Methods for Producing Fabs and IgG Bispecific Antibodies

Abstract

Methods for producing Fabs and IgG bi-specific antibodies comprising expressing nucleic acids encoding designed residues in the CH1/CL interface are provided. Also provided are Fabs and IgG bi-specific antibodies produced according to the provided methods as well as nucleic acids, vectors and host cells encoding the same.

We claim: 
     
         1 . A first and second fragment, antigen binding (Fab) comprising:
 a first heavy chain variable domain (V H ), a first human IgG heavy chain constant (C H 1) domain having an alanine at residue 145 according to Kabat Numbering and an alanine or a glycine at residue 188 according to Kabat Numbering, a first light chain variable (V L ) domain, and a first human light chain kappa constant (Ck) domain having an arginine at residue 131 according to Kabat Numbering and an isoleucine at residue 176 according to Kabat Numbering; and   a second heavy chain variable (V H ) domain, a second human constant C H 1 domain, a second light chain variable (V L ) domain, and a second human light chain kappa (Ck) domain, wherein   each of the first V H  domain and the first V L  domain comprise three complementarity determining regions (CDRs) which direct binding to a first antigen,   each of the second V H  domain and the second V L  domain comprise three CDRs which direct binding to a second antigen that differs from the first antigen, and   (i) the second human C H 1 domain has the wild-type human IgG C H 1 sequence and the second human Ck domain has the wild type human Ck sequence,   (ii) the second human C H 1 domain comprises an isoleucine at residue 188 according to Kabat Numbering and the second human Ck domain has an alanine or a glycine at residue 176 according to Kabat Numbering, or   (iii) the second human C H 1 domain comprises a glutamic acid at residue 221 according to Kabat Numbering and the second human Ck domain has lysine at residue 123 according to Kabat Numbering.   
     
     
         2 . The first and second fragment, antigen binding (Fab) according to  claim 1 , wherein the first human C H 1 domain further comprises a glutamic acid at residue 221 according to Kabat Numbering and the human Ck domain further comprises a lysine at residue 123 according to Kabat Numbering and wherein the second human C H 1 domain has the wild-type human IgG C H 1 sequence and the second human Ck domain has the wild-type human Ck sequence. 
     
     
         3 . The first and second fragment, antigen binding (Fab) according to  claim 1 , wherein
 (a) the first V H  domain comprises a glutamic acid at residue 62 and a lysine at residue 39 according to Kabat Numbering,   (b) the first V L  domain is kappa isotype and comprises an arginine at residue 1 and an aspartic acid at residue 38 according to Kabat Numbering,   (c) the second V H  domain comprises a tyrosine at residue 39 and an arginine at residue 105 according to Kabat Numbering, and   (d) the second V L  domain is kappa isotype and comprises an arginine at residue 38 and an aspartic acid at residue 42 according to Kabat Numbering.   
     
     
         4 . The first and second fragment, antigen binding (Fab) according to  claim 1 , wherein
 (a) the first V H  domain comprises a tyrosine at residue 39 and an arginine at residue 105 according to Kabat Numbering,   (b) the first V L  domain is kappa isotype and comprises an arginine at residue 38 and an aspartic acid at residue 42 according to Kabat Numbering,   (c) the second V H  domain comprises a glutamic acid at residue 62 and a lysine at residue 39 according to Kabat Numbering, and   (d) the second V L  domain is kappa isotype and comprises an arginine at residue 1 and an aspartic acid at residue 38 according to Kabat Numbering.   
     
     
         5 . The first and second fragment, antigen binding (Fab) according to  claim 1 , wherein each of the first and second human C H 1 domains are individually IgG1 or IgG4 isotype. 
     
     
         6 . The first and second fragment, antigen binding (Fab) according to  claim 5 , wherein each of the first and second human C H 1 domains are IgG1 isotype. 
     
     
         7 . The first and second fragment, antigen binding (Fab) according to  claim 5 , wherein each of the first and second human C H 1 domains are IgG4 isotype. 
     
