Transferrin receptor binding polypeptides and uses thereof

文档序号：1131756 发布日期：2020-10-02 浏览：15次中文

阅读说明：本技术 转铁蛋白受体结合性多肽和其用途 (Transferrin receptor binding polypeptides and uses thereof ) 是由马克·S·丹尼斯米哈利斯·卡里奥利斯关艾欣亚当·P·西尔弗曼扎卡里·K·斯威尼乔于 2019-01-10 设计创作，主要内容包括：本公开总体上涉及如下Fc多肽二聚体,所述Fc多肽二聚体含有非天然转铁蛋白受体(TfR)结合位点,不大体上消耗体内网织红细胞,但保持结合于Fcg受体(FcgR)。本公开还涉及一种如下Fc多肽二聚体,所述Fc多肽二聚体含有所述Fc多肽中的一者上的特异性结合TfR的非天然位点；对含有所述TfR结合位点的所述Fc多肽的当结合于TfR时减少FcgR结合的一个修饰或多个修饰,其中另一Fc多肽不含TfR结合位点,但保持FcgR结合。(The present disclosure relates generally to Fc polypeptide dimers that contain a non-native transferrin receptor (TfR) binding site, do not substantially deplete in vivo erythrocytes, but remain bound to Fcg receptor (FcgR). The present disclosure also relates to an Fc polypeptide dimer comprising a non-native site on one of the Fc polypeptides that specifically binds a TfR; one or more modifications to said Fc polypeptide containing said TfR binding site that reduces FcgR binding when bound to TfR, wherein another Fc polypeptide does not contain a TfR binding site, but retains FcgR binding.)

1. A modified Fc polypeptide dimer, or dimer fragment thereof, that:

(a) specifically binds to TfR;

(b) capable of binding to Fc γ receptors (Fc γ R); and is

2. A modified Fc polypeptide dimer, or dimer fragment thereof, comprising:

(a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising (i) a TfR binding site and (ii) one or more amino acid modifications that decrease fcyr binding when bound to a TfR; and

(b) A second Fc polypeptide that does not comprise a TfR binding site or any modification that reduces FcyR binding.

3. The modified Fc polypeptide dimer of claim 1 or 2, wherein the TfR binding site comprises a modified CH3 domain.

4. The modified Fc polypeptide dimer of claim 3, wherein the modified CH3 domain is derived from a human IgG1, IgG2, IgG3, or IgG4 CH3 domain.

5. The modified Fc polypeptide dimer of claim 3 or 4, wherein the modified CH3 domain comprises five, six, seven, eight, or nine substitutions in a set of amino acid positions according to EU numbering comprising 384, 386, 387, 388, 389, 390, 413, 416, and 421.

6. The modified Fc polypeptide dimer of claim 5, wherein the modified CH3 domain further comprises one, two, three, or four substitutions at positions comprising 380, 391, 392, and 415.

7. The modified Fc polypeptide dimer of claim 5 or 6, wherein the modified CH3 domain further comprises one, two, or three substitutions at positions comprising 414, 424, and 426.

8. The modified Fc polypeptide dimer of any one of claims 1 to 7, wherein the modified Fc polypeptide dimer binds to the top domain of TfR.

9. The modified Fc polypeptide dimer of claim 8, wherein the modified Fc polypeptide dimer binds to TfR without inhibiting the binding of transferrin to TfR.

10. The modified Fc polypeptide dimer of claim 8 or 9, wherein the modified Fc polypeptide dimer binds to an epitope comprising amino acid 208 of TfR.

11. The modified Fc polypeptide dimer of any one of claims 5 to 10, wherein the modified CH3 domain comprises a Trp at position 388.

12. The modified Fc polypeptide dimer of any one of claims 5 to 11, wherein the modified CH3 domain comprises an aromatic amino acid at position 421.

13. The modified Fc polypeptide dimer of claim 12, wherein the aromatic amino acid at position 421 is Trp or Phe.

14. The modified Fc polypeptide dimer of any one of claims 5 to 10, wherein the modified CH3 domain comprises at least one position selected from: position 384 is Leu, Tyr, Met or Val; position 386 is Leu, Thr, His, or Pro; val, Pro, or an acidic amino acid at position 387; position 388 is Trp; position 389 Val, Ser, or Ala; position 413 Glu, Ala, Ser, Leu, Thr, or Pro; position 416 is Thr or an acidic amino acid; and position 421 is Trp, Tyr, His or Phe.

15. The modified Fc polypeptide dimer of claim 14, wherein the modified CH3 domain comprises two, three, four, five, six, seven, or eight positions selected from: position 384 is Leu, Tyr, Met or Val; position 386 is Leu, Thr, His, or Pro; val, Pro, or an acidic amino acid at position 387; position 388 is Trp; position 389 Val, Ser, or Ala; position 413 Glu, Ala, Ser, Leu, Thr, or Pro; position 416 is Thr or an acidic amino acid; and position 421 is Trp, Tyr, His or Phe.

16. The modified Fc polypeptide dimer of any one of claims 5 to 15, wherein the modified CH3 domain comprises a Leu or Met at position 384; leu, His or Pro at position 386; val at position 387; a Trp at position 388; val or Ala at position 389; pro at position 413; thr at position 416; and/or Trp at location 421.

17. The modified Fc polypeptide dimer of claim 16, wherein the modified CH3 domain further comprises a Ser, Thr, Gln, or Phe at position 391.

18. The modified Fc polypeptide dimer of claim 16 or 17, wherein the modified CH3 domain further comprises Trp, Tyr, Leu, or Gln at position 380.

19. The modified Fc polypeptide dimer of any one of claims 16 to 18, wherein the modified CH3 domain further comprises Gln, Phe, or His at position 392.

20. The modified Fc polypeptide dimer of claim 16 or 17, wherein the modified CH3 domain further comprises a Trp at position 380 and/or a Gln at position 392.

21. The modified Fc polypeptide dimer of any one of claims 14 to 20, wherein the modified CH3 domain further comprises one, two, or three positions selected from: position 414 is Lys, Arg, Gly, or Pro; position 424 is Ser, Thr, Glu, or Lys; and position 426 Ser, Trp or Gly.

22. The modified Fc polypeptide dimer of any one of claims 5 to 15, wherein the modified CH3 domain comprises Tyr at position 384, Thr at position 386, Glu or Val at position 387, Trp at position 388, Ser at position 389, Ser or Thr at position 413, Glu at position 416, and/or Phe at position 421.

23. The modified Fc polypeptide dimer of claim 22, wherein the modified CH3 domain further comprises Trp, Tyr, Leu, or Gln at position 380.

24. The modified Fc polypeptide dimer of claim 22 or 23, wherein the modified CH3 domain further comprises a Glu at position 415.

25. The modified Fc polypeptide dimer of claim 22, wherein the modified CH3 domain further comprises a Trp at position 380 and/or a Glu at position 415.

26. The modified Fc polypeptide dimer of any one of claims 22 to 25, wherein the modified CH3 domain comprises an Asn at position 390.

27. The modified Fc polypeptide dimer of any one of claims 6 to 10, wherein the modified CH3 domain comprises one or more of the following substitutions: trp at position 380; thr at position 386; a Trp at position 388; val at position 389; ser or Thr at position 413; a Glu at position 415; and/or Phe at position 421.

28. The modified Fc polypeptide dimer of any one of claims 5 to 27, wherein the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 111 and 217 of any one of SEQ ID NOs 4-29 and 64-127.

29. The modified Fc polypeptide dimer of claim 28, wherein the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 111-217 of any one of SEQ ID NOs 66, 68, 94, 107-109, 119, and 268-270.

30. The modified Fc polypeptide dimer of any one of claims 5 to 27, wherein the modified CH3 domain has at least 85% identity to amino acid 111 and 217 of SEQ ID NO 1, with the proviso that the percentage identity does not comprise the set of positions 384, 386, 387, 388, 389, 390, 413, 416 and 421 according to EU numbering.

31. The modified Fc polypeptide dimer of any one of claims 5 to 30, wherein the modified CH3 domain comprises amino acids 154-160 and/or 183-191 of any one of SEQ ID NOS 4-29 and 125-127.

32. The modified Fc polypeptide dimer of any one of claims 6 to 10, wherein the modified CH3 domain comprises at least one position selected from: position 380 is Trp, Leu or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

33. The modified Fc polypeptide dimer of claim 32, wherein the modified CH3 domain comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from: position 380 is Trp, Leu or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

34. The modified Fc polypeptide dimer of claim 33, wherein the modified CH3 domain comprises the following 11 positions: position 380 is Trp, Leu or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

35. The modified Fc polypeptide dimer of claim 33 or 34, wherein the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 111-217 of any one of SEQ ID NOS 4-29 and 64-127.

36. The modified Fc polypeptide dimer of claim 35, wherein the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 111-217 of any one of SEQ ID NOs 66, 68, 94, 107-109, 119, and 268-270.

37. The modified Fc polypeptide dimer of claim 35, wherein no deletion or substitution occurs at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the positions corresponding to positions 380, 384, 386, 384, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426 according to the EU numbering scheme.

38. The modified Fc polypeptide dimer of claim 3, wherein the modified CH3 domain comprises the sequence of any one of SEQ ID NOS 38-61 and 131-173.

39. The modified Fc polypeptide dimer of any one of claims 5 to 37, wherein the modified CH3 domain further comprises (i) a Trp at position 366 or (ii) a Ser at position 366, an Ala at position 368, and a Val at position 407 according to the EU numbering scheme.

40. The modified Fc polypeptide dimer of any one of claims 5 to 39, wherein the corresponding unmodified CH3 domain is a human IgG1, IgG2, IgG3, or IgG4 CH3 domain.

41. The modified Fc polypeptide dimer of any one of claims 2 to 40, wherein the amino acid modification that reduces Fc γ R binding when binding to TfR comprises Ala at position 234 and position 235 according to the EU numbering scheme.

42. The modified Fc polypeptide dimer of any one of claims 2 to 41, wherein the first Fc polypeptide and/or the second Fc polypeptide comprises an amino acid modification that increases serum half-life.

43. The modified Fc-polypeptide dimer of claim 42, wherein the amino acid modification to increase serum half-life comprises (i) a Leu at position 428 and a Ser at position 434, or (ii) a Ser or Ala at position 434, according to the EU numbering scheme.

44. The modified Fc polypeptide dimer of any one of claims 1 to 43, wherein the first Fc polypeptide and/or the second Fc polypeptide is further fused to a Fab.

45. A modified Fc polypeptide dimer according to any one of claims 2 to 44, wherein the first Fc polypeptide comprises knob mutation T366W and the second Fc polypeptide comprises hole mutations T366S, L368A, and Y407V according to the EU numbering scheme.

46. A modified Fc polypeptide dimer according to any one of claims 2 to 44, wherein the first Fc polypeptide comprises hole mutations T366S, L368A, and Y407V and the second Fc polypeptide comprises knob mutation T366W according to the EU numbering scheme.

47. A modified Fc polypeptide dimer, comprising:

(a) a first Fc polypeptide that specifically binds TfR, said first Fc polypeptide comprising a TfR binding site, amino acid modifications L234A and L235A according to the EU numbering scheme, and a knob mutation T366W, and

(b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding.

48. The modified Fc polypeptide dimer of claim 47, wherein the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 178, 190, 202, 214, 226, 238, 252, 286, 298, and 310.

49. The modified Fc polypeptide dimer of claim 47 or 48, wherein the second Fc polypeptide comprises the sequence of SEQ ID NO: 397.

50. A modified Fc polypeptide dimer, comprising:

(b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding.

51. The modified Fc polypeptide dimer of claim 50, wherein the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 323, 330, 337, 344, 351, 358, 365, 372, 379, and 386.

52. The modified Fc polypeptide dimer of claim 50 or 51, wherein the second Fc polypeptide comprises the sequence of SEQ ID NO: 397.

53. A modified Fc polypeptide dimer, comprising:

(b) A second Fc polypeptide comprising hole mutations T366S, L368A and Y407V and the amino acid modification N434S with or without M428L according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding.

54. The modified Fc polypeptide dimer of claim 53, wherein the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 178, 190, 202, 214, 226, 238, 252, 286, 298, and 310.

55. The modified Fc polypeptide dimer of claim 53 or 54, wherein the second Fc polypeptide comprises the sequence of SEQ ID NO 407.

56. A modified Fc polypeptide dimer, comprising:

(b) a second Fc polypeptide comprising hole mutations T366S, L368A and Y407V and the amino acid modification N434S with or without M428L according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding.

57. The modified Fc polypeptide dimer of claim 56, wherein the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 323, 330, 337, 344, 351, 358, 365, 372, 379, and 386.

58. The modified Fc polypeptide dimer of claim 56 or 57, wherein the second Fc polypeptide comprises the sequence of seq id NO 407.

59. A modified Fc polypeptide dimer, comprising:

(a) a first Fc polypeptide that specifically binds TfR, said first Fc polypeptide comprising a TfR binding site, amino acid modifications L234A and L235A and hole mutations T366S, L368A and Y407V according to the EU numbering scheme, and

(b) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding.

60. The modified Fc polypeptide dimer of claim 59, wherein the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 184, 196, 208, 220, 232, 244, 280, 292, 304, and 316.

61. The modified Fc polypeptide dimer of claim 59 or 60, wherein the second Fc polypeptide comprises the sequence of SEQ ID NO: 391.

62. A modified Fc polypeptide dimer, comprising:

(a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A and L235A; hole mutations T366S, L368A, and Y407V; and amino acid modifications N434S with or without M428L, and

(b) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding.

63. The modified Fc polypeptide dimer of claim 62, wherein the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 326, 333, 340, 347, 354, 361, 368, 375, 382, and 389.

64. The modified Fc polypeptide dimer of claim 62 or 63, wherein the second Fc polypeptide comprises the sequence of SEQ ID NO: 391.

65. A modified Fc polypeptide dimer, comprising:

(b) A second Fc polypeptide comprising knob mutation T366W and amino acid modification N434S with or without M428L according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

66. The modified Fc polypeptide dimer of claim 65, wherein the first Fc polypeptide comprises the sequence of any one of SEQ id nos 184, 196, 208, 220, 232, 244, 280, 292, 304, and 316.

67. The modified Fc polypeptide dimer of claim 65 or 66, wherein the second Fc polypeptide comprises the sequence of seq id No. 404.

68. A modified Fc polypeptide dimer, comprising:

(b) a second Fc polypeptide comprising knob mutation T366W and amino acid modification N434S with or without M428L according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

69. The modified Fc polypeptide dimer of claim 68, wherein the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 326, 333, 340, 347, 354, 361, 368, 375, 382, and 389.

70. The modified Fc polypeptide dimer of claim 68 or 69, wherein the second Fc polypeptide comprises the sequence of seq id No. 404.

71. The modified Fc polypeptide dimer of any one of claims 1 to 70, wherein the modified Fc polypeptide dimer does not substantially deplete reticulocytes.

72. The modified Fc polypeptide dimer of claim 71, wherein the amount of reticulocytes consumed after administration of the modified Fc polypeptide dimer is less than the amount of reticulocytes consumed after administration of a control.

73. The modified Fc polypeptide dimer of claim 72, wherein the amount of reticulocytes consumed following administration of the modified Fc polypeptide dimer is less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1% of the amount of reticulocytes consumed following administration of a control.

