Optimized Anti-CD3 Bispecific Antibodies and Uses Thereof

Abstract

The present invention provides antibodies that bind to CD3 with weak or no detectable binding affinity and methods of using the same. According to certain embodiments, the antibodies of the invention bind human CD3 with low affinity and induce human T cell proliferation and hence induce T cell-mediated killing of tumor cells with high efficacy. According to certain embodiments, the present invention provides bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds human CD3 with weak or no detectable binding affinity in an in vitro assay, and a second antigen-binding molecule that specifically binds human tumor-associated antigen. In certain embodiments, the bispecific antigen-binding molecules of the present invention are capable of inhibiting the growth of tumors expressing target antigen, such as PSMA. 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 or other diseases where immunotherapy, i.e. effector cell immunomodulation is warranted.

What is claimed: 
     
         1 . A cytotoxic composition comprising a bispecific antigen-binding molecule that (i) specifically binds an effector cell with an EC 50  of greater than about 100 nM and ii) specifically binds a target tumor cell, wherein the EC 50  is a binding affinity value measured in an in vitro FACS binding assay. 
     
     
         2 . The composition of  claim 1 , wherein the antigen-binding molecule specifically binds human CD3 with an EC 50  of greater than 500 nM or greater than about 1 μM. 
     
     
         3 . The composition of  claim 1 , wherein the bispecific antigen-binding molecule comprises a first antigen-binding fragment (Fab1), derived from a first antibody, that is not capable of specifically binding to an effector cell, as measured in an in vitro FACS binding assay or an in vitro surface plasmon resonance binding assay. 
     
     
         4 . The composition of  claim 1 , wherein the bispecific antigen-binding molecule exhibits no detectable binding to an effector cell or to CD3 antigen. 
     
     
         5 . The composition of any one of  claims 1 - 4 , wherein the bispecific antigen-binding molecule comprises a second antigen-binding fragment (Fab2) derived from a second antibody that specifically binds the target tumor cell with an EC 50  value of less than about 50 nM. 
     
     
         6 . The composition of any one of  claims 1 - 5 , wherein the bispecific antigen-binding molecule specifically binds each of human CD3 and cynomolgus CD3 with an EC 50  value of greater than about 500 nM, or greater than about 1 μM. 
     
     
         7 . The composition of any one of  claims 1 - 6 , wherein the target tumor cell is a human tumor cell. 
     
     
         8 . The composition of any one of  claims 1 - 7 , wherein the bispecific antigen-binding molecule induces T cell-mediated tumor cell killing with an EC 50  value of less than about 1.3 nM, as measured in an in vitro T cell-mediated tumor cell killing assay. 
     
     
         9 . The composition of any one of  claims 1 - 8 , wherein the target tumor cell expresses a tumor-associated antigen selected from the group consisting of AFP, ALK, BAGE proteins, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3. 
     
     
         10 . The composition of any one of  claims 1 - 9 , wherein the tumor-associated antigen is CD20, EGFRvIII, PSMA (FOLH1), STEAP2, or MUC16. 
     
     
         11 . The composition of any one of  claims 1 - 10 , wherein the bispecific antigen-binding molecule comprises a first heavy chain comprising: a CDR1 comprising amino acid residues 1-7 of SEQ ID NO:178, a CDR2 comprising amino acid residues 1-7 of SEQ ID NO:179, and/or a CDR3 comprising amino acid residues 4-11 of SEQ ID NO:180. 
     
     
         12 . The composition of  claim 11 , wherein the first heavy chain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12 or 20. 
     
     
         13 . The composition of any one of  claim 11  or  12 , wherein the first heavy chain comprises a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 or 54. 
     
     
         14 . The composition of any one of  claims 11  to  13 , wherein the first heavy chain comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 136, SEQ ID NO: 144, or SEQ ID NO: 152. 
     
     
         15 . The composition of any one of  claims 11  to  14 , wherein the first heavy chain comprises variable domain framework regions having an amino acid sequence selected from FR1 (SEQ ID NO: 174), FR2 (SEQ ID NO: 175), FR3 (SEQ ID NO: 176), and FR4 (SEQ ID NO: 177). 
     
     
         16 . The composition of any one of  claims 1 - 11 , wherein the bispecific antigen-binding molecule comprises a HCVR and LCVR amino acid sequence pair (HCVR/LCVR) selected from the group consisting of: SEQ ID NOs: 2/162; 10/162; 18/162; 26/162; 34/162; 42/162; 50/162; 58/162; 66/162; 74/162; 82/162; 90/162; 98/162; 106/162; 114/162; 122/162; 130/162; 138/162; 146/162. 
     
