Fc mutants with modified functional activity

Abstract

Disclosed is a polypeptide including a mutated Fc region and having functional activity, mediated by the Fc region, that is modified compared with that of a parent polypeptide. The Fc region includes at least one combination of 2 mutations, the combination being selected from among one mutation selected from among a first set of mutations, and at least one mutation selected from among a second set of mutations, and provided that mutation (i) does not take place on the same amino acid as mutation (ii). Also disclosed are use of the polypeptide, compositions including the same, and methods for preparing the polypeptide.

1 . Polypeptide comprising a mutated Fc region and having functional activity. mediated by the Fc region, that is modified compared with that of a parent polypeptide, wherein said Fc region comprises at least one combination of 2 mutations, said combination being selected from among:
 (i) one mutation selected from among 307N, 326E, 326T, 334N, 334R, 352L, 378V, 378T, 394P, 396L, 397M, 421T; and   (ii) at least one mutation selected from among 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 397I, 397M, 398P, 405V, 405L, 410R, 412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N,   the numbering being that of the EU Index or Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).   
     
     
         2 . Polypeptide according to  claim 1 , wherein the mutation (i) is selected from among 378V, 396L and 397M. 
     
     
         3 . Polypeptide according to  claim 2 , further comprising a mutation selected from among 248E, 326T, 333G and 423Y. 
     
     
         4 . Polypeptide according to  claim 1 , wherein said functional activity mediated by the Fc region is selected from among antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cell phagocytosis (ADCP), and a combination of at least two of these activities. 
     
     
         5 . Polypeptide according to  claim 1 , having functional activity mediated by the Fc region that is increased compared with that of the parent polypeptide, preferably by a ratio of at least 2, preferably higher than 5, preferably higher than 10, preferably higher than 15, preferably higher than 20, preferably higher than 25, preferably higher than 30. 
     
     
         6 . Polypeptide according to  claim 1 , having functional activity mediated by the Fc region that is reduced compared with that of the parent polypeptide, preferably by a ratio of at least 2, preferably higher than 5, preferably higher than 10, preferably higher than 15, preferably higher than 20, preferably higher than 25, preferably higher than 30. 
     
     
         7 . Polypeptide according to  claim 1 , wherein said mutated Fc region has modified affinity for at least one of the receptors (FcRs) of the Fc region selected from among the C1q complement and FcgRIIIa (CD16a), FcgRIIa (CD32a) and FcgRIIb (CD32b) receptors. 
     
     
         8 . Polypeptide according to  claim 1 , wherein said mutated Fc region comprises 2 to mutations compared with the parent polypeptide, preferably 2 to 10 mutations. 
     
     
         9 . Polypeptide according to  claim 1 , wherein said mutated Fc region has improved affinity for the C1q complement, and comprises at least one combination of 2 mutations, said combination comprising:
 i) one mutation selected from among 378V, 378T, 396L, 421T, 334R and 326E; and   ii) at least one mutation selected from among 361H, 290E, 316D, 248E, 410R, 421T, 334R, 394P, 307P, 447N, 378V, 284L, 421T, 396L, 286I, 315D and 397M, the numbering being that of the EU Index or Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).   
     
     
         10 . Polypeptide according to  claim 1 , wherein said mutated Fc region has improved affinity for the FcgRIIIa receptor (CD16a), and comprises at least one combination of 2 mutations, said combination comprising:
 i) one mutation selected from among 378V, 326E, 397M, 334N and 396L; and   ii) at least one mutation selected from among 316D, 397M, 334N, 248E, 231V, 246R, 336T, 421T, 361H, 366A, 439R, 290E, 394P, 307P, 378V, 378T, 286I, 286Y and 298N,   the numbering being that of the EU Index or Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).   
     
     
         11 . Polypeptide according to  claim 1 , wherein said mutated Fc region has increased affinity for the FcgRIIa receptor (CD32a), and comprises at least one combination of 2 mutations, said combination comprising:
 i) one mutation selected from among 378V, 326E, 397M, 307N, 394P, 326T, 396L and 334N; and   ii) at least one mutation selected from among: 316D, 334R, 334N, 323I, 231V, 246R, 336T, 378T, 286Y, 286I, 352S, 383R, 359A, 421T, 361H, 315D, 366A, 290E, 307P and 439R,   the numbering being that of the EU Index or Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).   
     
     
         12 . Polypeptide according to  claim 1 , wherein said mutated Fc region has increased affinity for the FcgRIIb receptor (CD32b), and comprises at least one combination of 2 mutations, said combination comprising:
 i) one mutation selected from among 326E, 326T, 378V, 397M, 352L, 394P, 396L and 421T; and   ii) at least one mutation selected from among 316D, 334R, 248E, 334N, 418P, 231V, 320E, 402D, 359A, 383R, 421T and 361H,   the numbering being that of the EU Index or Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).   
     