74. The modified Fc polypeptide dimer of claim 71, wherein the amount of reticulocytes remaining after administration of the modified Fc polypeptide dimer is greater than the amount of reticulocytes remaining after administration of a control.

75. The modified Fc polypeptide dimer of claim 74, wherein the amount of reticulocytes remaining after administration of the modified Fc polypeptide dimer is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater than the amount of reticulocytes remaining after administration of a control.

76. The modified Fc polypeptide dimer of any one of claims 1 to 70, wherein the modified Fc polypeptide dimer does not substantially deplete reticulocytes in bone marrow.

77. The modified Fc polypeptide dimer of claim 76, wherein the amount of reticulocytes consumed in the bone marrow after administration of the modified Fc polypeptide dimer is less than the amount of reticulocytes consumed in the bone marrow after administration of a control.

78. The modified Fc polypeptide dimer of claim 77, wherein the amount of reticulocytes consumed in the bone marrow after administration of the modified Fc polypeptide dimer is less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1% of the amount of reticulocytes consumed in the bone marrow after administration of a control.

79. The modified Fc polypeptide dimer of claim 76, wherein the amount of reticulocytes remaining in the bone marrow after administration of the modified Fc polypeptide dimer is greater than the amount of reticulocytes remaining in the bone marrow after administration of a control.

80. The modified Fc polypeptide dimer of claim 79, wherein the amount of reticulocytes remaining in the bone marrow after administration of the modified Fc polypeptide dimer is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater than the amount of reticulocytes remaining in the bone marrow after administration of a control.

81. The modified Fc polypeptide dimer of any one of claims 72 to 75 and 77 to 80, wherein the control is a corresponding TfR-binding Fc dimer with complete effector function and/or without a mutation that reduces fcyr binding.

82. An Fc polypeptide dimer-Fab fusion protein capable of active transport across the BBB, comprising:

(a) an antibody variable region capable of binding an antigen, or an antigen-binding fragment thereof; and

(b) A modified Fc polypeptide dimer comprising (i) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site and one or more amino acid modifications that reduce fcyr binding when bound to a TfR, and (ii) a second Fc polypeptide that does not contain a TfR binding site or any modifications that reduce fcyr binding.

83. The Fc polypeptide dimer-Fab fusion protein of claim 82, wherein the amino acid modification that reduces fcyr binding when binding to TfR comprises Ala at position 234 and at position 235 according to the EU numbering scheme.

84. The Fc polypeptide dimer-Fab fusion protein of claim 82 or 83, wherein the first Fc polypeptide and/or the second Fc polypeptide comprises an amino acid modification that increases serum half-life.

85. The Fc polypeptide dimer-Fab fusion protein of claim 84, wherein the amino acid modification to increase serum half-life comprises (i) a Leu at position 428 and a Ser at position 434, or (ii) a Ser or Ala at position 434, according to the EU numbering scheme.

86. The Fc polypeptide dimer-Fab fusion protein of any one of claims 82 to 85, wherein the antibody variable region sequence comprises a Fab domain.

87. The Fc polypeptide dimer-Fab fusion protein of any one of claims 82 to 86, wherein the antibody variable region sequence comprises two antibody variable region heavy chains and two antibody variable region light chains, or fragments thereof, respectively.

88. A pharmaceutical composition comprising the modified Fc polypeptide dimer of any one of claims 1-81 and a pharmaceutically acceptable carrier.

89. A pharmaceutical composition comprising the Fc polypeptide dimer-Fab fusion protein of any one of claims 82-87 and a pharmaceutically acceptable carrier.

90. A method of transcytosis of a composition across an endothelium, said method comprising contacting said endothelium with a composition comprising a modified Fc polypeptide dimer of any one of claims 1-81.

91. A method of transcytosing a composition across an endothelium, said method comprising contacting said endothelium with a composition comprising an Fc polypeptide dimer-Fab fusion protein of any one of claims 82-87.

92. The method of claim 90 or 91, wherein the endothelium is the BBB.

Technical Field

The present disclosure relates to modified Fc polypeptide dimers that bind to transferrin receptor (TfR) and that induce at least one effector functional activity (e.g., antibody-dependent cellular cytotoxicity (ADCC)) without causing substantial depletion of reticulocytes.

Background

TfR has been proposed as a receptor-mediated transcytosis of therapeutic agents across the Blood Brain Barrier (BBB). While TfR is expressed on endothelial cells that form the BBB, TfR is also expressed on other cell types, including reticulocytes. Previous work has shown that anti-TfR antibodies can deplete reticulocytes from the circulation.

Because reticulocyte depletion is mediated by effector function activity, this toxicity can be overcome by forming modifications that reduce or eliminate effector function. However, this approach precludes the use of therapeutic agents where effector function is desired or required.

Therefore, a means of delivering an effector function positive therapeutic agent to the brain without causing reticulocyte depletion would be advantageous.

Disclosure of Invention

We have developed Fc polypeptides modified to bind to TfR. These Fc polypeptides are capable of active transport into the brain by receptor-mediated endocytic transport through binding to TfR at the BBB. Because Fc polypeptides are capable of inducing effector functional activities, including ADCC, by binding to Fc γ receptors (Fc γ rs) on immune cells, and because tfrs are expressed on reticulocytes, simultaneous binding of these polypeptides to reticulocytes and Fc γ rs can lead to depletion of the reticulocytes. Although effector function can be reduced or eliminated by introducing mutations into the Fc polypeptide, in some therapeutic applications this is undesirable.

The present disclosure is based on the development of modified Fc polypeptide dimers that bind TfR, cross the BBB, and retain effector functional activity but do not cause substantial TfR-dependent toxicity, including depletion of reticulocytes. Such dimers may be engineered as described herein.

In one aspect, the present disclosure provides a modified Fc polypeptide dimer, or dimer fragment thereof, that: (a) comprises a TfR binding site that specifically binds to TfR; (b) capable of binding to Fc γ receptors (Fc γ R); and (c) does not substantially consume in vivo reticulocytes.

In one aspect, the present disclosure provides a modified Fc polypeptide dimer, or dimer fragment thereof, comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising (i) a TfR binding site and (ii) one or more amino acid modifications that, e.g., reduce fcyr binding when bound to a TfR (e.g., but with limited or no fcyr binding reduction when not bound to a TfR); and (b) a second Fc polypeptide that does not contain a TfR binding site or any modification that reduces fcyr binding.

In some embodiments of this aspect, the TfR binding site comprises a modified CH3 domain. In some embodiments, the modified CH3 domain is derived from a human IgG1, IgG2, IgG3, or IgG4 CH3 domain. In particular embodiments, the modified CH3 domain comprises five, six, seven, eight, or nine substitutions in a set of amino acid positions comprising 384, 386, 387, 388, 389, 390, 413, 416, and 421 according to EU numbering. In particular embodiments, the modified CH3 domain further comprises one, two, three, or four substitutions at positions including 380, 391, 392, and 415.

In some embodiments, the modified CH3 domain further comprises one, two, or three substitutions at positions including 414, 424, and 426.

In some embodiments, the modified Fc polypeptide dimer binds to the top domain of a TfR. In some embodiments, the modified Fc polypeptide dimer binds to TfR without inhibiting the binding of transferrin to TfR. In particular embodiments, the modified Fc polypeptide dimer binds to an epitope comprising amino acid 208 of TfR.

In some embodiments, the modified CH3 domain comprises a Trp at position 388. In some embodiments, the modified CH3 domain comprises an aromatic amino acid at position 421. In particular embodiments, the aromatic amino acid at position 421 is Trp or Phe.

In some embodiments of this aspect, the modified CH3 domain comprises at least one position selected from: position 384 is Leu, Tyr, Met or Val; position 386 is Leu, Thr, His, or Pro; val, Pro, or an acidic amino acid at position 387; position 388 is Trp; position 389 Val, Ser, or Ala; position 413 Glu, Ala, Ser, Leu, Thr, or Pro; position 416 is Thr or an acidic amino acid; and position 421 is Trp, Tyr, His or Phe.

In some embodiments of this aspect, the modified CH3 domain comprises two, three, four, five, six, seven, or eight positions selected from: position 384 is Leu, Tyr, Met or Val; position 386 is Leu, Thr, His, or Pro; val, Pro, or an acidic amino acid at position 387; position 388 is Trp; position 389 Val, Ser, or Ala; position 413 Glu, Ala, Ser, Leu, Thr, or Pro; position 416 is Thr or an acidic amino acid; and position 421 is Trp, Tyr, His or Phe.

In some embodiments of this aspect, the modified CH3 domain comprises a Leu or Met at position 384; leu, His or Pro at position 386; val at position 387; a Trp at position 388; val or Ala at position 389; pro at position 413; thr at position 416; and/or Trp at location 421.

In some embodiments, the modified CH3 domain further comprises Ser, Thr, Gln, or Phe at position 391. In some embodiments, the modified CH3 domain further comprises Trp, Tyr, Leu, or Gln at position 380. In some embodiments, the modified CH3 domain further comprises Gln, Phe, or His at position 392. In some embodiments, the modified CH3 domain further comprises a Trp at position 380 and/or a Gln at position 392.

In some embodiments, the modified CH3 domain further comprises one, two, or three positions selected from: position 414 is Lys, Arg, Gly, or Pro; position 424 is Ser, Thr, Glu, or Lys; and position 426 Ser, Trp or Gly.

In some embodiments, the modified CH3 domain comprises a Tyr at position 384, a Thr at position 386, a Glu or Val at position 387, a Trp at position 388, a Ser at position 389, a Ser or Thr at position 413, a Glu at position 416, and/or a Phe at position 421. In some embodiments, the modified CH3 domain further comprises Trp, Tyr, Leu, or Gln at position 380. In some embodiments, the modified CH3 domain further comprises a Glu at position 415. In some embodiments, the modified CH3 domain further comprises a Trp at position 380 and/or a Glu at position 415. In some embodiments, the modified CH3 domain comprises an Asn at position 390.

In some embodiments, the modified CH3 domain comprises one or more of the following substitutions: trp at position 380; thr at position 386; a Trp at position 388; val at position 389; ser or Thr at position 413; a Glu at position 415; and/or Phe at position 421.

In some embodiments, the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 111-217 of any one of SEQ ID NOs 4-29 and 64-127. In particular embodiments, the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 111-217 of any one of SEQ ID NOs 66, 68, 94, 107-109, 119, and 268-270.

In some embodiments, the modified CH3 domain has at least 85% identity to amino acids 111-217 of SEQ ID No. 1, with the proviso that the percentage identity does not include the set of positions 384, 386, 387, 388, 389, 390, 413, 416 and 421 according to EU numbering.

In some embodiments, the modified CH3 domain comprises amino acids 154-160 and/or 183-191 of any one of SEQ ID NOs 4-29 and 125-127.

In some embodiments, the modified CH3 domain comprises at least one position selected from: position 380 is Trp, Leu or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe. In some embodiments, the modified CH3 domain comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from: position 380 is Trp, Leu or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

In some embodiments, the modified CH3 domain comprises the following 11 positions: position 380 is Trp, Leu or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe.

In some embodiments, the deletion or substitution is absent at a residue at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the positions corresponding to positions 380, 384, 386, 384, 388, 389, 390, 391, 392, 413, 414, 415, 416, 421, 424, and 426 according to EU numbering scheme.

In some embodiments, the modified CH3 domain comprises the sequence of any one of SEQ ID NOs 38-61 and 131-173.

In some embodiments of this aspect, the modified CH3 domain further comprises (i) a Trp at position 366 or (ii) a Ser at position 366, an Ala at position 368, and a Val at position 407 according to the EU numbering scheme.

In some embodiments of this aspect, the corresponding unmodified CH3 domain is a human IgG1, IgG2, IgG3, or IgG4 CH3 domain.

In some embodiments of this aspect, the amino acid modification that decreases Fc γ R binding, e.g., when binding to TfR, comprises Ala at position 234 and at position 235 according to EU numbering scheme. In some embodiments, the amino acid modification that reduces Fc γ R binding, e.g., when binding to TfR, further comprises Gly at position 329 according to the EU numbering scheme.

In some embodiments of this aspect, the first Fc polypeptide and/or the second Fc polypeptide comprises an amino acid modification that increases serum stability (e.g., serum half-life). In some embodiments, the amino acid modification that increases serum stability (e.g., serum half-life) comprises Tyr at position 252, Thr at position 254, and Glu at position 256 according to the EU numbering scheme. In some embodiments, the amino acid modification that increases serum stability (e.g., serum half-life) comprises (i) a Leu at position 428 and a Ser at position 434, or (ii) a Ser or Ala at position 434, according to EU numbering scheme.

In some embodiments of this aspect, the modified Fc polypeptide dimer is further fused to a Fab. In some embodiments, the first Fc polypeptide and/or the second Fc polypeptide is further fused to a Fab.