     
         17 . The composition of any one of  claims 1 - 11 , wherein the bispecific antigen binding molecule comprises a first heavy chain comprising a HCVR comprising HCDR1-HCDR2-HCDR3 domains having the amino acid sequences of SEQ ID NOs: 178-179-180, respectively. 
     
     
         18 . A method of making the composition of any one of  claims 1 - 17 , the method comprising:
 a. identifying the amino acid sequence of a first heavy chain derived from a first antibody that specifically binds CD3 with an EC 50  value of less than about 40 nM,   b. modifying selected amino acid residues in the heavy chain variable region of the first antibody to produce a modified antibody,   c. pairing the modified antibody with a second heavy chain derived from a second antibody that specifically binds a target tumor antigen to produce a bispecific antibody,   d. testing the bispecific antibody in a binding affinity assay, and if the binding affinity to CD3 has an EC 50  value of greater than about 500 nM, then   e. preparing a composition comprising the bispecific antibody and a pharmaceutically acceptable carrier or diluent.   
     
     
         19 . The method of  claim 18 , wherein the first antibody heavy chain comprises a first C H 3 domain and the second antibody comprises a second C H 3 domain suitable for bispecific pairing and isolation. 
     
     
         20 . The method of  claim 19 , wherein the first and second C H 3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. 
     
     
         21 . A pharmaceutical composition comprising the composition of any one of  claims 1 - 17  or made by the method of any one of  claims 18 - 20 , comprising a pharmaceutically acceptable carrier or diluent. 
     
     
         22 . A method for treating cancer in a subject, the method comprising administering to the subject the pharmaceutical composition of  claim 21 . 
     
     
         23 . The method of  claim 22 , wherein the cancer is selected from the group consisting of: pancreatic cancer, melanoma, glioblastoma, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, melanomaglioma, breast cancer, squamous cell carcinoma, esophageal cancer, clear cell renal cell carcinoma, chromophobe renal cell carcinoma, renal oncocytoma, renal transitional cell carcinoma, urothelial carcinoma, adenocarcinoma, or small cell carcinoma. 
     
     
         24 . The method of  claim 22  or  23 , wherein the subject is afflicted with a tumor that is resistant to, or incompletely responsive to monospecific therapy alone. 
     
     
         25 . A method for mediating tumor cell lysis, or suppressing tumor growth in a subject, the method comprising administering to the subject a therapeutic amount of the composition of  claim 21 . 
     
     
         26 . The method of  claim 25 , wherein the amount is sufficient to reduce tumor burden, produce tumor regression, inhibit tumor growth or reduce tumor development in the subject. 
     
     
         27 . The method of  claim 26 , wherein subject is afflicted with a tumor expressing a tumor-associated antigen selected from the group consisting of AFP, ALK, BAGE proteins, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3. 
     
     
         28 . Use of a cytotoxic bispecific antibody having reduced clearance for suppression of tumor growth in a subject, wherein the bispecific antibody comprises no detectable binding affinity to an effector cell or weak binding affinity to an effector cell at least greater than 200 EC 50  or K D , and specifically binds with high affinity to a target tumor cell, wherein binding affinity is measured in an in vitro FACS binding assay or in vitro surface plasmon resonance assay. 
     
     
         29 . A method of producing a cytotoxic bispecific antibody, comprising:
 a. identifying a first human antibody or antigen-binding fragment thereof that interacts with an effector cell antigen from multiple species;   b. identifying the germline amino acid residues of the heavy chain variable region (HCVR) of the first human antibody;   c. comparing the amino acid sequence of the HCVR of the first human antibody to the amino acid sequence of the corresponding germline HCVR;   d. identifying amino acids within a modified region of the HCVR of the first human antibody; whereby a modified region in the first human antibody displays at least one amino acid modification by substitution, deletion or addition of a single amino acid residue compared to the same region in the germline HCVR;   e. producing a plurality of modified antibodies each comprising at least one modified region of the HCVR;   f. screening each of the plurality of modified antibodies for monovalent affinity to the effector cell antigen;   g. selecting those modified antibodies that exhibit weaker binding affinity for the effector cell antigen compared to the first human antibody; and   h. pairing a selected antibody with a second antibody that interacts with a tumor-associated antigen to produce a cytotoxic bispecific antibody.