     
         8 . An IgG bispecific antibody comprising:
 a first Fab comprising a first V H  domain, a first human IgG constant region comprising a first human C H 1 domain having an alanine at residue 145 according to Kabat Numbering and an alanine or a glycine at residue 188 according to Kabat Numbering, a first V L  domain, and a first human Ck domain comprising an arginine at residue 131 according to Kabat Numbering and an isoleucine at residue 176 according to Kabat Numbering; and   a second Fab comprising a second V H  domain, a second human V L  domain, and a human IgG constant region comprising a second human C H 1 domain and a second human Ck domain, wherein   each of the first V H  domain and the first V L  domain comprise three CDRs which direct binding to a first antigen,   each of the second heavy chain and light chain variable domains comprise three CDRs which direct binding to a second antigen that differs from the first antigen, and   (i) the second human C H 1 domain having a wild-type human IgG C H 1 sequence and the second human Ck domain having a wild type human Ck sequence,   (ii) the second human C H 1 domain comprises an isoleucine at residue 188 according to Kabat Numbering and the second human Ck domain has an alanine or a glycine at residue 176 according to Kabat Numbering, or   (iii) the second human IgG heavy chain constant region comprises a second human C H 1 domain that comprises a glutamic acid at residue 221 according to Kabat Numbering and the second human Ck domain has lysine at residue 123 according to Kabat Numbering.   
     
     
         9 . The IgG bispecific antibody according to  claim 8 , wherein the first human C H 1 domain further comprises glutamic acid at residue 221 according to Kabat Numbering and the first human Ck domain further comprises a lysine at residue 123 according to Kabat Numbering, wherein the second human C H 1 domain has the wild-type human IgG C H 1 sequence and the second human Ck domain has the wild-type human Ck sequence. 
     
     
         10 . The IgG bispecific antibody according to  claim 8 , wherein
 (a) the first V H  domain comprises a glutamic acid at residue 62 and a lysine at residue 39 according to Kabat Numbering,   (b) the first V L  domain is kappa isotype and comprises an arginine at residue 1 and an aspartic acid at residue 38 according to Kabat Numbering,   (c) the second V H  domain comprises a tyrosine at residue 39 and an arginine at residue 105 according to Kabat Numbering, and   (d) the second V L  domain is kappa isotype and comprises an arginine at residue 38 and an aspartic acid at residue 42 according to Kabat Numbering.   
     
     
         11 . The IgG bispecific antibody according to  claim 8 , wherein
 (a) the first V H  domain comprises a tyrosine at residue 39 and an arginine at residue 105 according to Kabat Numbering,   (b) the first V L  domain is kappa isotype and comprises an arginine at residue 38 and an aspartic acid at residue 42 according to Kabat Numbering;   (c) the second V H  domain comprises a glutamic acid at residue 62 and a lysine at residue 39 according to Kabat Numbering, and   (d) the second V L  domain is kappa isotype and comprises an arginine at residue 1 and an aspartic acid at residue 38 according to Kabat Numbering.   
     
     
         12 . The IgG bispecific antibody according to  claim 8 , wherein one of the first or second human IgG constant regions comprises a CH3 domain comprising an alanine at residue 407 according to EU Index Numbering; and the other of the first or second human IgG constant regions comprises a CH3 domain comprising a valine or methionine at residue 366 and a valine at residue 409 according to EU Index Numbering. 
     
     
         13 . The IgG bispecific antibody according to  claim 8 , wherein one of the first or second human IgG constant regions comprises a C H 3 domain having an alanine at residue 407, a methionine at residue 399, and an aspartic acid at residue 360 according to EU Index Numbering; and the other of the first or second human IgG constant regions comprising a CH3 domain having a valine at residue 366, a valine at residue 409, and an arginine at residues 345 and 347 according to EU Index Numbering. 
     