In some embodiments, the first Fc polypeptide comprises knob mutation T366W and the second Fc polypeptide comprises hole mutations T366S, L368A, and Y407V according to the EU numbering scheme. In some embodiments, the first Fc polypeptide comprises hole mutations T366S, L368A, and Y407V and the second Fc polypeptide comprises knob mutation T366W according to the EU numbering scheme.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds TfR, said first Fc polypeptide comprising a TfR binding site, amino acid modifications L234A and L235A, and knob mutation T366W according to EU numbering scheme, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 178, 190, 202, 214, 226, 238, 252, 286, 298, and 310. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 397.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; and a knob mutation, T366W, and (b) a second Fc polypeptide comprising hole mutations, T366S, L368A, and Y407V according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 179, 191, 203, 215, 227, 239, 275, 287, 299, and 311. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 397.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds TfR, said first Fc polypeptide comprising a TfR binding site, amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modifications M252Y, S254T, and T256E according to EU numbering scheme, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 181, 193, 205, 217, 229, 241, 277, 289, 301, and 313. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 397.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds TfR, which first Fc polypeptide comprises a TfR binding site, amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modification N434S with or without M428L according to EU numbering, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to EU numbering, and does not comprise a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 323, 330, 337, 344, 351, 358, 365, 372, 379, and 386. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 397.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; pestle mutation T366W; and amino acid modifications M252Y, S254T and T256E, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A and Y407V according to the EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 182, 194, 206, 218, 230, 242, 278, 290, 302, and 314. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 397.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; pestle mutation T366W; and amino acid modification N434S with or without M428L, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 324, 331, 338, 345, 352, 359, 366, 373, 380, and 387. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 397.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds TfR, said first Fc polypeptide comprising a TfR binding site, amino acid modifications L234A and L235A and knob mutation T366W according to EU numbering scheme, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A and Y407V and amino acid modifications M252Y, S254T and T256E according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 178, 190, 202, 214, 226, 238, 252, 286, 298, and 310. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 400.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds TfR, said first Fc polypeptide comprising a TfR binding site, amino acid modifications L234A and L235A and knob mutation T366W according to EU numbering scheme, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A and Y407V and amino acid modification N434S with or without M428L according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 178, 190, 202, 214, 226, 238, 252, 286, 298, and 310. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO 407.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; and a knob mutation T366W, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V and amino acid modifications M252Y, S254T, and T256E according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 179, 191, 203, 215, 227, 239, 275, 287, 299, and 311. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 400.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; and a knob mutation T366W, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V and amino acid modifications N434S with or without M428L according to EU numbering scheme, and not containing a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 179, 191, 203, 215, 227, 239, 275, 287, 299, and 311. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO 407.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds TfR, said first Fc polypeptide comprising a TfR binding site, amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modifications M252Y, S254T, and T256E according to EU numbering scheme, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, and amino acid modifications M252Y, S254T, and T256E according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 181, 193, 205, 217, 229, 241, 277, 289, 301, and 313. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 400.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds TfR, which first Fc polypeptide comprises a TfR binding site, amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modification N434S with or without M428L according to EU numbering scheme, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, and amino acid modification N434S with or without M428L according to EU numbering scheme, and which does not comprise a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 323, 330, 337, 344, 351, 358, 365, 372, 379, and 386. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO 407.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; pestle mutation T366W; and amino acid modifications M252Y, S254T and T256E, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A and Y407V and amino acid modifications M252Y, S254T and T256E according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 182, 194, 206, 218, 230, 242, 278, 290, 302, and 314. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 400.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; pestle mutation T366W; and amino acid modification N434S with or without addition of M428L, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V and amino acid modification N434S with or without addition of M428L according to the EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 324, 331, 338, 345, 352, 359, 366, 373, 380, and 387. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO 407.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds TfR, which first Fc polypeptide comprises a TfR binding site, amino acid modifications L234A and L235A and hole mutations T366S, L368A and Y407V according to EU numbering scheme, and (b) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering scheme and is free of a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 184, 196, 208, 220, 232, 244, 280, 292, 304, and 316. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 391.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; and hole mutations T366S, L368A, and Y407V, and (b) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 185, 197, 209, 221, 233, 245, 281, 293, 305, and 317. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 391.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A and L235A; hole mutations T366S, L368A, and Y407V; and amino acid modifications M252Y, S254T, and T256E, and (b) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 187, 199, 211, 223, 235, 247, 283, 295, 307, and 319. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 391.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A and L235A; hole mutations T366S, L368A, and Y407V; and amino acid modification N434S with or without M428L, and (b) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering scheme and not comprising a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 326, 333, 340, 347, 354, 361, 368, 375, 382, and 389. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 391.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; hole mutations T366S, L368A, and Y407V; and amino acid modifications M252Y, S254T, and T256E, and (b) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 188, 200, 212, 224, 236, 248, 284, 296, 308, and 320. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 391.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; hole mutations T366S, L368A, and Y407V; and amino acid modification N434S with or without M428L, and (b) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering scheme and not comprising a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 327, 334, 341, 348, 355, 362, 369, 376, 383, and 390. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 391.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds TfR, which first Fc polypeptide comprises a TfR binding site, amino acid modifications L234A and L235A and hole mutations T366S, L368A and Y407V according to EU numbering scheme, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modifications M252Y, S254T and T256E according to EU numbering scheme, and does not comprise a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 184, 196, 208, 220, 232, 244, 280, 292, 304, and 316. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO 394.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds TfR, which first Fc polypeptide comprises a TfR binding site, amino acid modifications L234A and L235A and hole mutations T366S, L368A and Y407V according to EU numbering scheme, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modification N434S with or without M428L according to EU numbering scheme, and does not comprise a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 184, 196, 208, 220, 232, 244, 280, 292, 304, and 316. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 404.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; and hole mutations T366S, L368A, and Y407V, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modifications M252Y, S254T, and T256E according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 185, 197, 209, 221, 233, 245, 281, 293, 305, and 317. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO 394.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; and hole mutations T366S, L368A, and Y407V, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modifications N434S with or without M428L according to the EU numbering scheme, and which does not contain a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 185, 197, 209, 221, 233, 245, 281, 293, 305, and 317. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO: 404.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A and L235A; hole mutations T366S, L368A and Y407V and amino acid modifications M252Y, S254T and T256E, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modifications M252Y, S254T and T256E according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 187, 199, 211, 223, 235, 247, 283, 295, 307, and 319. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO 394.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A and L235A; hole mutations T366S, L368A, and Y407V; and amino acid modification N434S with or without M428L, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modification N434S with or without M428L according to EU numbering scheme, and which does not contain a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 326, 333, 340, 347, 354, 361, 368, 375, 382, and 389. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ id No. 404.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; hole mutations T366S, L368A, and Y407V; and amino acid modifications M252Y, S254T, and T256E, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modifications M252Y, S254T, and T256E according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 188, 200, 212, 224, 236, 248, 284, 296, 308, and 320. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO 394.

In another aspect, the present disclosure provides a modified Fc polypeptide dimer comprising: (a) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; hole mutations T366S, L368A, and Y407V; and amino acid modification N434S with or without M428L, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modification N434S with or without M428L according to EU numbering scheme, and which does not contain a TfR binding site or any modification that reduces Fc γ R binding. In some embodiments, the first Fc polypeptide comprises the sequence of any one of SEQ ID NOs 327, 334, 341, 348, 355, 362, 369, 376, 383, and 390. In some embodiments, the second Fc polypeptide comprises the sequence of SEQ ID NO 404.

In any aspect of the modified Fc polypeptide dimers described herein, the modified Fc polypeptide dimers do not substantially consume reticulocytes (e.g., circulating reticulocytes). In some embodiments, the amount of reticulocytes consumed after administration of the modified Fc polypeptide dimer is less than the amount of reticulocytes consumed after administration of the control. In some embodiments, the amount of reticulocytes consumed following administration of the modified Fc polypeptide dimer is less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1% of the amount of reticulocytes consumed following administration of the control. In some embodiments, the amount of reticulocytes remaining after administration of the modified Fc polypeptide dimer is greater than the amount of reticulocytes remaining after administration of the control. In some embodiments, the amount of reticulocytes remaining after administration of the modified Fc polypeptide dimer is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater than the amount of reticulocytes remaining after administration of the control.

In any aspect of the modified Fc polypeptide dimers described herein, the modified Fc polypeptide dimers do not substantially deplete reticulocytes in bone marrow. In some embodiments, the amount of reticulocytes consumed in the bone marrow after administration of the modified Fc polypeptide dimer is less than the amount of reticulocytes consumed in the bone marrow after administration of the control. In some embodiments, the amount of reticulocytes consumed in bone marrow after administration of the modified Fc polypeptide dimer is less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1% of the amount of reticulocytes consumed in bone marrow after administration of the control. In some embodiments, the amount of reticulocytes remaining in the bone marrow after administration of the modified Fc polypeptide dimer is greater than the amount of reticulocytes remaining in the bone marrow after administration of the control. In some embodiments, the amount of reticulocytes remaining in the bone marrow after administration of the modified Fc polypeptide dimer is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater than the amount of reticulocytes remaining in the bone marrow after administration of the control.

In some embodiments, the control is a corresponding TfR-binding Fc dimer (i.e., having the same mutation causing TfR binding as the modified Fc polypeptide dimer described above) with complete effector function and/or without a mutation that reduces fcyr binding.

In another aspect, the present disclosure provides an Fc polypeptide dimer-Fab fusion protein capable of active transport across the BBB, the Fc polypeptide dimer-Fab fusion protein comprising: (a) an antibody variable region capable of binding an antigen, or an antigen-binding fragment thereof; and (b) a modified Fc polypeptide dimer comprising (i) a first Fc polypeptide that specifically binds a TfR, said first Fc polypeptide comprising a TfR binding site and one or more amino acid modifications that, e.g., reduce fcyr binding when bound to a TfR (e.g., but with limited or no fcyr binding reduction when not bound to a TfR), and (ii) a second Fc polypeptide that does not contain a TfR binding site or any modification that reduces fcyr binding.

In some embodiments of this aspect, the amino acid modification that decreases Fc γ R binding, e.g., when binding to TfR, comprises Ala at position 234 and at position 235 according to EU numbering scheme. In particular embodiments, the amino acid modification that reduces Fc γ R binding, e.g., when binding to TfR, further comprises Gly at position 329 according to the EU numbering scheme.

In some embodiments of this aspect, the antibody variable region sequence comprises a Fab domain. In some embodiments, the antibody variable region sequence comprises two antibody variable region heavy chains and two antibody variable region light chains, or fragments thereof.

In some embodiments, the Fc polypeptide or Fc polypeptide dimer is fucose-deficient or nonfucosylated (e.g., as described herein).

In another aspect, the present disclosure provides a pharmaceutical composition comprising a modified Fc polypeptide dimer as described herein and a pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides a pharmaceutical composition comprising an Fc polypeptide dimer-Fab fusion protein described herein and a pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides a method of transcytosis of a composition across an endothelium, comprising contacting the endothelium with a composition comprising a modified Fc polypeptide dimer as described herein. In some embodiments, the endothelium is the BBB.

In another aspect, the present disclosure provides a method of transcytosis of a composition across an endothelium, the method comprising contacting the endothelium with a composition comprising an Fc polypeptide dimer-Fab fusion protein described herein. In some embodiments, the endothelium is the BBB.

Drawings

Fig. 1A and 1B are diagrams illustrating the following cases: in human TfR knock-in (TfR)^ms/huKI) mice did not deplete reticulocytes in blood (fig. 1A) or bone marrow (fig. 1B) of TfR-binding Fc polypeptide dimers fused to anti-BACE 1 Fab, which were modified on both Fc polypeptides of the dimer with L234A and L235A (LALA) mutations (numbered with reference to the EU numbering scheme) to reduce Fc γ R binding.

Fig. 2A to 2D are diagrams showing the following cases: in human TfR knock-in (TfR) ^ms/huKI) mice were not depleted of blood by modified Fc polypeptide dimers fused to anti-BACE 1Fab, where TfR-binding Fc polypeptide dimers had a LALA mutation in cis configuration relative to the TfR binding site ("cis-LALA") (fig. 2A: at 25 mg/kg; FIG. 2C: at 50 mg/kg) or bone marrow (fig. 2B: at 25 mg/kg; FIG. 2D: at 50 mg/kg), while a similarly modified Fc polypeptide dimer fused to an anti-BACE 1Fab, wherein the Fc polypeptide dimer has a LALA mutation in trans relative to the TfR binding site, depletes reticulocytes in blood and bone marrow.

Fig. 3A and 3B are diagrams illustrating the following cases: the cis-LALA modified Fc polypeptide dimer fused to anti-BACE 1Fab (fig. 3A: ch3 c.35.21; fig. 3B: ch3c.35.23) and the modified Fc polypeptide with LALA mutations on both Fc polypeptides fused to anti-BACE 1Fab did not induce TfR mediated ADCC, whereas the hIgG1 with a TfR binding site but no LALA mutation induced ADCC on Ramos cells (Ramos cells) expressing endogenous TfR.

Fig. 4 is a diagram showing the following cases: TfR-binding Fc polypeptide dimer (ch3c.35.21) had no effect on TfR-mediated complement-dependent cytotoxicity (CDC) activity, while anti-TfR control antibody Ab204 induced CDC in CHO-hTfR cells.

Fig. 5 is a diagram showing the following cases: cis-LALA modified Fc polypeptide dimer fused to anti-BACE 1 Fab in primary human microglia induced pSyk protein levels similar to those observed in TfR binding polypeptide in the case of wild-type hIgG1, while modified Fc polypeptide dimer with LALA mutations on both Fc polypeptides fused to anti-BACE 1 Fab did not induce pSyk.

Fig. 6A and 6B are diagrams illustrating the following cases: hIgG1 with cis-LALA Fc polypeptide dimer and mCD20 Fab binding site elicited ADCC similar to anti-mCD 20 antibody and hIgG1 with TfR binding site and mCD20 Fab binding site (fig. 6A). Similarly, hIgG1 with cis-LALA Fc polypeptide dimer and hCD20 Fab binding site elicited Fab-mediated CDC to the same extent as anti-hCD 20 and hIgG1 with TfR binding site and hCD20 Fab binding site (fig. 6B).

Fig. 7A and 7B are diagrams illustrating the following cases: hIgG1 with cis-LALA Fc polypeptide dimer and mCD20 Fab binding site elicited robust B cell depletion similar to anti-mCD 20 antibody and hIgG1 with TfR binding site and mCD20 Fab binding site (fig. 7A and 7B). These results demonstrate that the cis-LALA modified Fc polypeptide dimer retains its Fc function and has Fab-mediated effector functions in vivo.

FIGS. 8A, 8B, and 8C are graphs demonstrating that mice treated with anti-A β with a TfR binding site in the case of cis-LALA Fc polypeptide dimer (CH3C.35.23.4) caused robust recruitment of microglia towards A β plaques (FIG. 8A: plaque area in case of microglia overlap%; FIG. 8B: same data corrected for control IgG) and were sized 30-125 μm²these results indicate that anti- Α β having cis-LALA Fc polypeptide dimer retains robust effector function against microglia recruitment and the ability to reduce some Α β plaques similar to anti- Α β.

Detailed Description

I. Introduction to the design reside in

The modified Fc polypeptide dimer comprising a TfR binding site is capable of crossing the BBB and transporting a therapeutic agent across the BBB. As described herein, these Fc polypeptide dimers, if not engineered to reduce effector function, can also deplete reticulocytes in vivo, as reticulocytes also express TfR. Reticulocyte depletion can be avoided by introducing modifications in the Fc polypeptide of the Fc polypeptide dimer that remove effector function, i.e., modifications that remove or reduce Fc γ receptor (Fc γ R) binding (e.g., substitutions numbered L234A and L235A (LALA) with reference to the EU numbering scheme). However, this approach is disadvantageous in situations where effector function is required when the Fab portion of the molecule binds to its target (e.g., a therapeutic target protein).

The present disclosure provides modified Fc polypeptide dimers that retain effector function but do not cause substantial reticulocyte depletion. These modified Fc polypeptide dimers are also referred to herein as "TfR-binding Fc polypeptide dimers that are positive for effector function". In some embodiments, only one of the two Fc polypeptides (but not both Fc polypeptides) of the TfR-binding Fc polypeptide dimer that is positive for effector function is modified to decrease effector function and bind TfR. The other Fc polypeptide of the modified Fc polypeptide dimer does not contain a TfR binding site or any modification that reduces effector function, but it may contain a mutation that enhances effector function. A TfR-binding Fc polypeptide dimer that is positive for effector function that contains a TfR binding site and a modification that reduces fcyr binding when bound to a TfR while the other Fc polypeptide does not contain a TfR binding site or reduces any modification of fcyr binding is said to have a cis configuration. The effect of these modified Fc polypeptide dimers having the cis configuration on reticulocytes was tested as described herein. These experiments demonstrate that by introducing both a TfR binding site and a mutation that reduces Fc γ R binding when bound to TfR into only one of the two polypeptides that form the modified Fc polypeptide dimer, the effector function following TfR binding can be reduced, resulting in TfR binding without substantial reticulocyte depletion.

As described in detail herein, modified Fc polypeptide dimers having different configurations are fused to fabs directed to the target of a therapeutic agent (e.g., CD20) to determine whether effector functions (e.g., ADCC and CDC) can be retained when the Fab binds its target and not TfR. As described in detail below, specific configurations of (a) modifications that reduce fcyr binding, e.g., when binding to TfR, and (b) modifications that cause TfR binding, in the modified Fc polypeptide dimer, when fused to Fab, can result in Fc polypeptide dimer-Fab fusions that still retain effector function (e.g., ADCC or CDC) but do not consume reticulocytes. This approach allows the use of TfR-mediated transport across the BBB while retaining effector function.

