Optimized Fc variants and methods for their generation

• US 20070003546A1
• Filed: 07/07/2006
• Published: 01/04/2007
• Est. Priority Date: 03/01/2002
• Status: Active Grant

• Current Assignee: Xencor, Inc.
• Original Assignee: Xencor, Inc.
• Inventors: Dang, Wei, Vafa, Omid, Doberstein, Stephen, Desjarlais, John, Hayes, Robert, Chirino, Arthur, Lazar, Gregory, Karki, Sher
• Granted Patent: US 7,662,925 B2
• Time in Patent Office: Days
• US Class Current: 424/133.100
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/283 against Fc-receptors, e.g. CD16, CD32, CD64 CD23 C07K16/2851
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 16/2893 against CD52
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 16/3015 Breast
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/71 Decreased effector function due to an Fc-modification
• CPC Class Codes: C07K 2317/72 Increased effector function due to an Fc-modification
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/734 Complement-dependent cytotoxicity [CDC]
• CPC Class Codes: C07K 2317/77 Internalization into the cell
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The present invention relates to optimized Fc variants, methods for their generation, and antibodies and Fc fusions comprising optimized Fc variants.

Claims

1-37. -37. (canceled)

38. A composition comprising a protein comprising an Fc variant region of an IgG polypeptide, wherein said Fc variant region comprises at least one engineered glycoform, wherein said Fc variant region comprises at least one amino acid modification in the Fc region of said IgG polypeptide at a position selected from the group consisting of:

221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 260, 262, 263, 264, 265, 266, 269, 270, 271, 273, 274, 275, 278, 281, 282, 284, 288, 291, 292, 293, 294, 295, 296, 297, 299, 300, 302, 303, 304, 313, 317, 318, 323, 324, 325, 327, 328, 329, 332, 335 and 336, wherein said Fc variant has greater affinity for an Fc ligand relative to said parent polypeptide, and wherein numbering is according to the EU index.

39. A composition according to claim 38 wherein said Fc variant region further comprises at least one amino acid modification in the Fc region of said IgG polypeptide at a position selected from the group consisting of:

255, 258, 267, 268, 272, 276, 280, 283, 285, 286, 290, 298, 301, 305, 320, 322, 326, 330, 331, 333, 334, and 337.