     
         14 . The IgG bispecific antibody according to  claim 8 , wherein one of the first or second human IgG constant regions comprises a C H 3 domain having an alanine at residue 407, a glycine at residue 356, an aspartic acid at residue 357, and a glutamine at residue 364 according to EU Index Numbering; and the other of the first or second human IgG constant regions comprises a C H 3 domain having a methionine at residue 366, a valine at residue 409, a serine at residue 349, and a tyrosine at residue 370 according to EU Index Numbering. 
     
     
         15 . The IgG bispecific antibody according to  claim 8 , wherein each of the first and second human C H 1 domains are IgG1 or IgG4 isotype. 
     
     
         16 . The IgG bispecific antibody according to  claim 15 , wherein each of the first human C H 1 domain and the second human C H 1 domain are IgG1 isotype. 
     
     
         17 . The IgG bispecific antibody according to  claim 15 , wherein each of the first human C H 1 domain and the second human C H 1 domain are IgG4 isotype. 
     
     
         18 . The IgG bispecific antibody according to  claim 8 , wherein each of the first V L  domain and the second V L  domain is human kappa isotype. 
     
     
         19 . A first and second fragment, antigen binding (Fab) comprising:
 (a) a first heavy chain variable domain (V H ) and a first human IgG heavy chain constant C H 1 (C H 1) domain having an alanine at residue 145 according to Kabat Numbering and a glutamic acid at residue 221 according to Kabat Numbering;   (b) a first light chain variable domain (V L ) and a first human light chain kappa constant (Ck) domain having an arginine at residue 131 according to Kabat Numbering and a lysine at residue 123 according to Kabat Numbering;   (c) a second heavy chain variable domain V H  and a second human IgG heavy chain constant C H 1 domain having a wild-type human IgG C H 1 sequence; and   (d) a second light chain variable V L  domain and a second human light chain kappa constant Ck domain having a wild type human Ck sequence,   
       wherein
 each of the first V H  domain and the V L  domain comprise three complementarity determining regions (CDRs) which direct binding to a first antigen, and 
 each of the second V H  and V L  domains comprise three CDRs which direct binding to a second antigen that differs from the first antigen. 
 
     
     
         20 . The first and second fragment, antigen binding (Fab) of  claim 19 , wherein,
 (a) the first V H  domain comprises a glutamic acid at residue 62 and a lysine at residue 39 according to Kabat Numbering,   (b) the first V L  domain is kappa isotype and comprises an arginine at residue 1 and an aspartic acid at residue 38 according to Kabat Numbering,   (c) the second V H  domain comprises a tyrosine at residue 39 and an arginine at residue 105 according to Kabat Numbering, and   (d) the second V L  domain is kappa isotype and comprises an arginine at residue 38 and an aspartic acid at residue 42 according to Kabat Numbering.   
     
     
         21 . The first and second fragment, antigen binding (Fab) according to  claim 19 , wherein
 (a) the first V H  domain comprises a tyrosine at residue 39 and an arginine at residue 105 according to Kabat Numbering,   (b) the first V L  domain is kappa isotype and comprises an arginine at residue 38 and an aspartic acid at residue 42 according to Kabat Numbering,   (c) the second V H  domain comprises a glutamic acid at residue 62 and a lysine at residue 39 according to Kabat Numbering, and   (d) the second V L  domain is kappa isotype and comprises an arginine at residue 1 and an aspartic acid at residue 38 according to Kabat Numbering.   
     
     
         22 . The first and second fragment, antigen binding (Fab) according to  claim 19 , wherein each of the first human C H 1 domain and the second human C H 1 domain are individually IgG1 or IgG4 isotype. 
     
     
         23 . The first and second fragment, antigen binding (Fab) of  claim 22 , wherein each of the first human C H 1 domain and the second human C H 1 domain are IgG1 isotype. 
     
     
         24 . The first and second fragment, antigen binding (Fab) of  claim 22 , wherein each of the first human C H 1 domain and the second human C H 1 domain are IgG4 isotype. 
     