Accordingly, the present disclosure is directed, in part, to modified Fc polypeptide dimers that have been engineered to bind TfR with reduced effector function (e.g., ADCC or CDC) when bound to TfR, but which retain effector function (e.g., ADCC or CDC) when fused to the therapeutic Fab and bound to the target antigen of the Fab.

Definition of

As used herein, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" can include two or more such molecules, and the like.

As used herein, the terms "about" and "approximately," when used to modify a numerical value or an amount specified in a range, indicate that the numerical value and reasonable deviations from the stated value (e.g., ± 20%, ± 10%, or ± 5%) known to those skilled in the art are within the intended meaning of the stated value.

As used herein, the term "Fc polypeptide" refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide that is characterized by an Ig fold as a structural domain. The Fc polypeptide contains a constant region sequence including at least a CH2 domain and/or a CH3 domain and may contain at least a portion of a hinge region. In general, Fc polypeptides do not contain variable regions.

By "modified Fc polypeptide" is meant an Fc polypeptide having at least one mutation, e.g., substitution, deletion, or insertion, as compared to the wild-type immunoglobulin heavy chain Fc polypeptide sequence, but which retains the overall Ig folding or structure of the native Fc polypeptide.

As used herein, the term "Fc polypeptide dimer" refers to a dimer of two Fc polypeptides. In some embodiments, the Fc polypeptide dimer is capable of binding an Fc receptor (e.g., fcyr). In the Fc polypeptide dimer, two Fc polypeptides dimerize via interaction between two CH3 antibody constant domains. In some embodiments, the two Fc polypeptides may also dimerize via one or more disulfide bonds formed between the hinge domains of the two dimerizing Fc domain monomers. The Fc polypeptide dimer may be a wild-type Fc polypeptide dimer or a modified Fc polypeptide dimer. Wild-type Fc polypeptide dimers are formed by dimerization of two wild-type Fc polypeptides. The Fc polypeptide dimer may be a heterodimer or a homodimer.

As used herein, the term "modified Fc polypeptide dimer" refers to an Fc polypeptide dimer that contains at least one modified Fc polypeptide. In some embodiments, the modified Fc polypeptide dimer contains two modified Fc polypeptides. The modified Fc polypeptide dimer may be a homodimer (i.e., containing two identical modified Fc polypeptides) or a heterodimer (i.e., containing two different Fc polypeptides, wherein at least one of the two Fc polypeptides is a modified Fc polypeptide).

As used herein, "transferrin receptor" or "TfR" refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is set forth in SEQ ID NO 63. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee, accession number XP _ 003310238.1; rhesus monkey, NP _ 001244232.1; dog, NP _ 001003111.1; bovine, NP _ 001193506.1; mouse, NP _ 035768.1; rat, NP _ 073203.1; and chicken, NP _ 990587.1). The term "transferrin receptor" also encompasses dual variants of exemplary reference sequences (e.g., human sequences) encoded by genes at the transferrin receptor protein 1 chromosomal locus. The full length TfR protein includes a short N-terminal intracellular domain, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and a tip domain. The top domain sequence of human transferrin receptor 1 is set forth in SEQ ID NO 31.

As used herein, the term "Fc γ receptor" or "Fc γ R" refers to a type of Fc receptor that is classified based on the type of antibody they recognize. Fc γ R includes several members: fc γ RI (CD64), Fc γ RIIA (CD32), Fc γ RIIB (CD32), Fc γ RIIIA (CD16a) and Fc γ RIIIB (CD16b), whose antibody affinities differ due to different molecular structures. Fc γ R binds to the Fc portion of antibodies of the IgG class and is important for inducing phagocytosis of opsonized microorganisms. Fc γ R is present on the cell surface of cells in the immune system. Fc γ R is responsible for eliciting immune system effector functions and is activated upon binding of the Fc portion of the antibody to the receptor. Fc γ R mediates immune functions, such as binding to antibodies attached to infected cells or invading pathogens, thereby stimulating phagocytic or cytotoxic cells to destroy microorganisms or infected cells by antibody-mediated phagocytosis or ADCC.

As used herein, the term "reduce Fc γ R binding" refers to a modified Fc polypeptide or modified Fc polypeptide dimer containing a mutation in the CH3 domain of the modified Fc polypeptide, wherein the mutation reduces the affinity of the modified Fc polypeptide for Fc γ R by 0.01% to 90% (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) compared to the affinity of an Fc polypeptide that does not contain the mutation that reduces Fc γ R binding (e.g., a wild-type Fc polypeptide dimer). For example, Surface Plasmon Resonance (SPR) methods (e.g., Biacore) can be used ^TMSystem) to measure Fc γ R binding. Alternatively, Fc γ R binding can be measured using a functional assay, e.g., an ADCC assay, such as the ADCC assay described herein (e.g., in vivo or in vitro assay of cell killing). A decrease in Fc γ R binding can be measured when the modified Fc polypeptide or modified Fc polypeptide dimer binds to TfR. In some embodiments, the modified Fc polypeptide or modified Fc polypeptide dimer may have reduced Fc γ R binding when bound to a TfR, but limited (e.g., less than 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1%) or no reduction when not bound to a TfR.

As further described herein, the modified Fc polypeptide dimer may contain: a first Fc polypeptide having both a TfR binding site and a mutation that reduces fcyr binding when bound to a TfR; and a second Fc polypeptide having neither a TfR binding site nor a mutation that reduces fcyr binding. Thus, upon TfR participation, the resulting asymmetric Fc polypeptide dimer with the first and second Fc polypeptides may have an overall reduced affinity for Fc γ R. In contrast, there may be a limited decrease (e.g., as described above) or no decrease in Fc γ R binding when not bound to TfR.

The term "FcRn" refers to a neonatal Fc receptor. Binding of Fc polypeptides to FcRn reduces clearance of Fc polypeptides and increases serum half-life. The human FcRn protein is a heterodimer consisting of a protein of about 50kDa in size and similar to the Major Histocompatibility (MHC) class I protein and β 2-microglobulin of about 15kDa in size.

As used herein, "FcRn binding site" refers to the region of an Fc polypeptide that binds to FcRn. In human IgG, the FcRn binding sites include L251, M252, I253, S254, R255, T256, M428, H433, N434, H435, and Y436 as numbered using the EU numbering scheme. These positions correspond to positions 21 to 26, 198 and 203 to 206 of SEQ ID NO: 1.

As used herein, a "native FcRn binding site" refers to a region of an Fc polypeptide that binds to FcRn and has the same amino acid sequence as the region of a naturally occurring Fc polypeptide that binds to FcRn.

As used herein, the term "does not substantially deplete in vivo reticulocytes" means that the reticulocyte reduction (e.g., bone marrow reticulocyte or circulating reticulocyte reduction) caused by an effector-positive TfR-binding Fc polypeptide dimer described herein or an Fc polypeptide dimer-Fab fusion protein containing an effector-positive TfR-binding Fc polypeptide dimer described herein is less (e.g., less than 80%, 75%, 70%, 65% >, or circulating reticulocyte reduction) than the reticulocyte reduction (e.g., bone marrow reticulocyte or circulating reticulocyte reduction) caused by a control (e.g., a corresponding TfR-binding Fc dimer with complete effector function and/or without mutations that reduce fcyr binding or an antibody containing a corresponding TfR-binding Fc dimer with complete effector function and/or without mutations that reduce fcyr binding) 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1%).

The term "does not substantially deplete reticulocytes in vivo" may also mean that the amount or percentage of reticulocytes remaining after administration of a TfR-binding Fc polypeptide dimer that is positive for effector function described herein or an Fc polypeptide dimer-Fab fusion protein described herein that contains a TfR-binding Fc polypeptide dimer that is positive for effector function (e.g., reticulocytes remaining in bone marrow or circulation) is greater (e.g., at least 1% greater) than the amount or percentage of reticulocytes remaining after administration of a control (e.g., a corresponding TfR-binding Fc dimer that has full effector function and/or does not contain mutations that reduce fcyr binding, or an antibody that contains a corresponding TfR-binding Fc dimer that has full effector function and/or does not contain mutations that reduce fcyr binding), e.g., reticulocytes remaining in bone marrow or circulation, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%).

The amount or percentage of reticulocyte depletion (e.g., reticulocyte depletion in bone marrow or in circulation) or the amount or percentage of reticulocytes remaining (e.g., reticulocytes remaining in bone marrow or in circulation) can be knocked-in (TfR) in a human TfR^ms/huKI) mice (e.g., human TfR apical domain knock-in mice (' hTFR) ^{Tip end}Knock-in mice ")), which are engineered to replace the mouse TfR with a human apical domain/mouse chimeric TfR protein; or in a non-human primate such as a cynomolgus monkey. As described herein, a modified Fc dimer or control (e.g., 25 to 50mg/kg, i.v.) can be administered (e.g., to a TfR)^ms/huKI mouse) and circulating reticulocytes can be measured by cytochemical reaction at 24h post-dose using the Advia 120 hematology system. As described herein, bone marrow reticulocytes can be measured using FACS sorting to determine the following populations: ter119⁺、hCD71^{Height of}And FSC^{Is low in}And (4) a group.

The terms "CH 3 domain" and "CH 2 domain" as used herein refer to immunoglobulin constant region domain polypeptides. In the case of IgG antibodies, the CH3 domain polypeptide refers to a segment of amino acids at about position 341 to about position 447 as numbered according to the EU numbering scheme, and the CH2 domain polypeptide refers to a segment of amino acids at about position 231 to about position 340 as numbered according to the EU numbering scheme. The CH2 and CH3 domain polypeptides may also be numbered according to the IMGT (imminogenetics) numbering scheme, with the CH2 domain numbered 1-110 and the CH3 domain numbered 1-107 according to the IMGT science chart numbering (IMGT website). The CH2 and CH3 domains are part of an immunoglobulin Fc region. In the case of an IgG antibody, the Fc region refers to a stretch of amino acids from about position 231 to about position 447 as numbered according to the EU numbering scheme. As used herein, the term "Fc region" may also include at least a portion of an antibody hinge region. An illustrative hinge region sequence is set forth in SEQ ID NO: 62.

The term "variable region" refers to a domain in the heavy or light chain of an antibody that is derived from a germline variable (V) gene, diversity (D) gene, or joining (J) genes (but not from constant (C μ and C) gene segments) and confers specificity for the antibody to bind to an antigen. Typically, an antibody variable region comprises four conserved "framework" regions interspersed with three hypervariable "complementarity determining regions".

The terms "wild-type", "native" and "naturally occurring" in reference to a CH3 or CH2 domain are used herein to refer to domains having sequences that occur in nature.

As used herein, the terms "mutant" and "variant" with respect to a mutant polypeptide or mutant polynucleotide are used interchangeably. Variants for a given wild-type CH3 or CH2 domain reference sequence may include naturally occurring duality variants. A "non-naturally" occurring CH3 or CH2 domain refers to a variant or mutant domain that does not occur in a cell in nature and that results from genetic modification of a native CH3 or CH2 domain polynucleotide or polypeptide, for example, using genetic engineering techniques or mutagenesis techniques. "variants" include any domain comprising at least one amino acid mutation relative to the wild type. Mutations may include substitutions, insertions, and deletions.

The term "amino acid" refers to both naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are subsequently modified, such as hydroxyproline, γ -carboxyglutamate, and O-phosphoserine. "amino acid analogs" refers to compounds having the same basic chemical structure as a naturally occurring amino acid (i.e., the alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "amino acid mimetics" refers to compounds that differ in structure from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid.

Naturally occurring alpha-amino acids include, but are not limited to, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (gin), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of naturally occurring alpha-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

Amino acids may be referred to herein by commonly known three-letter symbols or by one-letter symbols as suggested by the IUPAC-IUB Biochemical Nomenclature Commission (IUPAC-IUB Biochemical Nomenclature Commission).

The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The amino acid polymer may comprise a complete L-amino acid, a complete D-amino acid, or a mixture of L and D amino acids.

The term "protein" as used herein refers to a dimer (i.e., two) or multimer (i.e., three or more) of a polypeptide or single-chain polypeptide. Single chain polypeptides of a protein may be linked by covalent bonds (e.g., disulfide bonds) or non-covalent interactions.

The term "conservative substitution," "conservative mutation," or "conservatively modified variant" refers to an alteration that allows an amino acid to be classified as another amino acid substitution with similar characteristics. Examples of conservative amino acid group classes defined in this way may include: "charged/polar group" includes Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H); the "aromatic group" includes Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and the "aliphatic group" includes Gly (glycine or G), Ala (alanine or a), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine or C). Within each group, subgroups may also be identified. For example, the group of charged or polar amino acids may be subdivided into subgroups comprising: "positively charged subgroup" comprising Lys, Arg and His; "negatively charged subgroup," comprising Glu and Asp; and "polar subgroups," comprising Asn and gin. In another example, aromatic or cyclic groups may be subdivided into subgroups including: "nitrogen ring subgroup" comprising Pro, His, and Trp; and the "phenyl subgroup," comprising Phe and Tyr. In another example, the aliphatic group can be subdivided into subgroups such as: "aliphatic nonpolar subgroup" comprising Val, Leu, Gly, and Ala; and "aliphatic, slightly polar subgroups" comprising Met, Ser, Thr and Cys. Examples of conservative mutation classes include amino acid substitutions of amino acids within the above subgroups, such as, but not limited to: lys for Arg or vice versa such that a positive charge can be maintained; glu for Asp or vice versa, such that a negative charge can be maintained; ser for Thr or vice versa, so that free-OH can be maintained; and Gln substitution of Asn or vice versa, so that free-NH ₂Can be maintained. In some embodiments, hydrophobic amino acids are substituted for naturally occurring hydrophobic amino acids in, for example, the active site to retain hydrophobicity.

The term "identical" or percent "identity," in the context of two or more polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same over a specified region, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more identity, when compared and aligned over a comparison window or specified region for maximum correspondence, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

For sequence comparison of polypeptides, typically one amino acid sequence serves as a reference sequence to which candidate sequences are compared. Alignment can be performed using various methods available to those skilled in the art (e.g., visual alignment) or using publicly available software using known algorithms to achieve optimal alignment. Such programs include the BLAST program, ALIGN-2(Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters for alignment to achieve optimal alignment can be determined by one skilled in the art. For sequence comparison of polypeptide sequences for the purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two protein sequences is used, and default parameters are used.

When the terms "corresponding to," "determined with reference to … …," or "numbered with reference to … …" are used in the context of identifying a given amino acid residue in a polypeptide sequence, it is meant that the residue position of the reference sequence is specified when the given amino acid sequence is optimally aligned and compared to the reference sequence. Thus, for example, when an amino acid residue in a polypeptide is aligned with an amino acid in SEQ ID NO:1 when optimally aligned with SEQ ID NO:1, the residue "corresponds to" an amino acid in the region of SEQ ID NO: 1. The polypeptide aligned with a reference sequence need not be the same length as the reference sequence.

As used herein, the term when referring to a polypeptide comprising a modified CH3 domain as described herein"specifically binds" or "selectively binds" to a target (e.g., TfR or fcyr) refers to a binding reaction that causes the polypeptide to bind to the target with greater affinity, greater avidity, and/or for a longer duration than it binds to a structurally different target. In typical embodiments, a polypeptide has at least 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greater affinity for a particular target (e.g., TfR or fcyr) when analyzed under the same affinity assay conditions as an unrelated target. As used herein, the term "specifically binds to a particular target (e.g., TfR or Fc γ R)", or "specific for a particular target (e.g., TfR or Fc γ R)" can be, for example, caused by the equilibrium dissociation constant K for the bound target _DIs for example 10^-4M or less (e.g. 10)^-5M、10^-6M、10^-7M、10^-8M、10^- ⁹M、10^-10M、10^-11M or 10^-12M) molecule display. In some embodiments, the modified CH3 domain polypeptide specifically binds to an epitope on TfR that is conserved across species (e.g., structurally conserved across species), e.g., conserved between non-human primate and human species (e.g., structurally conserved between non-human primate and human species). In some embodiments, the polypeptide can exclusively bind to human TfR.

