Heterodimeric antibody fc-containing proteins and methods for production thereof

Abstract

Novel heterodimeric antibody-Fc-containing proteins, such as bispecific antibodies, and novel methods for producing such proteins

1 . An in vitro method for generating a heterodimeric protein, said method comprising the following steps:
 a) providing a first homodimeric protein comprising an Fc region of an immunoglobulin, said Fc region comprising a first CH3 region,   b) providing a second homodimeric protein comprising an Fc region of an immunoglobulin, said Fc region comprising a second CH3 region,   wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions,   c) incubating said first protein together with said second protein under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide-bond isomerization, and   d) obtaining said heterodimeric protein.   
     
     
         2 . The in vitro method according to  claim 1 , wherein said first homodimeric protein and said second homodimeric protein are selected from the group consisting of (i) an Fc region, (ii) an antibody, (iii) a fusion protein comprising an Fc region, and (iv) a Fc region conjugated to a prodrug, peptide, drug or a toxin. 
     
     
         3 . The in vitro method according to  claim 1 , wherein said first and/or second homodimeric protein is a full-length antibody. 
     
     
         4 . (canceled) 
     
     
         5 . The in vitro method according to  claim 1 , wherein said first and second homodimeric proteins are both antibodies and bind different epitopes. 
     
     
         6 . The in vitro method according to  claim 1 , wherein the Fc region of the first homodimeric protein is of an isotype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 and wherein the Fc region of the second homodimeric protein is of an isotype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. 
     
     
         7 - 9 . (canceled) 
     
     
         10 . The in vitro method according to  claim 1 , wherein the heterodimeric interaction between said first and second proteins in the resulting heterodimeric protein is
 (a) such that no Fab-arm exchange can occur at 0.5 mM GSH under the conditions described in Example 13, and/or   (b) such that no Fab-arm exchange occurs in vivo in mice under the conditions described in Example 14.   
     
     
         11 . (canceled) 
     
     
         12 . The in vitro method according to  claim 1 , wherein the heterodimeric interaction between said first and second proteins in the resulting heterodimeric protein is more than two times stronger, such as more than three times stronger, e.g. more than five times stronger than the strongest of the two homodimeric interactions, e.g. when determined as described in Example 30. 
     
     
         13 . The in vitro method according to  claim 1 , wherein the sequences of said first and second CH3 regions are such that:
 (a) the dissociation constants of the heterodimeric interaction between said first and second proteins in the resulting heterodimeric protein is below 0.05 micromolar when assayed as described in Example 30 and/or   (b) the dissociation constants of both homodimeric interactions are above 0.01 micromolar, such as above 0.05 micromolar, preferably between 0.01 and 10 micromolar, such as between 0.05 and 10 micromolar, more preferably between 0.01 and 5, such as between 0.05 and 5 micromolar, even more preferably between 0.01 and 1 micromolar, such as between 0.05 and 1 micromolar, between 0.01 and 0.5 or between 0.01 and 0.1 micromolar when assayed as described in Example 21.   
     
     
         14 . (canceled) 
     
     
         15 . The in vitro method according to  claim 1 , wherein the sequences of said first and second CH3 regions contain amino acid substitutions at non-identical positions. 
     
     
         16 . (canceled) 
     
     
         17 . The in vitro method according to  claim 1 , wherein said first homodimeric protein has no more than one amino acid substitution in the CH3 region, and the second homodimeric protein has no more than one amino acid substitution in the CH3 region relative to the wild-type CH3 regions. 
     
     
         18 . The in vitro method according to  claim 1 , wherein said first homodimeric protein has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409, and said second homodimeric protein has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409, and wherein said first homodimeric protein and said second homodimeric protein is not substituted in the same positions. 
     
