Readily Isolated Bispecific Antibodies with Native Immunoglobulin Format

Abstract

A bispecific antibody format providing ease of isolation is provided, comprising immunoglobulin heavy chain variable domains that are differentially modified in the CH3 domain, wherein the differential modifications are non-immunogenic or substantially non-immunogenic with respect to the CH3 modifications, and at least one of the modifications results in a differential affinity for the bispecific antibody for an affinity reagent such as Protein A, and the bispecific antibody is isolable from a disrupted cell, from medium, or from a mixture of antibodies based on its affinity for Protein A.

1 - 19 . (canceled) 
     
     
         20 . An IgG bispecific antibody that comprises a binding specificity toward a tumor antigen and a T-cell antigen comprising:
 a. a first polypeptide that comprises a first CH2 region and a first CH3 region, and   b. a second polypeptide that comprises a second CH2 region and a second CH3 region,   wherein there is at least one amino acid sequence modification in the first CH2 region or first CH3 region and the second CH2 region or second CH3 region that decreases the affinity of said IgG bispecific antibody to a human Fcγ receptor.   
     
     
         21 . The bispecific antibody of  claim 20 , wherein the T-cell antigen is CD3. 
     
     
         22 . The bispecific antibody of  claim 20 , where the IgG bispecific antibody is human or humanized. 
     
     
         23 . The bispecific antibody of  claim 20 , that is a human IgG selected from IgG1, IgG2 and IgG4. 
     
     
         24 . The bispecific antibody of  claim 20 , wherein the human Fcγ receptor is selected from FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and FcγRIIIB. 
     
     
         25 . The bispecific antibody of  claim 20 , wherein the decreased affinity of the IgG bispecific antibody to a human Fcγ receptor is assessed by a surface plasmon resonance technique. 
     
     
         26 . The bispecific antibody of  claim 21 , wherein the first and second CH3 regions comprise an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO: 5. 
     
     
         27 . The bispecific antibody of  claim 21 , where the first and second CH3 regions have amino acids sequences that are different from one another. 
     
     
         28 . The bispecific antibody of  claim 21 , where the first and second CH3 regions have amino acids sequences that are the same. 
     
     
         29 . The bispecific antibody of  claim 20 , wherein said modification includes modifications and combinations thereof, with reference to EU numbering of an immunoglobulin constant region, at one or more of the following amino acid positions: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, 383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438 and 439. 
     
     
         30 . The bispecific antibody of  claim 29 , wherein the modification is at position 238. 
     
     
         31 . The bispecific antibody of  claim 29 , wherein the modification is at position 239. 
     
     
         32 . The bispecific antibody of  claim 29 , wherein the modification is at position 265. 
     
     
         33 . The bispecific antibody of  claim 29 , wherein the modification is at position 267. 
     
     
         34 . The bispecific antibody of  claim 29 , wherein the modification is at position 269. 
     
     
         35 . The bispecific antibody of  claim 29 , wherein the modification is at position 270. 
     
     
         36 . The bispecific antibody of  claim 29 , wherein the modification is at position 295. 
     
     
         37 . The bispecific antibody of  claim 29 , wherein the modification is at position 296. 
     
     
         38 . The bispecific antibody of  claim 29 , wherein the modification is at position 297. 
     
     
         39 . The bispecific antibody of  claim 29 , wherein the modification is at position 298. 
     
     
         40 . The bispecific antibody of  claim 29 , wherein the modification is at position 327. 
     
     
         41 . The bispecific antibody of  claim 29 , wherein the modification is at position 328. 
     
     
         42 . The bispecific antibody of  claim 29 , wherein the modification is at position 329. 
     
     
         43 . The bispecific antibody of  claim 29 , wherein the modification is at position 330. 
     
     
         44 . The bispecific antibody of  claim 29 , wherein the modification is at position 331. 
     
     
         45 . The bispecific antibody of  claim 29 , wherein the modification is at position 332. 
     
     
         46 . The bispecific antibody of  claim 29 , that has a binding specificity to CD20 and CD3. 
     
     
         47 . An IgG bispecific antibody that comprises a binding specificity toward a tumor antigen and a T-cell antigen comprising:
 a. a first polypeptide that comprises a first CH2 region and a first CH3 region, and   b. a second polypeptide that comprises a second CH2 region and a second CH3 region,   wherein there is at least one amino acid sequence modification in the first CH2 region or first CH3 region and the second CH2 region or second CH3 region that decreases the affinity of said IgG bispecific antibody to a human Fcγ receptor;   wherein the T-cell antigen is CD3;   wherein said modification includes modifications and combinations thereof, with reference to EU numbering of an immunoglobulin constant region, at one or more of the following amino acid positions: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, 383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438 and 439.   
     
     
         48 . An IgG bispecific antibody that comprises a binding specificity toward a tumor antigen and a T-cell antigen comprising:
 a. a first polypeptide that comprises a first CH2 region and a first CH3 region, and   b. a second polypeptide that comprises a second CH2 region and a second CH3 region,   wherein there is at least one amino acid sequence modification in the first CH2 region or first CH3 region and the second CH2 region or second CH3 region that decreases the affinity of said IgG bispecific antibody to a human Fcγ receptor;   wherein the T-cell antigen is CD3;   wherein said modification includes a modification, with reference to EU numbering of an immunoglobulin constant region, of the amino acid at position 297.   
     
     
         49 . The IgG bispecific antibody of  claim 48 , wherein the IgG bispecific antibody is IgG2. 
     
     
         50 . An IgG bispecific antibody that comprises a binding specificity toward a tumor antigen and a T-cell antigen comprising:
 a. a first polypeptide that comprises a first CH2 region and a first CH3 region, and   b. a second polypeptide that comprises a second CH2 region and a second CH3 region,   wherein there is at least one amino acid sequence modification in the first CH2 region and the second CH2 region that decreases the affinity of said IgG bispecific antibody to a human Fcγ receptor;   wherein the first and second CH3 regions comprise an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO: 5;   wherein the T-cell antigen is CD3;   wherein said modification includes modifications and combinations thereof, with reference to EU numbering of an immunoglobulin constant region, at one or more of the following amino acid positions: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339 and 340.   
     
     
         51 . A pharmaceutical composition, wherein the pharmaceutical composition comprises as an active ingredient the bispecific antibody of  claim 20 ,  claim 48 , or  claim 50 .