The term "binding affinity" as used herein refers to the strength of a non-covalent interaction between two molecules, e.g., a single binding site on a polypeptide and a target to which it binds (e.g., TfR). Thus, for example, the term can refer to a 1:1 interaction between a polypeptide and its target unless otherwise indicated or clear from the context. Binding affinity can be measured by the equilibrium dissociation constant (K)_D) To quantify, the equilibrium dissociation constant refers to the dissociation rate constant (k)_dTime of day^-1) Divided by the association rate constant (k)_aTime of day^-1M^-1). K can be determined by measuring the kinetics of complex formation and dissociation_DE.g. using Surface Plasmon Resonance (SPR) methods, e.g. Biacore^TMA system; kinetic exclusion analysis, such as And BioLayer interferometry (e.g., using

A platform). As used herein, "binding affinity" includes not only formal binding affinities, such as those that reflect 1:1 interactions between a polypeptide and its target, but also includes the calculated Ks that may reflect affinity binding_D' apparent affinity to be obtained.

TfR binding Fc polypeptides

This section describes the production of modified Fc polypeptides that bind to TfR and are capable of transport across the Blood Brain Barrier (BBB).

CH3 TfR binding polypeptides

In some embodiments, the modified Fc polypeptide contains a modified human Ig CH3 domain, such as an IgG CH3 domain. The CH3 domain may belong to any IgG subtype, i.e. from IgG1, IgG2, IgG3 or IgG 4. In the case of IgG antibodies, the CH3 domain refers to a segment of amino acids from about position 341 to about position 447 as numbered according to the EU numbering scheme. Unless otherwise indicated, the positions in the CH3 domain for the purpose of identifying the corresponding set of amino acid positions for TfR binding are determined with reference to the EU numbering scheme, amino acids 111-217 of SEQ ID No. 3 or SEQ ID No. 1. Substitutions are also determined with reference to the EU numbering scheme or SEQ ID NO:1, i.e., amino acids are considered as substitutions relative to the corresponding amino acid position in the EU numbering scheme or SEQ ID NO: 1.

As indicated above, the collection of residues in the CH3 domain that can be modified are numbered herein with reference to the EU numbering scheme or SEQ ID NO 1. Any CH3 domain (e.g., IgG1, IgG2, IgG3, or IgG4 CH3 domain) can have modifications, e.g., amino acid substitutions, in one or more of the collection of residues corresponding to the positions indicated in the EU numbering scheme or SEQ ID NO: 1. The position in each of the IgG1, IgG2, IgG3, and IgG4 sequences corresponding to any given position of the EU numbering scheme or SEQ id no:1 can be readily determined.

Those skilled in the art will appreciate that the CH3 domains of other immunoglobulin isotypes (e.g., IgM, IgA, IgE, IgD, etc.) may be similarly modified by identifying the amino acids in those domains that correspond to the amino acid positions described herein. Corresponding domains of immunoglobulins from other species (e.g., non-human primates, monkeys, mice, rats, rabbits, dogs, pigs, chickens, etc.) may also be modified.

In one embodiment, a modified CH3 domain polypeptide that specifically binds TfR binds to the top domain of TfR at an epitope comprising position 208 of the full length human TfR sequence (SEQ ID NO:63), which corresponds to position 11 of the human TfR top domain sequence set forth in SEQ ID NO: 31. SEQ ID NO:31 corresponds to amino acids 198-378 of human TfR-1 uniform protein (uniprotein) sequence P02786(SEQ ID NO: 63). In some embodiments, the modified CH3 domain polypeptide binds to the top domain of a TfR at an epitope comprising positions 158, 188, 199, 207, 208, 209, 210, 211, 212, 213, 214, 215, and/or 294 of the full length human TfR sequence (SEQ ID NO: 63). The modified CH3 domain polypeptide can bind to TfR without blocking or otherwise inhibiting the binding of transferrin to the receptor. In some embodiments, the binding of transferrin to TfR is not substantially inhibited. In some embodiments, the binding of transferrin to TfR is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%). In some embodiments, the binding of transferrin to TfR is inhibited by less than about 20% (e.g., less than about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%). Illustrative CH3 domain polypeptides exhibiting this binding specificity include polypeptides having amino acid substitutions at positions 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 according to the EU numbering scheme.

CH3 TfR binding set: 384. 386, 387, 388, 389, 390, 413, 416 and 421

In some embodiments, the modified CH3 domain polypeptide comprises one, two, three, four, five, six, seven, eight, nine, ten, or eleven substitutions in a set comprising amino acid positions 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 (set CH3C) according to the EU numbering scheme. Illustrative substitutions that may be introduced at these positions are shown in table 3. Additional substitutions are shown in table 4. In some embodiments, the amino acids at positions 388 and/or 421 are aromatic amino acids, such as Trp, Phe, or Tyr. In some embodiments, the amino acid at position 388 is Trp. In some embodiments, the amino acid at position 388 is Gly. In some embodiments, the aromatic amino acid at position 421 is Trp or Phe.

In certain embodiments, the modified CH3 domain polypeptide comprises one, two, three, four, five, six, seven, eight, nine, ten, or eleven positions selected from: glu, Leu, Ser, Val, Trp, Tyr, or Gln at position 380; leu, Tyr, Phe, Trp, Met, Pro, or Val at position 384; leu, Thr, His, Pro, Asn, Val, or Phe at position 386; val, Pro, Ile or an acidic amino acid at position 387; a Trp at position 388; an aliphatic amino acid at position 389, Gly, Ser, Thr, or Asn; gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, Asp, Glu, Asn, Arg, or Thr at position 390; an acidic amino acid at position 413, Ala, Ser, Leu, Thr, Pro, Ile, or His; glu, Ser, Asp, Gly, Thr, Pro, Gln or Arg at position 415; thr, Arg, Asn, or acidic amino acid at position 416; and/or an aromatic amino acid, His or Lys at position 421.

In some embodiments, a modified CH3 domain polypeptide that specifically binds to TfR comprises at least one of the following positions with substitutions (according to EU numbering scheme): leu, Tyr, Met, or Val at position 384; leu, Thr, His or Pro at position 386; val, Pro, or acidic amino acid at position 387; an aromatic amino acid at position 388 (e.g., Trp or Gly) (e.g., Trp); val, Ser, or Ala at position 389; an acidic amino acid at position 413, Ala, Ser, Leu, Thr, or Pro; thr or an acidic amino acid at position 416; or Trp, Tyr, His or Phe at position 421. In some embodiments, the modified CH3 domain polypeptides may comprise conservative substitutions, such as amino acids in the same charge grouping, hydrophobic grouping, side chain loop structure grouping (e.g., aromatic amino acids), or size grouping and/or polar or non-polar grouping of specified amino acids at one or more positions in the collection. Thus, for example, Ile can be present at location 384, 386, and/or location 413. In some embodiments, the acidic amino acid at position one, two, or each of positions 387, 413, and 416 is Glu. In other embodiments, the acidic amino acid at one, both, or each of positions 387, 413, and 416 is Asp. In some embodiments, two, three, four, five, six, seven or all eight of positions 384, 386, 387, 388, 389, 413, 416 and 421 have an amino acid substitution as specified in this paragraph.

In some embodiments, the CH3 domain polypeptide having a modification in set CH3C comprises a native Asn at position 390. In some embodiments, the modified CH3 domain polypeptide comprises Gly, His, gin, Leu, Lys, Val, Phe, Ser, Ala, or Asp at position 390. In some embodiments, the modified CH3 domain polypeptide further comprises one, two, three, or four substitutions at positions 380, 391, 392 and 415. In some embodiments, Trp, Tyr, Leu, or Gln may be present at position 380. In some embodiments, Ser, Thr, Gln, or Phe may be present at position 391. In some embodiments, Gln, Phe or His may be present at position 392. In some embodiments, Glu may be present at position 415.

In certain embodiments, the modified CH3 domain polypeptide comprises two, three, four, five, six, seven, eight, nine, or ten positions selected from: trp, Leu, or Glu at position 380; tyr or Phe at position 384; thr at position 386; a Glu at position 387; a Trp at position 388; ser, Ala, Val, or Asn at position 389; ser or Asn at position 390; thr or Ser at position 413; glu or Ser at position 415; a Glu at position 416; and/or Phe at position 421. In some embodiments, the modified CH3 domain polypeptide includes all eleven positions: trp, Leu, or Glu at position 380; tyr or Phe at position 384; thr at position 386; a Glu at position 387; a Trp at position 388; ser, Ala, Val, or Asn at position 389; ser or Asn at position 390; thr or Ser at position 413; glu or Ser at position 415; a Glu at position 416; and/or Phe at position 421.

In certain embodiments, the modified CH3 domain polypeptide comprises a Leu or Met at position 384; leu, His or Pro at position 386; val at position 387; a Trp at position 388; val or Ala at position 389; pro at position 413; thr at position 416; and/or Trp at location 421. In some embodiments, the modified CH3 domain polypeptide further comprises a Ser, Thr, Gln, or Phe at position 391. In some embodiments, the modified CH3 domain polypeptide further comprises Trp, Tyr, Leu, or Gln at position 380 and/or Gln, Phe, or His at position 392. In some embodiments, Trp is present at position 380 and/or Gln is present at position 392. In some embodiments, the modified CH3 domain polypeptide does not have a Trp at position 380.

In other embodiments, the modified CH3 domain polypeptide comprises Tyr at position 384; thr at position 386; glu or Val at position 387; a Trp at position 388; ser at position 389; ser or Thr at position 413; a Glu at position 416; and/or Phe at position 421. In some embodiments, the modified CH3 domain polypeptide comprises a native Asn at position 390. In certain embodiments, the modified CH3 domain polypeptide further comprises a Trp, Tyr, Leu, or Gln at position 380; and/or Glu at position 415. In some embodiments, the modified CH3 domain polypeptide further comprises a Trp at position 380 and/or a Glu at position 415.

In other embodiments, the modified CH3 domain further comprises one, two, or three positions selected from: position 414 is Lys, Arg, Gly, or Pro; position 424 is Ser, Thr, Glu, or Lys; and position 426 Ser, Trp or Gly.

In some embodiments, a modified CH3 domain polypeptide that specifically binds TfR has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 111-217 of any one of SEQ ID NOS 4-29, 64-127, and 268-274 (e.g., SEQ ID NOS 66, 68, 94, 107-109, 119, and 268-270). In some embodiments, such modified CH3 domain polypeptides comprise amino acids 154-160 and/or 183-191 of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270). In some embodiments, such modified CH3 domain polypeptides comprise amino acids 150-160 and/or 183-191 of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270). In some embodiments, the modified CH3 domain polypeptide comprises amino acids 150-160 and/or 183-196 of any one of SEQ ID NOs: 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs: 66, 68, 94, 107-109, 119, and 268-270).

In some embodiments, the modified CH3 domain polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 111-217 of SEQ ID No. 1, with the proviso that the percentage identity does not include the set of positions 154, 156, 157, 158, 159, 160, 183, 186, and 191 (according to the EU numbering scheme, positions 384, 386, 387, 388, 389, 390, 413, 416, and 421) of SEQ ID No. 1. In some embodiments, the modified CH3 domain polypeptide comprises amino acids 154-160 and/or amino acids 183-191 as set forth in any one of SEQ ID NOs 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs 66, 68, 94, 107-109, 119, and 268-270).

In some embodiments, the modified CH3 domain polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs 66, 68, 94, 107-109, 119, and 268-270), with the proviso that position 150, 154, 156, 157, 158, 159, 160, 161, 162, 183, 184, 185, 186, 191, 194, and 196 (according to the EU numbering scheme, position 380, 384, 388, 389, 390, 391, 384, 388, 389, 390, 268, 94, 107-109, 119, and 268-270) corresponds to any one of SEQ ID NOs 4-29, 64-127, at least 85% identity, at least 90% identity, at least 80% at least 85% identity, at least 90% or at least 95% identity to any one of SEQ ID NOs 4-29, 392. 413, 414, 415, 416, 421, 424, and 426), at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen of the positions are not deleted or substituted.

In some embodiments, the modified CH3 domain polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs 66, 68, 94, 107-109, 119, and 268-270) and further comprises at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen of the following positions: trp, Tyr, Leu, Gln, or Glu at position 380; leu, Tyr, Met, or Val at position 384; leu, Thr, His or Pro at position 386; val, Pro, or acidic amino acid at position 387; an aromatic amino acid at position 388 (e.g., Trp); val, Ser, or Ala at position 389; ser or Asn at position 390; ser, Thr, Gln or Phe at position 391; gln, Phe or His at position 392; an acidic amino acid at position 413, Ala, Ser, Leu, Thr, or Pro; lys, Arg, Gly, or Pro at position 414; glu or Ser at position 415; thr or an acidic amino acid at position 416; trp, Tyr, His, or Phe at position 421; ser, Thr, Glu or Lys at position 424; and Ser, Trp, or Gly at position 426.

In some embodiments, a TfR binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs 38-52. In other embodiments, a TfR binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs 38-52, but with one or two amino acids substituted. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs 38-52, but wherein three amino acids are substituted.

In some embodiments, a TfR binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs 53-61. In other embodiments, a TfR binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs 53-61, but in which one or two amino acids are substituted. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs 53-61, but wherein three or four amino acids are substituted.

In some embodiments, the TfR binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs 131-167. In other embodiments, a TfR binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs 131-167, but with one or two amino acids substituted. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs 131-167, but wherein three amino acids are substituted.

In some embodiments, a TfR binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs 58, 60, and 168-173. In other embodiments, a TfR binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs 58, 60, and 168-173, but with one or two amino acids substituted. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs 58, 60, and 168-173, but wherein three or four amino acids are substituted.

In other embodiments, a TfR binding polypeptide comprises amino acids 157-194, amino acids 153-194, or amino acids 153-199 of any one of SEQ ID NOS 4-29, 64-127, and 268-274 (e.g., SEQ ID NOS: 66, 68, 94, 107-109, 119, and 268-270). In other embodiments, the polypeptide comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to amino acids 157-194 of any one of SEQ ID NOS 4-29, 64-127, and 268-274 (e.g., SEQ ID NOS: 66, 68, 94, 107-109, 119, and 268-270), or to amino acids 153-194, or to amino acids 153-199 of any one of SEQ ID NOS 4-29, 64-127, and 268-274 (e.g., SEQ ID NOS: 66, 68, 94, 107-109, 119, and 268-270).

In some embodiments, the polypeptide comprises any one of SEQ ID NOs 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs 66, 68, 94, 107-109, 119, and 268-270). In other embodiments, the polypeptide can have at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs 4-29, 64-127, and 268-274 (e.g., SEQ ID NOs 66, 68, 94, 107-109, 119, and 268-270).

FcRn binding site

The polypeptides described herein that can be transported across the BBB may additionally comprise an FcRn binding site. In some embodiments, the FcRn binding site is within a modified Fc polypeptide or fragment thereof.

