Bispecific antibodies

Abstract

The present invention relates to engineered heteromultimeric proteins, and more specifically, to methods for producing and purifying heterodimeric proteins, such as bispecific antibodies and. Methods for producing and purifying such engineered heterodimeric proteins and their use in diagnostics and therapeutics are also provided. The present invention also relates to a humanized antibody that specifically binds human TrkB and methods for producing and using the antibody to, inter alia, treat a hearing loss disorder.

1 . A heterodimeric protein, comprising
 (i) a first C H 1 domain (C H 1) and a first C L  domain (C L ), the first C H 1 and the first C L  interacting together at a first C H C L  interface to form a first C H C L  domain (C H C L );   (ii) a second C H 1 domain (C H 1) and a second C L  domain (C L ), the second C H 1 and the second C L  interacting together at a second C H C L  interface to form a second C H C L  domain (C H C L );   wherein the first C H 1 is engineered to differ from the second C H 1 by at least one C H 1 mutant residue in the first C H 1; and the first C L  is engineered to differ from the second C L  by at least one C L  mutant residue in the first C L ;   
       such that the C H 1 mutant residue and the C L  mutant residue of the first C H C L  interact with each other in preference to the corresponding residue positions on the second C H C L , the interacting mutant residues of the first C H 1 and first C L  thereby forming a first complementary residue set,
 and wherein the first C H 1 is attached to a first variable heavy domain (V H ), and the first C L  is attached to a first variable light domain (V L ), and the second C H 1 is attached to a second V H , and the second C L  is attached to a second V L , such that when combined, the first V H , first V L , first C H  and first C L  together form a first Fab, and when combined, the second V H , second V L , second C H 1, and second C L  form a second Fab, 
 and wherein preferential formation of the first Fab and the second Fab does not rely on complementary pairing of the variable domains. 
 
     
     
         2 - 24 . (canceled) 
     
     
         25 . An isolated antibody that specifically binds human TrkB, wherein the antibody comprises a V H  region comprising the amino acid of SEQ ID NO:51 and a V H  region comprising the amino acid sequence of SEQ ID NO:53. 
     
     
         26 - 29 . (canceled) 
     
     
         30 . A heterodimeric protein, comprising
 (i) a first C H 1 domain (C H 1) and a first C L  domain (C L ), the first C H 1 and the first C L  interacting together at a first C H C L  interface to form a first C H C L  domain (C H C L );   (ii) a second C H 1 domain (C H 1) and a second C L  domain (C L ), the second C H 1 and the second C L  interacting together at a second C H C L  interface to form a second C H C L  domain (C H C L );   wherein the first C H 1 is engineered to differ from the second C H 1 by at least one C H 1 mutant residue in the first C H 1; and   wherein the first C L  is engineered to differ from the second C L  by at least one C L  mutant residue in the first C L ;   such that the at least one C H 1 mutant residue in the first C H 1 and the at least one C L  mutant residue in the first C L  interact with each other in preference to a corresponding at least one C H 1 mutant residue in the second C H 1 and at least one C L  mutant residue in the second C L ;   wherein the interacting mutant residues of the first C H 1 and first C L  thereby form a first complementary residue set;   wherein the location of the first complementary residue set is selected from the group consisting of:   (i) C H 1-188 and C L -178;   (ii) C H 1-143 and C L -178;   (iii) C H 1-143 and C L -131;   (iv) C H 1-145 and C L -131;   (v) C H 1-179 and C L -131; and   (vi) C H 1-188 and C L -133   wherein the at least one C H 1 mutant residue at position C H 1-188 is selected from the group consisting of D, E, G, H, K, R, W and Y;   wherein the at least one C H 1 mutant residue at position C H 1-143 is selected from the group consisting of D, E, H, K, R, S and T;   Docket No. PC71995B wherein the at least one C H 1 mutant residue at position C H 1-145 is selected from the group consisting of D, E and S;   wherein the at least one C H 1 mutant residue at position C H 1-179 is selected from the group consisting of D and E;   wherein the at least one C L  mutant residue at position C L -178 is selected from the group consisting of D, E, G, K, R and S;   wherein the at least one C L  mutant residue at position C L -131 is selected from the group consisting of D, E, H and R;   wherein the at least one C L  mutant residue at position C L -133 is selected from the group consisting of M, Q and S;   wherein the first C H 1 is attached to a first variable heavy domain (V H ), and the first C L  is attached to a first variable light domain (V L ), and the second C H 1 is attached to a second V H , and the second C L  is attached to a second V L , such that when combined, the first V H , first V L , first C H 1 and first C L  together form a first Fab, and when combined, the second V H , second V L , second C H 1, and second C L  form a second Fab;   and wherein preferential formation of the first Fab and the second Fab does not rely on complementary pairing of the variable domains.   
     