     
         19 . The in vitro method according to  claim 1 , wherein
 (a) said first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405 and 407,   (b) said first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein has an amino acid other than Phe at position 405,   (c) said first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein has an amino acid other than Phe, Arg or Gly at position 405,   (d) said first homodimeric protein comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein comprises an amino acid other than Phe at position 405 and a Lys at position 409,   (e) said first homodimeric protein comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein comprises an amino acid other than Phe, Arg or Gly at position 405 and a Lys at position 409,   (f) said first homodimeric protein comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein comprises a Leu at position 405 and a Lys at position 409,   (g) said first homodimeric protein comprises a Phe at position 405 and an Arg at position 409 and said second homodimeric protein comprises an amino acid other than Phe, Arg or Gly at position 405 and a Lys at position 409,   (h) said first homodimeric protein comprises Phe at position 405 and an Arg at position 409 and said second homodimeric protein comprises a Leu at position 405 and a Lys at position 409,   (i) said first homodimeric protein comprises an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405,   (j) said first homodimeric protein comprises an Arg at position 409 and said second homodimeric protein comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405,   (k) said first homodimeric protein comprises a Lys at position 370, a Phe at position 405 and an Arg at position 409 and said second homodimeric protein comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405,   (l) said first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407,   (m) said first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407,   (n) said first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein has a Gly, Leu, Met, Asn or Trp at position 407,   (o) said first homodimeric protein has a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a Lys at position 409,   (p) said first homodimeric protein has a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys at position 409,   (q) said first homodimeric protein has a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein has a Gly, Leu, Met, Asn or Trp at position 407 and a Lys at position 409,   (r) said first homodimeric protein has a Tyr at position 407 and an Arg at position 409 and said second homodimeric protein has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a Lys at position 409,   (s) said first homodimeric protein has a Tyr at position 407 and an Arg at position 409 and said second homodimeric protein has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys at position 409,   (t) said first homodimeric protein has a Tyr at position 407 and an Arg at position 409 and said second homodimeric protein has a Gly, Leu, Met, Asn or Trp at position 407 and a Lys at position 409,   (u) said first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has
 (i) an amino acid other than Phe, Leu and Met at position 368, or 
 (ii) a Trp at position 370, or 
 (iii) an amino acid other than Asp, Cys, Pro, Glu or Gln at position 399, 
   (v) said first homodimeric protein has an Arg, Ala, His or Gly at position 409, and the second homodimeric protein has
 (i) a Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or 
 (ii) a Trp at position 370, or 
 (iii) an Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at position 399, or 
   (w) said first homodimeric protein has an Arg at position 409, and the second homodimeric protein has
 (i) an Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or 
 (ii) a Trp at position 370, or 
 (iii) a Phe, His, Lys, Arg or Tyr at position 399. 
   
     
     
         20 - 42 . (canceled) 
     
     
         43 . The in vitro method according to  claim 1 , wherein
 (a) neither said first nor said second homodimeric protein comprises a Cys-Pro-Ser-Cys sequence in the hinge region, or   (b) both said first and said second homodimeric protein comprise a Cys-Pro-Pro-Cys sequence in the hinge region.   
     
     
         44 . (canceled) 
     
     
         45 . The in vitro method according to  claim 1 , wherein said first and second homodimeric proteins, except for any specified mutations, are human antibodies. 
     
     
         46 . The in vitro method according to  claim 1 , wherein said first and second homodimeric proteins are heavy-chain antibodies. 
     
     
         47 . The in vitro method according to  claim 1 , wherein both said first and said second homodimeric proteins further comprise a light chain, wherein, optionally, said light chains are different. 
     
     
         48 - 49 . (canceled) 
     
     
         50 . The in vitro method according to  claim 1 , wherein said first and second homodimeric proteins provided in step a) and b) are purified. 
     
     
         51 . The in vitro method according to  claim 1 , wherein said first and/or second homodimeric protein is conjugated to a drug, a prodrug or a toxin or contains an acceptor group for the same. 
     
     
         52 - 58 . (canceled) 
     
     
         59 . The in vitro method according to  claim 1 , wherein the reducing conditions in step c) comprise the addition of a reducing agent, e.g. a reducing agent selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine or chemical derivatives thereof. 
     
     
         60 . The in vitro method according to  claim 1 , wherein step c)
 (i) is performed under reducing conditions with a redox potential between −150 and −600 mV, such as between −250 and −400 mV and/or   (ii) comprises incubation for at least 90 min at a temperature of at least 20° C. in the presence of at least 25 mM 2-mercaptoethylamine or in the presence of at least 0.5 mM dithiothreitol.   
     