In some embodiments, the FcRn binding site comprises a native FcRn binding site. In some embodiments, the FcRn binding site does not comprise an amino acid change relative to the amino acid sequence of the native FcRn binding site. In some embodiments, the native FcRn binding site is an IgG binding site, e.g., a human IgG binding site. In some embodiments, the FcRn binding site comprises a modification that alters FcRn binding.

In some embodiments, the FcRn binding site has one or more amino acid residues that are mutated (e.g., substituted), wherein the one or more mutations increase serum half-life or do not substantially decrease serum half-life (i.e., reduce serum half-life by no more than 25% when compared to a corresponding protein having a wild-type residue at the mutated position when analyzed under the same conditions). In some embodiments, the FcRn binding site has one or more amino acid residues substituted at positions 21 to 26, 198 and 203 to 206, wherein the positions are determined with reference to SEQ ID No. 1.

In some embodiments, the FcRn binding site comprises one or more mutations relative to the native human IgG sequence that extend the serum half-life of the modified polypeptide. In some embodiments, a mutation (e.g., a substitution) is introduced at one or more of positions 14-27, 49-54, 77-87, 153-160, and 198-205 (which positions correspond to positions 244-257, 279-284, 307-317, 383-390, and 428-435 using EU numbering) as determined with reference to SEQ id no: 1. In some embodiments, one or more mutations are introduced at position 21, 22, 24, 25, 26, 77, 78, 79, 81, 82, 84, 155, 156, 157, 159, 198, 203, 204 or 206 (which positions correspond to positions 251, 252, 254, 255, 256, 307, 308, 309, 311, 312, 314, 385, 386, 387, 389, 428, 433, 434 or 436 using EU numbering) as determined with reference to SEQ ID NO: 1. In some embodiments, the mutation is introduced into one, two or three of positions 22, 24 and 25 (which correspond to positions 252, 254 and 256 using EU numbering) as determined with reference to SEQ ID NO: 1. In some embodiments, the mutations are M22Y, S24T, and T26E as numbered with reference to SEQ ID NO: 1. In some embodiments, the modified Fc polypeptides described herein further comprise mutations M22Y, S24T, and T26E. In some embodiments, the mutation is introduced into one or both of positions 198 and 204 (which correspond to positions 428 and 434 using EU numbering) as determined with reference to SEQ ID NO: 1. In some embodiments, the mutations are M198L and N204S as numbered with reference to SEQ ID NO: 1. In some embodiments, the modified Fc polypeptides described herein further comprise mutation N204S with or without the addition of M198L. In some embodiments, the modified Fc polypeptide comprises substitutions at one, two or all three of positions T307, E380 and N434 (which positions correspond to T77, E150 and N204 as numbered with reference to SEQ ID NO: 1) according to EU numbering. In some embodiments, the mutations are T307Q and N434A (SEQ ID NO:1, T77Q and N204A). In some embodiments, the modified Fc polypeptide comprises mutations T307A, E380A, and N434A (SEQ ID NO:1, T77A, E150A, and N204A). In some embodiments, the modified Fc polypeptide comprises substitutions at positions T250 and M428 (which positions correspond to T20 and M198 as numbered with reference to SEQ ID NO: 1). In some embodiments, the Fc polypeptide comprises mutations T250Q and/or M428L (SEQ ID NOs: 1, T20Q, and M198L). In some embodiments, the modified Fc polypeptide comprises substitutions at positions M428 and N434 (which correspond to M198 and N204 as numbered with reference to SEQ ID NO: 1). In some embodiments, the modified Fc polypeptide comprises substitutions M428L and N434S (the positions correspond to M198L and N204S as numbered with reference to SEQ id no: 1). In some embodiments, the modified Fc polypeptide comprises a N434S or N434A substitution (the substitution corresponding to N204S or N204A as numbered with reference to SEQ ID NO: 1).

Mutations that reduce effector function or reduce Fc γ R binding

An Fc polypeptide modified to bind TfR and initiate transport across the BBB as provided herein may also comprise additional mutations to reduce effector function. As described herein, by introducing both a TfR binding site and a mutation that reduces fcyr binding into the same Fc polypeptide of an Fc polypeptide dimer, effector function following TfR binding can be reduced, resulting in TfR binding without substantial reticulocyte depletion, but effector function (e.g., ADCC or CDC) is retained when the Fc polypeptide dimer is fused to a therapeutic Fab and bound to a target antigen of the Fab.

In some embodiments, the Fc polypeptide comprising the modified CH3 domain has effector function, i.e., the ability to induce certain biological functions upon binding to an Fc receptor expressed on effector cells that mediate effector function. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, Natural Killer (NK) cells, and cytotoxic T cells.

Examples of effector functions include, but are not limited to, C1q binding and CDC, Fc receptor binding, ADCC, antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B-cell receptors), and B-cell activation. Effector functions may vary with antibody class. For example, native human IgG1 and IgG3 antibodies can elicit ADCC and CDC activity upon binding to the appropriate Fc receptors present on cells of the immune system; and native human IgG1, IgG2, IgG3, and IgG4 can elicit ADCP function upon binding to the appropriate Fc receptor present on immune cells.

In some embodiments, an Fc polypeptide having a TfR binding site as described herein may comprise an additional modification that reduces effector function (i.e., reduces effector function following TfR binding). It is desirable that the Fc polypeptide dimer has reduced effector function following TfR binding, as this results in reduced reticulocyte depletion due to reticulocytes also having TfR on the cell surface. As described in detail herein, an Fc polypeptide dimer having a cis configuration (i.e., an Fc polypeptide dimer having both a TfR binding site and a mutation that reduces effector function on the same Fc polypeptide of the Fc polypeptide dimer) exhibits TfR binding without substantial depletion of reticulocytes, but retains effector function (e.g., ADCC or CDC) when the Fc polypeptide dimer is fused to a therapeutic Fab and binds to a target antigen of the Fab. Having effector functions when the Fc polypeptide dimer is fused to a therapeutic Fab that binds to a target antigen of the Fab is desirable, for example, in cancer therapeutics (e.g., brain cancer therapeutics).

Illustrative Fc polypeptide mutations that modulate effector function include, but are not limited to, substitutions in the CH2 domain, e.g., at positions corresponding to positions 4 and 5 of seq id NO:1 (positions 234 and 235 according to the EU numbering scheme). In some embodiments, the substitution in the modified CH2 domain comprises Ala at positions 4 and 5 of SEQ ID NO: 1. In some embodiments, the substitution in the modified CH2 domain comprises Ala at positions 4 and 5 and Gly at position 99 of SEQ ID NO: 1.

Additional Fc polypeptide mutations that modulate effector function include, but are not limited to, one or more substitutions at positions 238, 265, 269, 270, 297, 327 and 329(EU numbering scheme, which positions correspond to positions 8, 35, 39, 40, 67, 97 and 99 as numbered with reference to SEQ ID NO: 1). Illustrative substitutions (as numbered with the EU numbering scheme) include the following: position 329 may have a mutation in which proline is substituted with glycine or arginine or an amino acid residue sufficiently large to disrupt the Fc/fcy receptor interface formed between proline 329 of Fc and tryptophan residues Trp 87 and Trp 110 of Fc γ RIII. Additional illustrative substitutions include S228P, E233P, L235E, N297A, N297D, and P331S. Multiple substitutions may also be present, for example L234A and L235A of the Fc region of human IgG 1; L234A, L235A and P329G of the Fc region of human IgG 1; S228P and L235E of the Fc region of human IgG 4; L234A and G237A of the Fc region of human IgG 1; L234A, L235A and G237A of the Fc region of human IgG 1; V234A and G237A of the Fc region of human IgG 2; L235A, G237A and E318A of the Fc region of human IgG 4; and S228P and L236E of the Fc region of human IgG 4. In some embodiments, the Fc polypeptide may have one or more amino acid substitutions that modulate ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region according to the EU numbering scheme.

In some embodiments, a polypeptide as described herein may have one or more amino acid substitutions that increase or decrease ADCC or may have mutations that alter C1q binding and/or CDC.

In particular embodiments, Fc polypeptides having a TfR binding site may be modified to reduce effector function, i.e., reduce Fc γ R binding. In some embodiments, an Fc polypeptide having a TfR binding site may comprise mutations L234A and L235A (EU numbering scheme, which correspond to positions 4 and 5 as numbered with reference to SEQ ID NO: 1). In other embodiments, an Fc polypeptide having a TfR binding site may comprise mutations L234A, L235A, and P329G (EU numbering scheme, which correspond to positions 4, 5, and 99 as numbered with reference to SEQ ID NO: 1).

Tfr-binding Fc polypeptide dimers positive for effector function

In certain aspects, the disclosure provides a TfR binding Fc polypeptide dimer that is positive for an effector function modified to bind to a TfR and have reduced fcyr binding when bound to a TfR, but limited or no reduction in fcyr binding when not bound to a TfR. These modified Fc polypeptide dimers may be fused to the therapeutic Fab to transport it across the BBB. These modified Fc polypeptide dimers have been demonstrated to have reduced effector function following TfR binding. When the modified Fc polypeptide dimer is fused to the Fab, the Fc polypeptide dimer retains effector function when the Fab is bound to its target (e.g., on a cancer cell). In this way, the TfR-binding Fc polypeptide dimers described herein that are positive for effector function are capable of transporting Fab across the BBB without substantial depletion of reticulocytes (which also contain TfR on the cell surface), and also serve their therapeutic purpose by exhibiting effector functions that can be disrupted by targeting extracellular aggregates (e.g., plaques) or certain diseased cells in the brain (e.g., cancer cells) upon binding of Fab to its target.

The effector-positive TfR-binding Fc polypeptide dimers described herein have a cis configuration, meaning that only one (but not both) Fc polypeptide in the Fc polypeptide dimer is modified to have a TfR binding site and a modification that reduces Fc γ R binding when binding to TfR. Another Fc polypeptide in the Fc polypeptide dimer does not contain a TfR binding site or a modification that substantially reduces fcyr binding. The trans configuration of a modified Fc polypeptide dimer means that one of the two Fc polypeptides in the Fc polypeptide dimer contains a TfR binding site, while the other Fc polypeptide contains a modification that reduces Fc γ R binding, for example, when binding to TfR. As demonstrated herein, a modified Fc polypeptide dimer having a cis rather than trans configuration is capable of reducing reticulocyte depletion in blood and bone marrow (see, e.g., fig. 2A-2D).

In one embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR and amino acid modifications L234A and L235A according to EU numbering scheme, and (b) a second Fc polypeptide that does not comprise a TfR binding site or any modification that reduces fcyr binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds TfR, amino acid modifications L234A and L235A and amino acid modification N434S with or without M428L according to EU numbering scheme, and (b) a second Fc polypeptide that does not contain a TfR binding site or any modification that reduces fcyr binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds TfR, amino acid modifications L234A and L235A and amino acid modification N434S plus or minus M428L according to EU numbering scheme, and (b) a second Fc polypeptide comprising amino acid modification N434S plus or minus M428L and lacking a TfR binding site or any modification that reduces fcyr binding.

In one embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds TfR, amino acid modifications L234A and L235A, and knob mutation T366W according to EU numbering scheme, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; and a knob mutation, T366W, and (b) a second Fc polypeptide comprising hole mutations, T366S, L368A, and Y407V according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds TfR, amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modifications M252Y, S254T, and T256E according to EU numbering scheme, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds TfR, amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modification N434S with or without M428L according to EU numbering scheme, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; pestle mutation T366W; and amino acid modifications M252Y, S254T and T256E, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A and Y407V according to the EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; pestle mutation T366W; and amino acid modification N434S with or without M428L, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V according to the EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds TfR, amino acid modifications L234A and L235A and knob mutation T366W according to EU numbering scheme, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A and Y407V and amino acid modifications M252Y, S254T and T256E according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds TfR, amino acid modifications L234A and L235A and knob mutation T366W according to EU numbering scheme, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A and Y407V and amino acid modification N434S with or without M428L according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; and a knob mutation T366W, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V and amino acid modifications M252Y, S254T, and T256E according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; and a knob mutation T366W, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V and amino acid modifications N434S with or without M428L according to EU numbering scheme, and not containing a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds TfR, amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modifications M252Y, S254T, and T256E according to EU numbering scheme, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, and amino acid modifications M252Y, S254T, and T256E according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds TfR, amino acid modifications L234A and L235A, knob mutation T366W, and amino acid modification N434S with or without M428L according to EU numbering scheme, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V, and amino acid modification N434S with or without M428L according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; pestle mutation T366W; and amino acid modifications M252Y, S254T and T256E, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A and Y407V and amino acid modifications M252Y, S254T and T256E according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; pestle mutation T366W; and amino acid modification N434S with or without addition of M428L, and (b) a second Fc polypeptide comprising hole mutations T366S, L368A, and Y407V and amino acid modification N434S with or without addition of M428L according to the EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds TfR, amino acid modifications L234A and L235A and hole mutations T366S, L368A and Y407V according to EU numbering scheme, and (b) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; and hole mutations T366S, L368A, and Y407V, and (b) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A and L235A; hole mutations T366S, L368A, and Y407V; and amino acid modifications M252Y, S254T, and T256E, and (b) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A and L235A; hole mutations T366S, L368A, and Y407V; and amino acid modification N434S with or without M428L, and (b) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering scheme and not comprising a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; hole mutations T366S, L368A, and Y407V; and amino acid modifications M252Y, S254T, and T256E, and (b) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering scheme and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; hole mutations T366S, L368A, and Y407V; and amino acid modification N434S with or without M428L, and (b) a second Fc polypeptide comprising a knob mutation T366W according to EU numbering scheme and not comprising a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds TfR, amino acid modifications L234A and L235A and hole mutations T366S, L368A and Y407V according to EU numbering scheme, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modifications M252Y, S254T and T256E according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds TfR, amino acid modifications L234A and L235A and hole mutations T366S, L368A and Y407V according to EU numbering scheme, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modification N434S with or without M428L according to EU numbering scheme, and which does not comprise a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; and hole mutations T366S, L368A, and Y407V, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modifications M252Y, S254T, and T256E according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; and hole mutations T366S, L368A, and Y407V, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modifications N434S with or without M428L according to the EU numbering scheme, and which does not contain a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A and L235A; hole mutations T366S, L368A, and Y407V; and amino acid modifications M252Y, S254T, and T256E, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modifications M252Y, S254T, and T256E according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A and L235A; hole mutations T366S, L368A, and Y407V; and amino acid modification N434S with or without M428L, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modification N434S with or without M428L according to EU numbering scheme, and which does not contain a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; hole mutations T366S, L368A, and Y407V; and amino acid modifications M252Y, S254T, and T256E, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modifications M252Y, S254T, and T256E according to EU numbering scheme, and lacking a TfR binding site or any modification that reduces Fc γ R binding.

In another embodiment, a TfR binding Fc polypeptide dimer that is positive for effector function comprises: (a) a first Fc polypeptide comprising a TfR binding site that specifically binds a TfR; amino acid modifications according to EU numbering scheme L234A, L235A and P329G; hole mutations T366S, L368A, and Y407V; and amino acid modification N434S with or without M428L, and (b) a second Fc polypeptide comprising a knob mutation T366W and amino acid modification N434S with or without M428L according to EU numbering scheme, and which does not contain a TfR binding site or any modification that reduces Fc γ R binding.

