, 1290, 1306, 1322 and 1333. 
     
     
         43 . A pharmaceutical composition comprising a bispecific antigen-binding molecule of any one of  claims 22  to  42  and a pharmaceutically acceptable carrier or diluent. 
     
     
         44 . A method for treating a B-cell cancer in a subject, the method comprising administering to the subject the pharmaceutical composition of  claim 43 . 
     
     
         45 . The method of  claim 44 , wherein the B-cell cancer is selected from the group consisting of: follicular lymphoma, B cell chronic lymphocytic leukemia, B cell lymphoblastic lymphoma, Hodgkin lymphoma, Non-Hodgkin lymphoma, diffuse large B cell lymphoma, marginal zone lymphoma, Mantle cell lymphoma, hairy cell leukemia and Burkitt lymphoma. 
     
     
         46 . The method of  claim 44  or  45 , wherein the subject is afflicted with a tumor that is resistant to, or incompletely responsive to anti-CD20 monospecific therapy alone. 
     
     
         47 . The method of  claim 46 , wherein the subject is afflicted with a tumor that is resistant to, or incompletely responsive to rituximab monotherapy. 
     
     
         48 . The method of any one of  claims 44  through  47 , wherein the subject has received an anti-CD20 monospecific antibody therapy at least 1 day to 1 year prior to the administration of the pharmaceutical composition. 
     
     
         49 . The method of  claim 48 , wherein the anti-CD20 monospecific therapy comprises or consists of an anti-CD20 mono-specific antibody. 
     
     
         50 . The method of  claim 49 , wherein the anti-CD20 mono-specific antibody is rituximab. 
     
     
         51 . A method for treating a B-cell cancer in a subject, the method comprising: (a) selecting a subject who is afflicted with a tumor that is resistant to, or incompletely responsive to anti-CD20 monospecific therapy alone; and (b) administering to the subject the pharmaceutical composition of  claim 43 . 
     
     
         52 . The method of  claim 51 , wherein the subject is selected on the basis of having a tumor that is resistant to, or incompletely responsive to rituximab monotherapy.