     
         25 . A bispecific antibody comprising:
 a first Fab comprising a first V H  domain, a first V L  domain, a first human IgG constant region comprising a first human C H 1 domain having an alanine at residue 145 according to Kabat Numbering and an alanine and a glutamic acid at residue 221 according to Kabat Numbering, and a first human Ck domain comprising an arginine at residue 131 according to Kabat Numbering and a lysine at residue 123 according to Kabat Numbering; and   a second Fab comprising a second V H  domain, a second V L  domain, and a human IgG constant region comprising a second human C H 1 domain having a wild-type human IgG C H 1 sequence and a second human Ck domain having a wild type human Ck sequence, wherein   each of the first V H  domain and the first V L  domain comprise three CDRs which direct binding to a first antigen, and   each of the second heavy chain variable domain and the second light chain variable domain comprise three CDRs which direct binding to a second antigen that differs from the first antigen.   
     
     
         26 . A bispecific antibody comprising according to  claim 25 , wherein,
 (a) the first V H  domain comprises a glutamic acid at residue 62 and a lysine at residue 39 according to Kabat Numbering,   (b) the first V L  domain is kappa isotype and comprises an arginine at residue 1 and an aspartic acid at residue 38 according to Kabat Numbering,   (c) the second V H  domain comprises a tyrosine at residue 39 and an arginine at residue 105 according to Kabat Numbering, and   (d) the second V L  domain is kappa isotype and comprises an arginine at residue 38 and an aspartic acid at residue 42 according to Kabat Numbering.   
     
     
         27 . The bispecific antibody according to  claim 25 , wherein
 (a) the first V H  domain comprises a tyrosine at residue 39 and an arginine at residue 105 according to Kabat Numbering,   (b) the first V L  domain is kappa isotype and comprises an arginine at residue 38 and an aspartic acid at residue 42 according to Kabat Numbering,   (c) the second V H  domain comprises a glutamic acid at residue 62 and a lysine at residue 39 according to Kabat Numbering, and   (d) the second V L  domain is kappa isotype and comprises an arginine at residue 1 and an aspartic acid at residue 38 according to Kabat Numbering.   
     
     
         28 . The bispecific antibody according to  claim 25 , wherein one of the first human IgG constant region or the second human IgG constant region comprises a C H 3 domain having an alanine at residue 407 with residue according to EU Index Numbering; and the other of the first human IgG constant region or the second human IgG constant region comprises a CH3 domain having a valine or methionine at residue 366 and a valine at residue 409 according to EU Index Numbering. 
     
     
         29 . The bispecific antibody according to  claim 25 , wherein one of the first human IgG constant region or the second human IgG constant region comprises a C H 3 domain having an alanine at residue 407, a methionine at residue 399, and an aspartic acid at residue 360 according to EU Index Numbering; and the other of the first human IgG constant region or the second human IgG constant region comprises a C H 3 domain having a valine at residue 366, a valine at residue 409, and an arginine at residues 345 and 347 according to EU Index Numbering. 
     
     
         30 . The bispecific antibody according to  claim 25 , wherein one of the first human IgG constant region or the second human IgG constant region comprises a C H 3 domain having an alanine at residue 407, a glycine at residue 356, an aspartic acid at residue 357, and a glutamine at residue 364 according to EU Index Numbering; and the other of the first human IgG constant region or the second human IgG constant region comprises a C H 3 domain having a methionine at residue 366, a valine at residue 409, a serine at residue 349, and a tyrosine at residue 370 according to EU Index Numbering. 
     
     
         31 . The bispecific antibody according to  claim 25 , wherein each of the first human C H 1 domain and the second human C H 1 domain are IgG1 or IgG4 isotype. 
     
     
         32 . The bispecific antibody according to  claim 31 , wherein each of the first human C H 1 domain and the second human C H 1 domains are IgG1 isotype. 
     
     
         33 . The bispecific antibody according to  claim 31 , wherein each of the first human C H 1 domain and the second human C H 1 domain are IgG4 isotype. 
     
     
         34 . The bispecific antibody according to  claim 25 , wherein each of the first V L  domain and the second V L  domain is human kappa isotype.