Measurement of Effector Functions or Fc Gamma R binding

Methods for analyzing binding affinity, binding kinetics, and cross-reactivity between Fc polypeptide dimers and Fc γ rs are known in the art. These methods include, but are not limited to, solid phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore)^TM(GE Healthcare, Piscataway, NJ)), kinetic exclusion analysis (e.g.

) Flow cytometry, Fluorescence Activated Cell Sorting (FACS), BioLayer interferometry (e.g.

(Forte Bio, Inc., Menlo Park, CA)) and Western blot analysis (Western blot analysis). In some embodiments, binding affinity and/or cross-reactivity is determined using ELISA. Methods for performing ELISA assays are known in the art. In some embodiments, Surface Plasmon Resonance (SPR) is used to determine binding affinity, binding kinetics, and/or cross-reactivity. In some embodiments, a kinetic exclusion assay is used to determine binding affinity, binding kinetics, and/or cross-reactivity. In some embodiments, binding affinity, binding kinetics, and/or cross-reactivity are determined using BioLayer interferometry analysis.

ADCC is a type of immune response in which antibodies bind to antigens on the surface of pathogenic or tumorigenic target cells and are identified for destruction by effector cells (e.g., peripheral blood mononuclear cells, such as Natural Killer (NK) cells, T cells, and B cells).Effector cells bearing Fc γ rs recognize and bind to the Fc region of antibodies that bind to target cells. The antibody thus confers specificity of killing of the target cells. CDC is initiated when the initiating component C1q of the classical complement pathway binds to the Fc region of an antibody that binds to a target. ADCC and CDC activities may be measured in standard in vivo or in vitro assays for cell killing. Methods for determining ADCC and CDC activity are available in the art. In some embodiments, the method may involve the use of radioactive materials (such as⁵¹Cr) or a fluorescent dye (such as calcein-AM) to label the target cells. The labeled cells can be incubated with antibodies and effector cells and killing of target cells by ADCC or CDC can be detected by the release of radioactivity or fluorescence.

Other assays for measuring ADCC and CDC activity include, for example, Lactate Dehydrogenase (LDH) release assays. When the cell membrane is damaged or destroyed in any way, the soluble and stable enzyme LDH in the cytoplasm is released into the surrounding cell space. The presence of this enzyme in the culture medium can be used as a marker for cell death. The relative amounts of live and dead cells within the medium can then be quantified by measuring the amount of LDH released using a colorimetric or fluorescent LDH cytotoxicity assay.

Additional mutations in the Fc region of a polypeptide comprising a modified CH3 domain

Fc polypeptides modified to bind TfR and initiate transport across the BBB as provided herein may also comprise additional mutations, for example to increase serum stability or serum half-life, modulate effector function, affect glycosylation, reduce immunogenicity in humans, and/or provide knob and hole heterodimerization of Fc polypeptides.

In some embodiments, a modified Fc polypeptide described herein has at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a corresponding wild-type Fc polypeptide (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide).

The modified Fc polypeptides described herein may also have other mutations introduced outside the specified set of amino acids, for example to affect glycosylation, increase serum half-life, or for the CH3 domain, provide knob and hole heterodimerization of the polypeptide comprising the modified CH3 domain. In general, the methods involve introducing a protuberance at the interface of the first polypeptide ("knob") and a corresponding cavity in the interface of the second polypeptide ("hole") such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and thus prevent homodimer formation. The protuberance is constructed by replacing a small amino acid side chain of the first polypeptide interface with a larger side chain (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protrusions are formed in the interface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). Such additional mutations are located at positions in the polypeptide that do not negatively affect the binding of the modified CH3 domain to TfR.

In one illustrative embodiment of the knob and hole dimerization process, the first Fc polypeptide subunit to be dimerized has a tryptophan in place of the native threonine at a position corresponding to position 136 of SEQ ID NO:1, and the second Fc polypeptide subunit of the dimer has a valine in place of the native tyrosine at a position corresponding to position 177 of SEQ ID NO: 1. The second subunit of the Fc polypeptide can further comprise a substitution wherein the native threonine at position corresponding to position 136 of SEQ ID NO:1 is substituted with serine and the native leucine at position corresponding to position 138 of SEQ ID NO:1 is substituted with alanine.

Modified Fc polypeptides as described herein may also be engineered to contain other modifications for heterodimerization, such as electrostatic engineering or hydrophobic patch modifications to naturally charged contact residues within the CH3-CH3 interface.

In some embodiments, modifications to enhance serum half-life may be introduced. For example, in some embodiments, a modified Fc polypeptide as described herein comprises a CH2 domain comprising: tyr at a position corresponding to position 22 of SEQ ID NO. 1, Thr at a position corresponding to position 24 of SEQ ID NO. 1, and Glu at a position corresponding to position 26 of SEQ ID NO. 1. Alternatively, the modified Fc polypeptide as described herein may comprise substitutions M198L and N204S as numbered with reference to SEQ id No. 1. Alternatively, the modified Fc polypeptide as described herein may comprise a substitution N204S or N204A as numbered with reference to seq id NO: 1.

Illustrative Fc Polypeptides comprising additional mutations

Fc polypeptides modified as described herein (e.g. clones ch3c.35.20.1, ch3c.35.23.2, ch3c.35.23.3, ch3c.35.23.4, ch3c.35.21.17.2, ch3c.35.23, ch3c.35.21, ch3c.35.20.1.1, ch3c.35.23.2.1 and ch3 c.35.23.1.1) may comprise additional mutations including a knob mutation (e.g. T136 23 as referenced to SEQ ID No. 1), a hole mutation (e.g. T136S, L138A and Y46177V as referenced to SEQ ID NO: 1), a mutation modulating effector function (e.g. L A, L5A and/or P99G (e.g. L4 and L5) and/or a mutation of L6323) and/or a serum half-life increasing mutation (e.g. m.g. 26) as referenced to SEQ ID No. S631) and/or No. 12 (e.g. S) and No. S2 (S) added to SEQ ID nos. 12).

In some embodiments, a modified Fc polypeptide as described herein (e.g., any of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1, and CH3C.35.23.1.1) can have a pestle mutation (e.g., T136W as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) and has at least 95% identity to any of SEQ ID NO:4-29, 64-127, and 268 (e.g., SEQ ID NO:66, SEQ ID NO: 68, SEQ ID NO: 107, 152, 268, and at least 95% identity to any of SEQ ID NO: 90, 95% or 268. In some embodiments, modified Fc polypeptides having the sequences of any one of SEQ ID NOS 4-29, 64-127 and 268-274 (e.g., SEQ ID NOS 66, 68, 94, 107-109, 119 and 268-270) can be modified to have a knob mutation and a mutation that modulates effector function.

In some embodiments, a modified Fc polypeptide as described herein (e.g., any of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1, and CH3C.35.23.1.1) can have a knob mutation (e.g., T136W as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., (i) a mutation such as M22Y, S24T, and T26E as referenced to SEQ ID NO:1, or (ii) a sequence that is at least about 70% identical to any of N204S or not M198L as referenced SEQ ID NO:1, plus at least about 75% identity to SEQ ID NO: 64, 268, or 109% identical to SEQ ID NO: 90, 268, or 85, or 95% identical to SEQ ID NO: 90. In some embodiments, modified Fc polypeptides having the sequences of any one of SEQ ID NOS 4-29, 64-127 and 268-274 (e.g., SEQ ID NOS 66, 68, 94, 107-109, 119 and 268-270) can be modified to have a knob mutation and a mutation that increases serum stability or serum half-life.

In some embodiments, a modified Fc polypeptide as described herein (e.g., any of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1, and CH3C.35.23.1.1) can have a pestle mutation (e.g., T136W as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A as referenced SEQ ID NO:1, L5A, and/or P99G (e.g., L4A and L5A)), a mutation that increases stability or serum half-life (e.g., (i) M22 as referenced SEQ ID: 1, L24S 26 as referenced SEQ ID NO: 26, and/or P99 (e.g., L4A as referenced SEQ ID NO: 26, S.g., SEQ ID NO: 268) and at least one of clones 120, 75, V.g., SEQ ID NO: 75, V.75, V.42, V.1, and V.1) as referenced SEQ ID NO: 75, and at least one of the sequence of the amino acids as depicted in SEQ ID NO:1, At least 90% identity or at least 95% identity. In some embodiments, modified Fc polypeptides having the sequences of any one of SEQ ID NOS 4-29, 64-127 and 268-274 (e.g., SEQ ID NOS 66, 68, 94, 107-109, 119 and 268-270) can be modified to have knob mutations, mutations that modulate effector function, and mutations that increase serum stability or serum half-life.

In some embodiments, a modified Fc polypeptide as described herein (e.g., any of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1, and CH3C.35.23.1.1) can have a mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates an effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A as referenced SEQ ID NO: 1), and has at least one percent identity with at least one of SEQ ID NO:4-29, 64-127, and SEQ ID NO: 68 (e.g., SEQ ID NO: 268, SEQ ID NO: 107, 268, 270, 268, 90, or 95% identity with SEQ ID NO: 90). In some embodiments, modified Fc polypeptides having the sequences of any one of SEQ ID NOs 4-29, 64-127 and 268-274 (e.g., SEQ ID NOs 66, 68, 94, 107-109, 119 and 268-270) may be modified to have hole mutations and mutations that modulate effector function.

In some embodiments, a modified Fc polypeptide as described herein (e.g., any of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1, and CH3C.35.23.1.1) may have a mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that increases serum stability or half-life (e.g., (i) M22Y, S24T, and T26E as referenced SEQ ID NO:1, or (ii) N204 5 as referenced SEQ ID NO:1 or not plus M198, and at least one of identity with SEQ ID NO: 75, 268, 270%, 85, 268, and 270% of the sequence of at least one of SEQ ID NO: 90, 268, and 270% of identity with SEQ ID NO: 90. In some embodiments, modified Fc polypeptides having the sequences of any one of SEQ ID NOS 4-29, 64-127 and 268-274 (e.g., SEQ ID NOS 66, 68, 94, 107-109, 119 and 268-270) can be modified to have hole mutations and mutations that increase serum stability or serum half-life.

In some embodiments, a modified Fc polypeptide as described herein (e.g., any of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.35.23.2.1 and CH3C.35.23.1.1) may have a mutation (e.g., T136S, L138A and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A and/or P99G (e.g., L464 and L5A as referenced SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., (i) as referenced SEQ ID NO: 1: 24, S70, S24, S70, S1, S70, S24, S1, S24, S70, S1, S5, S70, at least 90% identity or at least 95% identity. In some embodiments, modified Fc polypeptides having the sequences of any one of SEQ ID NOS 4-29, 64-127 and 268-274 (e.g., SEQ ID NOS 66, 68, 94, 107-109, 119 and 268-270) can be modified to have hole mutations, mutations that modulate effector function, and mutations that increase serum stability or serum half-life.

Clone CH3C.35.20.1

In some embodiments, clone CH3C.35.20.1 may have a knob mutation (e.g., T136W as numbered with reference to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 177. In some embodiments, clone CH3C.35.20.1 with a knob mutation has the sequence of SEQ ID NO: 177.

In some embodiments, clone CH3C.35.20.1 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:178 or 179. In some embodiments, clone CH3C.35.20.1, having a knob mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO:178 or 179.

In some embodiments, clone ch3c.35.20.1 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 180. In some embodiments, clone CH3C.35.20.1, having a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 180.

In some embodiments, clone ch3c.35.20.1 may have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered by SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 322. In some embodiments, clone CH3C.35.20.1, with a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 322.

In some embodiments, clone ch3c.35.20.1 can have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered by SEQ ID NO: 1), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO:181 or 182. In some embodiments, clone CH3C.35.20.1, having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:181 or 182.

In some embodiments, clone ch3c.35.20.1 can have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered by SEQ ID NO:1 plus or minus M198L), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO:323 or 324. In some embodiments, clone CH3C.35.20.1, having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:323 or 324.

In some embodiments, clone ch3c.35.20.1 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 183. In some embodiments, clone CH3C.35.20.1 with a hole mutation has the sequence of SEQ ID NO: 183.

In some embodiments, clone ch3c.35.20.1 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:184 or 185. In some embodiments, the clone CH3C.35.20.1 with a hole mutation and a mutation that modulates effector function has the sequence of SEQ ID NO 184 or 185.

In some embodiments, clone ch3c.35.20.1 may have hole mutations (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 186. In some embodiments, clone CH3C.35.20.1, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 186.

In some embodiments, clone ch3c.35.20.1 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 325. In some embodiments, clone ch3c.35.20.1, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID No. 325.

In some embodiments, clone ch3c.35.20.1 may have a hole mutation (e.g., as referenced to T136S, L138A, and Y177V of SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) of SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E of SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:187 or 188. In some embodiments, clone CH3C.35.20.1, having a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 187 or 188.

In some embodiments, clone ch3c.35.20.1 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:326 or 327. In some embodiments, clone CH3C.35.20.1, having a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 326 or 327.

Clone CH3C.35.23.2

In some embodiments, clone CH3C.35.23.2 may have a knob mutation (e.g., T136W as numbered with reference to SEQ ID NO: 1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 189. In some embodiments, clone CH3C.35.23.2 with a knob mutation has the sequence of SEQ ID NO: 189.

In some embodiments, clone CH3C.35.23.2 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:190 or 191. In some embodiments, clone CH3C.35.23.2, having a knob mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO 190 or 191.

In some embodiments, clone ch3c.35.23.2 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 192. In some embodiments, clone CH3C.35.23.2, with a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 192.

In some embodiments, clone CH3C.35.23.2 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 329. In some embodiments, clone CH3C.35.23.2, having a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 329.

In some embodiments, clone CH3C.35.23.2 may have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered by SEQ ID NO: 1), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO:193 or 194. In some embodiments, clone CH3C.35.23.2, having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:193 or 194.

In some embodiments, clone ch3c.35.23.2 can have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered by SEQ ID NO:1 plus or minus M198L), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO:330 or 331. In some embodiments, clone CH3C.35.23.2, having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:330 or 331.

In some embodiments, clone CH3C.35.23.2 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 195. In some embodiments, clone CH3C.35.23.2 with a hole mutation has the sequence of SEQ ID NO: 195.

In some embodiments, clone CH3C.35.23.2 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:196 or 197. In some embodiments, the clone CH3C.35.23.2 with a hole mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:196 or 197.

In some embodiments, clone CH3C.35.23.2 may have hole mutations (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 198. In some embodiments, clone CH3C.35.23.2, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 198.

In some embodiments, clone ch3c.35.23.2 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 332. In some embodiments, clone CH3C.35.23.2, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 332.

In some embodiments, clone CH3C.35.23.2 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:199 or 200. In some embodiments, clone CH3C.35.23.2, having a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:199 or 200.

In some embodiments, clone ch3c.35.23.2 can have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:333 or 334. In some embodiments, clone CH3C.35.23.2, which has a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:333 or 334.

Clone CH3C.35.23.3

In some embodiments, clone CH3C.35.23.3 may have a knob mutation (e.g., T136W as numbered with reference to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 201. In some embodiments, clone CH3C.35.23.3 with a knob mutation has the sequence of SEQ ID NO: 201.

In some embodiments, clone CH3C.35.23.3 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:202 or 203. In some embodiments, clone CH3C.35.23.3, having a knob mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO 202 or 203.