     
         31 . The heterodimeric protein of  claim 30 , wherein the solvent accessible surface area of the first complementary residue set is less than 225 Å 2  as measured using a 2.5 Å probe. 
     
     
         32 . The heterodimeric protein of  claim 30 ,
 wherein the second C H 1 is engineered to differ from the first C H 1 by at least one C H 1 mutant residue in the second C H 1;   wherein the second C L  is engineered to differ from the first C L  by at least one C L  mutant residue in the second C L ;   such that the at least one C H 1 mutant residue in the second C H 1 and the at least one C L  mutant residue in the second C L  interact with each other in preference to the corresponding at least one C H 1 mutant residue in the first C H 1 and the at least one C L  mutant residue in the first C L ; and   wherein the interacting mutant residues of the second C H 1 and second C L  thereby form a second complementary residue set.   
     
     
         33 . The heterodimeric protein of  claim 32 , wherein the solvent accessible surface area of the second complementary residue set is less than 225 Å 2  as measured using a 2.5 Å probe 
     
     
         34 . The heterodimeric protein of  claim 30 , wherein formation of the first C H C L  and the second C H C L  preferentially occur over formation of a C H C L  comprised of either the first C H 1 and the second C L , or the second C H 1 and the first C L , by at least about 4-fold. 
     
     
         35 . The heterodimeric protein of  claim 32 , wherein at least one of the C L  domains is a kappa domain. 
     
     
         36 . The heterodimeric protein of  claim 32 ,
 wherein the first complementary residue set comprises a positively or negatively charged residue in the first C H 1 or the first C L , and either a polar residue, or an oppositely charged residue in the other domain; and   wherein the second complementary residue set comprises a positively or negatively charged residue in the second C H 1 or the second C L , and either a polar residue, or an oppositely charged residue in the other domain.   
     
     
         37 . The heterodimeric protein of  claim 32 , wherein the first and the second complementary residue sets are selected from two of the following groups:
 (i) C H 1-188E, C L -178K, C H 1-143E;   (ii) C H 1-188K, C L -178D, C H 1-143D;   (iii) C H 1-143K, C L -178D;   (iv) C H 1-143D, C L -178R;   (v) C H 1-143K, C L -178D;   (vi) C H 1-143D, C L -178K;   (vii) C H 1-143D, C L -178K, C L -176M;   (viii) C H 1-143E, C L -131R;   (ix) C H 1-143R, C L -131E, C H 1-186A;   (x) C H 1-188W, C H 1-143S, C L -133S, C L -178S, C L -131D;   (xi) C H 1-188W, C H 1-143S, C L -133S, C L -178G;   (xii) C H 1-188W, C H 1-143S, C L -133Q, C L -178G, C L -188H;   (xiii) C H 1-188W, C H 1-143S, C L -133M, C L -178G, C L -176G;   (xiv) C H 1-145E, C H 1-122C, C H 1-230C, C L -131H, C L -123C, C L -214S;   (xv) C H 1-143H, C H 1-179D, C H 1-186E, C H 1-174C, C H 1-190I, C H 1-230S, C L -131H, C L -135I, C L -176C, C L -214S;   (xvi) C H 1-145S, C H 1-186E, C H 1-139C, C H 1-230S, C L -131H, C L -116C, C L -214S;   (xvii) C H 1-188W, C H 1-143S, C H 1-174C, C H 1-230S, C L -133S, C L -178S, C L -131D, C L -176C, C L -214S;   C H 1-188W, C H 1-143S, C H 1-122C, C H 1-230S, C L -133M, C L -178G, C L -176G, C L -123C, C L -214S;   (xix) C H 1-188W, C H 1-143S, C H 1-122C, C H 1-139C, C H 1-174C, C H 1-230S, C L -133S, C L -178S, C L -131D, C L -116C, C L -123C, C L -176C, C L -214S;   (xx) C H 1-143R, C L -131E;   (xxi) C H 1-145E, C L -131H;   (xxii) C H 1-143H, C H 1-179D, C H 1-186E, C L -131H;   (xxiii) C H 1-145S, C H 1-186E, C L -131H;   (xxiv) C H 1-143S, C L -131D, C H 1-188W, C L -133S, C L -178S;   (xxv) C H 1-143S, C H 1-188W, C L -133M, C L -176G, C L -178G;   (xxvi) C H 1-143S, C L -131D, C H 1-188W, C L -133S, C L -176C;   (xxvii) C H 1-143S, C H 1-188W, C L -133M, C L -178G, C L -176G;   (xxviii) C H 1-143S, C H 1-188W, C L -131D;   (xxix) C H 1-143S, C L -131D, C H 1-188W, C L -133S, C L -178S, C H 1-174C, C H 1-230S, C L -176C, C L -214S;   (xxx) C H 1-143T, C H 1-188W, C L -131D; and   (xxxi) C H 1-143T, C H 1-188W, C L -131E.   
     