40. A composition according to claim 38, wherein said amino acid modification is selected from the group consisting of:

221 K, 221Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 227E, 227G, 227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231E, 231G, 231 K, 231P, 231Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G, 233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W, 233Y, 234A, 234D, 234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N, 234P, 234Q, 234R, 234S, 234T, 234V, 234W, 234Y, 235A, 235D, 235E, 235F, 235G, 235H, 235I, 235K, 235M, 235N, 235P, 235Q, 235R, 235S, 235T, 235V, 235W, 235Y, 236A, 236D, 236E, 236F, 236H, 236I, 236K, 236L, 236M, 236N, 236P, 236Q, 236R, 236S, 236T, 236V, 236W, 236Y, 237D, 237E, 237F, 237H, 237I, 237K, 237L, 237M, 237N, 237P, 237Q, 237R, 237S, 237T, 237V, 237W, 237Y, 238D, 238E, 238F, 238G, 238H, 238I, 238K, 238L, 238M, 238N, 238Q, 238R, 238S, 238T, 238V, 238W, 238Y, 239D, 239E, 239F, 239G, 239H, 239I, 239K, 239L, 239M, 239N, 239P, 239Q, 239Q, 239R, 239T, 239V, 239W, 239Y, 240A, 240I, 240M, 240T, 241D, 241E, 241E, 241L, 241R, 241S, 241W, 241Y, 241Y, 243E, 243H, 243L, 243Q, 243R, 243W, 243Y, 244H, 245A, 246D, 246E, 246H, 246Y, 247G, 247V, 249H, 249Q, 249Y, 260D, 260E, 260H, 260Y, 262A, 262E, 262E, 262F, 262I, 262T, 263A, 263I, 263M, 263T, 264A, 264D, 264E, 264E, 264F, 264G, 264H, 264I, 264K, 264L, 264M, 264N, 264P, 264Q, 264R, 264R, 264S, 264T, 264W, 264W, 264Y, 265F, 265F, 265G, 265H, 265H, 265I, 265I, 265K, 265L, 265L, 265M, 265N, 265P, 265Q, 265Q, 265R, 265S, 265T, 265T, 265V, 265V, 265W, 265Y, 265Y, 266A, 266I, 266M, 266T, 269F, 269G, 269H, 269I, 269K, 269L, 269M, 269N, 269P, 269R, 269S, 269T, 269V, 269W, 269Y, 270F, 270G, 270H, 270I, 270L, 270M, 270P, 270Q, 270R, 270S, 270T, 270W, 270Y, 271A, 271D, 271E, 271F, 271G, 271H, 271I, 271K, 271L, 271M, 271N, 271Q, 271R, 271S, 271T, 271V, 271W, 271Y, 273I, 274D, 274E, 274F, 274G, 274H, 274I, 274L, 274M, 274N, 274P, 274R, 274S, 274T, 274V, 274W, 274Y, 275L, 275W, 278D, 278E, 278G, 278H, 278I, 278K, 278L, 278M, 278N, 278P, 278Q, 278R, 278S, 278T, 278V, 278W, 281D, 281K, 281P, 281Y, 282E, 282G, 282K, 282P, 282Y, 284E, 284L, 284N, 284T, 284Y, 288D, 288E, 288Y, 291D, 291E, 291G, 291H, 291I, 291Q, 291T, 292D, 292E, 292T, 292Y, 293F, 293G, 293H, 293I, 293L, 293M, 293N, 293P, 293R, 293S, 293T, 293V, 293W, 293Y, 294F, 294G, 294H, 294I, 294K, 294L, 294M, 294P, 294R, 294S, 294T, 294V, 294W, 294Y, 295D, 295E, 295F, 295G, 295H, 295I, 295M, 295N, 295P, 295R, 295S, 295T, 295V, 295W, 295Y, 296A, 296D, 296E, 296G, 296H, 296I, 296K, 296L, 296M, 296N, 296Q, 296R, 296S, 296T, 296V, 297D, 297E, 297F, 297G, 297H, 297I, 297K, 297L, 297M, 297P, 297Q, 297R, 297S, 297T, 297V, 297W, 297Y, 299A, 299D, 299E, 299E, 299F, 299F, 299G, 299H, 299I, 299K, 299L, 299L, 299M, 299N, 299P, 299Q, 299R, 299S, 299V, 299W, 299Y, 300A, 300D, 300E, 300G, 300H, 300K, 300M, 300N, 300P, 300Q, 300R, 300S, 300T, 300V, 300W, 302I, 303D, 303E, 303Y, 304D, 304H, 304L, 304N, 304T, 313F, 317E, 317Q, 318H, 318L, 318Q, 318R, 318Y, 323I, 324D, 324F, 324G, 324H, 324I, 324L, 324M, 324P, 324R, 324T, 324V, 324W, 324Y, 325A, 325D, 325E, 325F, 325G, 325H, 325I, 325K, 325L, 325M, 325P, 325Q, 325R, 325S, 325T, 325V, 325W, 325Y, 327D, 327E, 327F, 327H, 327I, 327K, 327L, 327M, 327N, 327P, 327R, 327S, 327T, 327V, 327W, 327Y, 328A, 328D, 328D, 328E, 328F, 328G, 328H, 328I, 328I, 328K, 328M, 328N, 328P, 328Q, 328Q, 328R, 328S, 328T, 328T, 328V, 328V, 328W, 328Y, 329D, 329E, 329F, 329G, 329H, 3291, 329K, 329L, 329M, 329N, 329Q, 329R, 329S, 329T, 329V, 329W, 329Y, 332A, 332D, 332E, 332F, 332G, 332H, 332K, 332L, 332M, 332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W, 332Y, 335D, 335F, 335G, 335H, 335I, 335L, 335M, 335N, 335P, 335R, 335S, 335V, 335W, 335Y, 336E, 336K and 336Y

41. A composition according to claim 39 wherein said amino acid modification is selected from the group consisting of:

255E, 255Y, 258H, 258S, 258Y, 267D, 267E, 267F, 267H, 267I, 267K, 267L, 267L, 267M, 267N, 267P, 267Q, 267Q, 267R, 267T, 267V, 267W, 267Y, 268D, 268E, 268F, 268G, 268I, 268K, 268L, 268M, 268P, 268Q, 268R, 268T, 268V, 268W, 272D, 272F, 272G, 272H, 272I, 272K, 272L, 272M, 272P, 272R, 272S, 272T, 272V, 272W, 272Y, 276D, 276E, 276F, 276G, 276H, 276I, 276L, 276M, 276P, 276R, 276S, 276T, 276V, 276W, 276Y, 280G, 280K, 280L, 280P, 280W, 283G, 283H, 283K, 283L, 283P, 283R, 283Y, 285D, 285E, 285K, 285Q, 285W, 285Y, 286E, 286G, 286P, 286Y, 290D, 290H, 290L, 290N, 290W, 298A, 298D, 298E, 298F, 298H, 298I, 298K, 298M, 298N, 298Q, 298R, 298T, 298W, 298Y, 301D, 301E, 301H, 301Y, 305E, 305T, 305Y, 320D, 320F, 320G, 320H, 320I, 320L, 320N, 320P, 320S, 320T, 320V, 320W, 320Y, 322D, 322F, 322G, 322H, 322I, 322P, 322S, 322T, 322V, 322W, 322Y, 326E, 326I, 326L, 326P, 326T, 330E, 330F, 330G, 330H, 330I, 330L, 330M, 330N, 330P, 330R, 330S, 330T, 330V, 330W, 330Y, 331D, 331F, 331H, 331I, 331L, 331M, 331Q, 331R, 331T, 331V, 331W, 331Y, 333A, 333F, 333H, 333I, 333L, 333M, 333P, 333T, 333Y, 334A, 334F, 334I, 334P, 334T, 337E, 337H, and 337N.