     
         13 . Polypeptide according to  claim 1 , wherein said mutated Fc region has increased CDC activity, and comprises at least one combination of 2 mutations, said combination comprising:
 i) one mutation selected from among 378V, 378T, 396L, 421T, 334R and 326E; and   ii) at least one mutation selected from among 361H, 290E, 316D, 248E, 410R, 421T, 334R, 394P, 307P, 447N, 378V, 284L, 421T, 396L, 286I, 315D and 397M, the numbering being that of the EU Index or Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).   
     
     
         14 . Polypeptide according to  claim 1 , wherein said mutated Fc region has increased ADCC activity, and comprises at least one combination of 2 mutations, said combination comprising:
 i) one mutation selected from among 378V, 326E, 397M, 334N and 396L; and   ii) at least one mutation selected from among 316D, 397M, 334N, 248E, 231V, 246R, 336T, 421T, 361H, 366A, 439R, 290E, 394P, 307P, 378V, 378T, 286I, 286Y and 298N,   the numbering being that of the EU Index or Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).   
     
     
         15 . Polypeptide according to  claim 1 , wherein said mutated Fc region comprises at least one combination of 3 mutations, said combination comprising:
 (i) one mutation selected from among 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and   (ii) at least 2 mutations selected from among 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 397I, 397M, 398P, 405V, 405L, 410R, 412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N, the numbering being that of the EU Index or Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii),   preferably said mutated Fc region comprises a combination of 4 mutations, said combination comprising at least one mutation (i) and at least 3 mutations (ii), and preferably a combination of 5 mutations, said combination comprising at least one mutation (i) and at least 4 mutations (ii).   
     
     
         16 . Polypeptide according to  claim 1 , wherein the parent polypeptide comprises a parent Fc region that is a human Fc region, preferably an Fc region of a human IgG1 or human IgG2. 
     
     
         17 . Polypeptide according to  claim 1 , wherein the polypeptide is selected from among an isolated Fc region, a sequence derived from an isolated Fc region, an antibody and a fusion protein comprising an Fc region. 
     
     
         18 . Polypeptide according to  claim 1 , wherein the polypeptide consists of an Fc region or it is an antibody. 
     
     
         19 . Polypeptide according to  claim 1 , wherein the polypeptide is produced in the milk of transgenic animals. 
     
     
         20 . Composition of polypeptides according to  claim 1 , wherein the purified polypeptides of said composition, on their Asn297 glycosylation site, have N-glycans with a fucosylation rate lower than 65%, preferably lower than 50%, more preferably lower than 40%. 
     
     
         21 . Composition of polypeptides according to  claim 20 , wherein the purified polypeptides of said composition, on their Asn297 glycosylation site, have a glycan structure of biantennary type with short chains, low sialylation, having non-intercalary terminal mannoses and/or terminal N-acetylglucosamines. 
     
     
         22 . Composition of polypeptides according to  claim 21 , wherein the purified polypeptides of said composition have a content higher than 60% for the G0+G1+G0F+G1F forms, the G0F+G1F forms being lower than 50%, preferably lower than 40%. 
     
     
         23 . Composition of polypeptides according to  claim 21 , wherein the purified polypeptides of said composition have a content higher than 60% for the G0+G1+G0F+G1F forms, the fucose content being lower than 65%. 
     
     
         24 . Pharmaceutical composition comprising (i) a polypeptide according to  claim 1 , and (ii) at least one pharmaceutically acceptable excipient. 
     
     
         25 . A method for treating a subject in need thereof, comprising the step of administering to said subject a polypeptide according to  claim 1 . 
     
     
         26 . Antibody according to  claim 18  directed against an antigen selected from among a tumour antigen, viral antigen, bacterial antigen, fungal antigen, a toxin, membrane-bound or circulating membrane, a membrane receptor. 
     
     
         27 . Method to produce a polypeptide comprising an Fc region and having functional activity, mediated by the Fc region, that is modified compared with that of a parent polypeptide, said method comprising a step to introduce a least one combination of 2 mutations, said combination being selected from among:
 (i) one mutation selected from among 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and   (ii) at least one mutation selected from among 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 397I, 397M, 398P, 405V, 405L, 410R, 412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N,
 the numbering being that of the EU Index or Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii). 
   
     
     
         28 . Method to increase the binding of a polypeptide comprising an Fc region to at least one of the receptors of (FcR) of the Fc region, selected from among the receptors C1q, FcgRIIIa (CD16a), FcgRIIa (CD32a) and FcgRIIb (CD32b), said method comprising a step to introduce at least one combination of 2 mutations, said combination being selected from among:
 (i) one mutation selected from among 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and   (ii) at least one mutation selected from among 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 397I, 397M, 398P, 405V, 405L, 410R, 412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N,   the numbering being that of the EU Index or Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).