     
         38 . The heterodimeric protein of  claim 37 , wherein the first and second complementary residue sets further comprise one or more mutations selected from the group consisting of: C H 1-143D, C H 1-145S, C H 1-186A, C H 1-186E, C H 1-188G, C H 1-143S, C H 1-190S, C H 1-190I, C L -133S, C L -135I, C L -176G, C L -176M, C L -178G and C L -178S. 
     
     
         39 . The heterodimeric protein of  claim 30 , comprising an engineered disulfide bond between the first C H 1 and the first C L , and/or the second C H 1 and the second C L . 
     
     
         40 . The heterodimeric protein of  claim 39 , wherein the engineered disulfide bond is located at one or more of the following positions: C H 1-122 and C L -123; C H 1-139 and C L -116; and C H 1-174 and C L -176. 
     
     
         41 . The heterodimeric protein of  claim 30 , wherein a wild type disulfide bond has been removed by mutating one or both of C H 1-C230 and C L -C214 to any residue except C, on the first C H C L  and/or second C H C L , and wherein the first and/or second C H 1-C230 and first, and/or second C L -C214 are mutated to S. 
     
     
         42 . An isolated nucleic acid encoding the heterodimeric protein of  claim 30 . 
     
     
         43 . The isolated nucleic acid of  claim 42 , wherein the isolated nucleic acid encodes the first C H 1, the first C L , the second C H 1, the second C L , the first V H , the first V L , the second V H , the second V L , or a combination thereof. 
     
     
         44 . A vector comprising the nucleic acid of  claim 42 . 
     
     
         45 . A cell comprising the nucleic acid of  claim 42 . 
     
     
         46 . A cell comprising the vector of  claim 44 . 
     
     
         47 . A method of making the heterodimeric protein of  claim 30 , comprising:
 (i) cotransfecting a cell line with one or more vectors to express the first C H 1 and the first C L  of the first C H C L  and the second C H 1 and the second C L  of the second C H C L ;   (ii) culturing the cell line under conditions to express the one or more vectors and that allow the first C H C L  and the second C H C L  to assemble; and   (iii) purifying the heterodimeric protein from the cell culture.   
     
     
         48 . A bispecific antibody comprising a heterodimeric protein, comprising
 (i) a first C H 1 domain (C H 1) and a first C L  domain (C L ), the first C H 1 and the first C L  interacting together at a first C H C L  interface to form a first C H C L  domain (C H C L );   (ii) a second C H 1 domain (C H 1) and a second C L  domain (C L ), the second C H 1 and the second C L  interacting together at a second C H C L  interface to form a second C H C L  domain (C H C L );   wherein the first C H C L  comprises C H 1-124K, C L -176D, C H 1-190S, and C L -133S; and the second C H C L  comprises C H 1-124E, C L -176K, C H 1-188G, and C L -133S.   
     
     
         49 . A pharmaceutical composition comprising the bispecific antibody of  claim 48  and a pharmaceutically acceptable carrier.