     
         61 . (canceled) 
     
     
         62 . The in vitro method according to  claim 1 , wherein step d) comprises removal of a reducing agent, e.g. by desalting. 
     
     
         63 . A method for the selection of a bispecific antibody having a desired property, said method comprising the steps of:
 a) providing a first set of homodimeric antibodies comprising antibodies with different variable regions, wherein said antibodies of said first set comprise identical first CH3 regions,   b) providing a second set of homodimeric antibodies comprising antibodies with different variable regions or identical variable regions, wherein said antibodies of said second set comprise identical second CH3 regions,   wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions,   c) incubating combinations of antibodies of said first set and of said second set under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide-bond isomerization, thus generating a set of bispecific antibodies,   d) optionally restoring the conditions to non-reducing,   e) assaying the resulting set of bispecific antibodies for a given desired property, and   f) selecting a bispecific antibody having the desired property.   
     
     
         64 - 65 . (canceled) 
     
     
         66 . A method for producing a heterodimeric protein, said method comprising the following steps:
 a) providing a first nucleic-acid construct encoding a first polypeptide comprising a first Fc region of an immunoglobulin, said first Fc region comprising a first CH3 region,   b) providing a second nucleic-acid construct encoding a second polypeptide comprising a second Fc region of an immunoglobulin, said second Fc region comprising a first CH3 region,
 wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions, and 
 wherein said first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein has an amino-acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405 and 407.
 and/or 
 
 wherein the sequences of said first and second CH3 regions are such that the dissociation constants of homodimeric interactions of each of the CH3 regions are between 0.01 and 10 micromolar, such as between 0.05 and 10 micromolar, more preferably between 0.01 and 5, such as between 0.05 and 5 micromolar, even more preferably between 0.01 and 1 micromolar, such as between 0.05 and 1 micromolar, between 0.01 and 0.5 or between 0.01 and 0.1 when assayed as described in Example 21. 
   c) co-expressing said first and second nucleic-acid constructs in a host cell, and   d) obtaining said heterodimeric protein from the cell culture.   
     
     
         67 - 70 . (canceled) 
     
     
         71 . An expression vector comprising the nucleic-acid constructs specified in  claim 66 . 
     
     
         72 . A host cell comprising the nucleic-acid constructs specified in  claim 66 . 
     
     
         73 . A heterodimeric protein obtained or obtainable by the method of  claim 1 . 
     
     
         74 . A heterodimeric protein comprising a first polypeptide comprising a first Fc region of an immunoglobulin, said first Fc region comprising a first CH3 region, and a second polypeptide comprising a second Fc region of an immunoglobulin, said second Fc region comprising a second CH3 region, wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions, and
 wherein said first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and said second homodimeric protein has an amino-acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405 and 407   and/or   wherein the sequences of said first and second CH3 regions are such that the dissociation constants of homodimeric interactions of each of the CH3 regions are between 0.01 and 10 micromolar, such as between 0.05 and 10 micromolar, more preferably between 0.01 and 5, such as between 0.05 and 5 micromolar, even more preferably between 0.01 and 1 micromolar, such as between 0.05 and 1 micromolar, between 0.01 and 0.5 or between 0.01 and 0.1 when assayed as described in Example 21.   
     
     
         75 - 80 . (canceled) 
     
     
         81 . A pharmaceutical composition comprising a heterodimeric protein according to  claim 73  and a pharmaceutically-acceptable carrier. 
     
     
         82 . A method for inhibiting growth and/or proliferation and/or for killing of tumor cells comprising administration of a heterodimeric protein according to  claim 73  to an individual in need thereof. 
     
     
         83 . A pharmaceutical composition comprising a heterodimeric protein according to  claim 74  and a pharmaceutically-acceptable carrier. 
     
     
         84 . A method for inhibiting growth and/or proliferation and/or for killing of tumor cells comprising administration of a heterodimeric protein according to  claim 74  to an individual in need thereof.