42. A composition according to claim 38, wherein said position is selected from the group consisting of:

221, 222, 223, 224, 225, 228, 230, 231, 232, 240, 244, 245, 247, 262, 263, 266, 271, 273, 275, 281, 284, 291, 299, 302, 304, 313, 323, 325, 328, 332, and 336.

43. A composition according to claim 42, wherein said amino acid modification is selected from the group consisting of:

221 K, 221Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231E, 231G, 231K, 231P, 231Y, 232E, 232G, 232K, 232Y, 240A, 240I, 240M, 240T, 244H, 245A, 247G, 247V, 262A, 262E, 262E, 262F, 262I, 262T, 263A, 263I, 263M, 263T, 266A, 266I, 266M, 266T, 271A, 271D, 271E, 271F, 271G, 271H, 271I, 271K, 271L, 271M, 271N, 271Q, 271R, 271S, 271T, 271V, 271W, 271Y, 273I, 275L, 275W, 281D, 281 K, 281P, 281Y, 284E, 284L, 284N, 284T, 284Y, 291D, 291E, 291G, 291H, 291I, 291Q, 291T, 299A, 299D, 299E, 299E, 299F, 299F, 299G, 299H, 299I, 299K, 299L, 299L, 299M, 299N, 299P, 299Q, 299R, 299S, 299V, 299W, 299Y, 302I, 304D, 304H, 304L, 304N, 304T, 313F, 323I, 325A, 325D, 325E, 325F, 325G, 325H, 325I, 325K, 325L, 325M, 325P, 325Q, 325R, 325S, 325T, 325V, 325W, 325Y, 328A, 328D, 328D, 328E, 328F, 328G, 328H, 328I, 328I, 328K, 328M, 328N, 328P, 328Q, 328Q, 328R, 328S, 328T, 328T, 328V, 328V, 328W, 328Y, 332A, 332D, 332E, 332F, 332G, 332H, 332K, 332L, 332M, 332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W, 332Y, 336E, 336K, and 336Y.

44. A composition according to claim 38, wherein said position is selected from the group consisting of:

230, 240, 244, 245, 247, 262, 263, 266, 273, 275, 299, 302, 313, 323, 325, 328, and 332.

45. A composition according to claim 44, wherein said amino acid modification is selected from the group consisting of:

230A, 240A, 240I, 240M, 240T, 244H, 245A, 247G, 247V, 262A, 262E, 262I, 262T, 263A, 263I, 263M, 263T, 266A, 266I, 266M, 266T, 273I, 275W, 299A, 299E, 299F, 299H, 299I, 299L, 299S, 299V, 302I, 313F, 323I, 325A, 325D, 325E, 325H, 325I, 325L, 325Q, 325T, 325V, 328A, 328D, 328E, 328F, 328H, 328I, 328M, 328N, 328Q, 328T, 328V, 332A, 332D, 332E, 332H, 332N, 332Q, 332T, and 332Y.

46. A composition according to claim 38, wherein said position is selected from the group consisting of 236, 239, 268, and 332.

47. A composition according to claim 46, wherein said Fc variant further comprises an amino acid modification at position 330.

48. A composition according to claim 46, wherein said Fc variant further comprises an amino acid modification at a position selected from the group consisting of:

298, 326, 333, and 334.

49. A composition according to claim 38, wherein said engineered glycoform is a reduced level of fucose relative to a native human IgG.

50. A composition according to claim 49, wherein said Fc variant has been produced in lec13 CHO cells or YB2/0 cells, and wherein said reduced fucose is relative to a native human IgG that has been produced in CHO cells.

51. A composition according to claim 49, wherein said Fc variant has been produced in cells wherein a glycosylation enzyme has been modified, and wherein said reduced fucose is relative to a native human IgG that has been produced in CHO cells.

52. A composition according to claim 50, wherein said glycosylation enzyme is FUT8 or GnTIII.

53. A composition according to claim 38, wherein said Fc ligand is Fcγ

RIIIa.

54. A composition according to claim 38, wherein said Fc variant mediates greater ADCC relative to said parent Fc polypeptide.

55. A composition according to claim 38, wherein said protein is an antibody.

56. A composition according to claim 38, wherein said protein is an Fc fusion.

57. A composition according to claim 55, wherein said antibody is specific for a target antigen selected from the group consisting of CD20, CD22, CD30, CD33, CD52, CD5, CD19, Her2/neu, EGFR, EpCAM, MUC1, GD3, CEA, CA 125, HLA-DR, TNFalpha, and VEGF.

58. A nucleic acid encoding the protein of claim 38.

59. An expression vector comprising the nucleic acid of claim 58.

60. A host cell comprising the nucleic acid of claim 58.

61. A host cell comprising the expression vector of claim 59.

62. A method of producing a protein comprising culturing the host cell of claim 60 or 61 under conditions suitable for expression of said nucleic acid.

63. The method according to claim 62 further comprising recovering said protein.

64. A pharmaceutical composition comprising a protein according to claim 38 and a pharmaceutically acceptable carrier.

65. A method of treating a mammal in needs of said treatment, comprising administering a protein of claim 38.