In some embodiments, clone ch3c.35.23.3 can have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 204. In some embodiments, clone CH3C.35.23.3, with a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 204.

In some embodiments, clone CH3C.35.23.3 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 336. In some embodiments, clone CH3C.35.23.3, having a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO. 336.

In some embodiments, clone ch3c.35.23.3 can have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered by SEQ ID NO: 1), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO:205 or 206. In some embodiments, clone CH3C.35.23.3, having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:205 or 206.

In some embodiments, clone ch3c.35.23.3 can have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered by SEQ ID NO:1 plus or minus M198L), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO:337 or 338. In some embodiments, clone CH3C.35.23.3, having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 337 or 338.

In some embodiments, clone ch3c.35.23.3 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 207. In some embodiments, clone CH3C.35.23.3 with a knob mutation has the sequence of SEQ ID NO: 207.

In some embodiments, clone CH3C.35.23.3 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:208 or 209. In some embodiments, clone CH3C.35.23.3, which has a hole mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO:208 or 209.

In some embodiments, clone CH3C.35.23.3 may have hole mutations (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 210. In some embodiments, clone CH3C.35.23.3, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 210.

In some embodiments, clone CH3C.35.23.3 may have hole mutations (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), mutations that increase serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 339. In some embodiments, clone CH3C.35.23.3, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 339.

In some embodiments, clone CH3C.35.23.3 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:211 or 212. In some embodiments, clone CH3C.35.23.3, which has a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 211 or 212.

In some embodiments, clone ch3c.35.23.3 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:340 or 341. In some embodiments, clone CH3C.35.23.3, which has a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:340 or 341.

Clone CH3C.35.23.4

In some embodiments, clone CH3C.35.23.4 may have a knob mutation (e.g., T136W as numbered with reference to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 213. In some embodiments, clone CH3C.35.23.4 with a knob mutation has the sequence of SEQ ID NO. 213.

In some embodiments, clone CH3C.35.23.4 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:214 or 215. In some embodiments, clone CH3C.35.23.4, having a knob mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO:214 or 215.

In some embodiments, clone ch3c.35.23.4 may have a knob mutation (e.g., T136W as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 216. In some embodiments, clone CH3C.35.23.4, having a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 216.

In some embodiments, clone ch3c.35.23.4 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 343. In some embodiments, clone CH3C.35.23.4, having a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 343.

In some embodiments, clone ch3c.35.23.4 can have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered by SEQ ID NO: 1), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO:217 or 218. In some embodiments, clone CH3C.35.23.4, which has a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 217 or 218.

In some embodiments, clone ch3c.35.23.4 can have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered by SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:344 or 345. In some embodiments, clone CH3C.35.23.4, which has a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 344 or 345.

In some embodiments, clone CH3C.35.23.4 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 219. In some embodiments, clone CH3C.35.23.4 with a hole mutation has the sequence of SEQ ID NO: 219.

In some embodiments, clone CH3C.35.23.4 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:220 or 221. In some embodiments, the clone CH3C.35.23.4 with a hole mutation and a mutation that modulates effector function has the sequence of SEQ ID NO 220 or 221.

In some embodiments, clone ch3c.35.23.4 may have hole mutations (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and have at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 222. In some embodiments, clone CH3C.35.23.4, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 222.

In some embodiments, clone ch3c.35.23.4 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 346. In some embodiments, clone ch3c.35.23.4, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID No. 346.

In some embodiments, clone CH3C.35.23.4 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:223 or 224. In some embodiments, clone CH3C.35.23.4, which has a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:223 or 224.

In some embodiments, clone CH3C.35.23.4 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:347 or 348. In some embodiments, clone CH3C.35.23.4, which has a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:347 or 348.

Clone CH3C.35.21.17.2

In some embodiments, clone CH3C.35.21.17.2 may have a knob mutation (e.g., T136W as numbered with reference to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 225. In some embodiments, clone CH3C.35.21.17.2 with a knob mutation has the sequence of SEQ ID NO: 225.

In some embodiments, clone CH3C.35.21.17.2 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:226 or 227. In some embodiments, clone CH3C.35.21.17.2, having a knob mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO:226 or 227.

In some embodiments, clone ch3c.35.21.17.2 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 228. In some embodiments, clone CH3C.35.21.17.2, with a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 228.

In some embodiments, clone ch3c.35.21.17.2 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 350. In some embodiments, clone CH3C.35.21.17.2, having a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 350.

In some embodiments, clone CH3C.35.21.17.2 may have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered by SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:229 or 230. In some embodiments, clone CH3C.35.21.17.2, which has a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 229 or 230.

In some embodiments, clone ch3c.35.21.17.2 can have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered by SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:351 or 352. In some embodiments, clone CH3C.35.21.17.2, having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 351 or 352.

In some embodiments, clone CH3C.35.21.17.2 may have hole mutations (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 231. In some embodiments, clone CH3C.35.21.17.2 with a hole mutation has the sequence of SEQ ID NO: 231.

In some embodiments, clone CH3C.35.21.17.2 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:232 or 233. In some embodiments, clone CH3C.35.21.17.2, which has a hole mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO 232 or 233.

In some embodiments, clone ch3c.35.21.17.2 can have hole mutations (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 234. In some embodiments, clone CH3C.35.21.17.2, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 234.

In some embodiments, clone ch3c.35.21.17.2 can have hole mutations (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), mutations that increase serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 353. In some embodiments, clone CH3C.35.21.17.2, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 353.

In some embodiments, clone CH3C.35.21.17.2 may have a hole mutation (e.g., as referenced to SEQ ID NO:1 numbered T136S, L138A, and Y177V), a mutation that modulates effector function (e.g., as referenced to SEQ ID NO:1 numbered L4A, L5A, and/or P99G (e.g., L4A and L5A)), a mutation that increases serum stability or serum half-life (e.g., as referenced to SEQ ID NO:1 numbered M22Y, S24T, and T26E), and has at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO:235 or 236. In some embodiments, clone CH3C.35.21.17.2, which has a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:235 or 236.

In some embodiments, clone ch3c.35.21.17.2 may have a hole mutation (e.g., as referenced to T136S, L138A, and Y177V numbered SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:354 or 355. In some embodiments, clone CH3C.35.21.17.2, which has a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:354 or 355.

Clone CH3C.35.23

In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T136W as numbered with reference to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 237. In some embodiments, clone CH3C.35.23 with a knob mutation has the sequence of SEQ ID NO: 237.

In some embodiments, clone CH3C.35.23 may have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered by SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:238 or 239. In some embodiments, clone CH3C.35.23, having a knob mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO:238 or 239.

In some embodiments, clone ch3c.35.23 can have a knob mutation (e.g., T136W as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 240. In some embodiments, clone CH3C.35.23, with a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 240.

In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 357. In some embodiments, clone CH3C.35.23, with a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 357.

In some embodiments, clone ch3c.35.23 can have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered SEQ ID NO: 1), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO:241 or 242. In some embodiments, clone CH3C.35.23, which has a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:241 or 242.

In some embodiments, clone ch3c.35.23 can have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered by SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered by SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:358 or 359. In some embodiments, clone CH3C.35.23, which has a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 358 or 359.

In some embodiments, clone CH3C.35.23 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 243. In some embodiments, clone CH3C.35.23 with a hole mutation has the sequence of SEQ ID NO: 243.

In some embodiments, clone CH3C.35.23 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO:244 or 245. In some embodiments, clone CH3C.35.23, which has a hole mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO 244 or 245.

In some embodiments, clone ch3c.35.23 can have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO: 246. In some embodiments, clone CH3C.35.23, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 246.

In some embodiments, clone ch3c.35.23 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 360. In some embodiments, clone ch3c.35.23, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 360.

In some embodiments, clone ch3c.35.23 can have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:247 or 248. In some embodiments, clone CH3C.35.23, which has a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO. 247 or 248.

In some embodiments, clone ch3c.35.23 can have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:361 or 362. In some embodiments, clone CH3C.35.23, which has a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:361 or 362.

Clone CH3C.35.21

In some embodiments, clone CH3C.35.21 may have a knob mutation (e.g., T136W as numbered with reference to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 250. In some embodiments, clone CH3C.35.21 with a knob mutation has the sequence of SEQ ID NO. 250.

In some embodiments, clone CH3C.35.21 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:252 or 275. In some embodiments, clone CH3C.35.21, having a knob mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO 252 or 275.

In some embodiments, clone ch3c.35.21 can have a knob mutation (e.g., T136W as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 276. In some embodiments, clone CH3C.35.21, having a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 276.

In some embodiments, clone ch3c.35.21 can have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 364. In some embodiments, clone CH3C.35.21, having a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 364.

In some embodiments, clone ch3c.35.21 can have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered SEQ ID NO: 1), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO:277 or 278. In some embodiments, clone CH3C.35.21, which has a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 277 or 278.

In some embodiments, clone ch3c.35.21 can have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered by SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered by SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:365 or 366. In some embodiments, clone CH3C.35.21, which has a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO. 365 or 366.

In some embodiments, clone CH3C.35.21 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO:279. In some embodiments, clone CH3C.35.21 with a hole mutation has the sequence of SEQ ID NO:279.

In some embodiments, clone CH3C.35.21 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO:280 or 281. In some embodiments, clone CH3C.35.21, which has a hole mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO:280 or 281.

In some embodiments, clone ch3c.35.21 can have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO: 282. In some embodiments, clone CH3C.35.21, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 282.

In some embodiments, clone ch3c.35.21 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 367. In some embodiments, clone CH3C.35.21, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 367.

In some embodiments, clone CH3C.35.21 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:283 or 284. In some embodiments, clone CH3C.35.21, which has a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:283 or 284.

In some embodiments, clone ch3c.35.21 can have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:368 or 369. In some embodiments, clone CH3C.35.21, which has a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 368 or 369.

Clone CH3C.35.20.1.1

In some embodiments, clone CH3C.35.20.1.1 may have a knob mutation (e.g., T136W as referenced to SEQ ID NO:1 numbering) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 285. In some embodiments, clone CH3C.35.20.1.1 with a knob mutation has the sequence of SEQ ID NO: 285.

In some embodiments, clone CH3C.35.20.1.1 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered SEQ ID NO: 1), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO:286 or 287. In some embodiments, clone CH3C.35.20.1.1, having a knob mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO 286 or 287.

In some embodiments, clone ch3c.35.20.1.1 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 288. In some embodiments, clone CH3C.35.20.1.1, having a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 288.

In some embodiments, clone ch3c.35.20.1.1 can have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 371. In some embodiments, clone CH3C.35.20.1.1, having a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 371.

In some embodiments, clone CH3C.35.20.1.1 may have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered by SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:289 or 290. In some embodiments, clone CH3C.35.20.1.1, having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 289 or 290.

In some embodiments, clone ch3c.35.20.1.1 can have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered by SEQ ID NO:1 plus or minus M198L), and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of SEQ ID NO:372 or 373. In some embodiments, clone CH3C.35.20.1.1, having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:372 or 373.

In some embodiments, clone CH3C.35.20.1.1 may have hole mutations (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 291. In some embodiments, clone CH3C.35.20.1.1 with a hole mutation has the sequence of SEQ ID NO: 291.

In some embodiments, clone CH3C.35.20.1.1 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:292 or 293. In some embodiments, the clone CH3C.35.20.1.1, having a hole mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO 292 or 293.

In some embodiments, clone CH3C.35.20.1.1 may have hole mutations (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 294. In some embodiments, clone CH3C.35.20.1.1, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 294.

In some embodiments, clone ch3c.35.20.1.1 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 374. In some embodiments, clone CH3C.35.20.1.1, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 374.

In some embodiments, clone ch3c.35.20.1.1 may have a hole mutation (e.g., as referenced to T136S, L138A, and Y177V of SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) of SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E of SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:295 or 296. In some embodiments, clone CH3C.35.20.1.1, having a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:295 or 296.

In some embodiments, clone ch3c.35.20.1.1 may have a hole mutation (e.g., as referenced to T136S, L138A, and Y177V numbered SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:375 or 376. In some embodiments, clone CH3C.35.20.1.1, having a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:375 or 376.

Clone CH3C.35.23.2.1

In some embodiments, clone ch3c.35.23.2.1 may have a knob mutation (e.g., T136W as numbered with reference to SEQ ID NO: 1) and have at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 297. In some embodiments, clone CH3C.35.23.2.1 with a knob mutation has the sequence of SEQ ID NO: 297.

In some embodiments, clone CH3C.35.23.2.1 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:298 or 299. In some embodiments, clone CH3C.35.23.2.1, having a knob mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO:298 or 299.

In some embodiments, clone ch3c.35.23.2.1 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 300. In some embodiments, clone CH3C.35.23.2.1, having a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO. 300.

In some embodiments, clone ch3c.35.23.2.1 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 378. In some embodiments, clone CH3C.35.23.2.1, having a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 378.

In some embodiments, clone CH3C.35.23.2.1 may have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered by SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:301 or 302. In some embodiments, clone CH3C.35.23.2.1, having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:301 or 302.

In some embodiments, clone ch3c.35.23.2.1 can have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered by SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:379 or 380. In some embodiments, clone CH3C.35.23.2.1, which has a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:379 or 380.

In some embodiments, clone ch3c.35.23.2.1 may have hole mutations (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO:1 numbering) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 303. In some embodiments, clone CH3C.35.23.2.1 with a hole mutation has the sequence of SEQ ID NO: 303.

In some embodiments, clone CH3C.35.23.2.1 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:304 or 305. In some embodiments, clone CH3C.35.23.2.1, having a hole mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO:304 or 305.

In some embodiments, clone CH3C.35.23.2.1 may have hole mutations (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 306. In some embodiments, clone CH3C.35.23.2.1, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 306.

In some embodiments, clone ch3c.35.23.2.1 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 381. In some embodiments, clone CH3C.35.23.2.1, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 381.

In some embodiments, clone ch3c.35.23.2.1 may have a hole mutation (e.g., as referenced to T136S, L138A, and Y177V of SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) of SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E of SEQ ID NO: 1), and has at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO:307 or 308. In some embodiments, clone CH3C.35.23.2.1, which has a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO 307 or 308.

In some embodiments, clone ch3c.35.23.2.1 may have a hole mutation (e.g., as referenced to T136S, L138A, and Y177V numbered SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:382 or 383. In some embodiments, clone CH3C.35.23.2.1, which has a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:382 or 383.

Clone CH3C.35.23.1.1

In some embodiments, clone CH3C.35.23.1.1 may have a knob mutation (e.g., T136W as numbered with reference to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 309. In some embodiments, clone CH3C.35.23.1.1 with a knob mutation has the sequence of SEQ ID NO: 309.

In some embodiments, clone CH3C.35.23.1.1 may have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered by SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:310 or 311. In some embodiments, clone CH3C.35.23.1.1, having a knob mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO:310 or 311.

In some embodiments, clone ch3c.35.23.1.1 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 312. In some embodiments, clone CH3C.35.23.1.1, having a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 312.

In some embodiments, clone CH3C.35.23.1.1 may have a knob mutation (e.g., as referenced to T136W numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 385. In some embodiments, clone CH3C.35.23.1.1, having a knob mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 385.

In some embodiments, clone CH3C.35.23.1.1 may have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E numbered by SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:313 or 314. In some embodiments, clone CH3C.35.23.1.1, having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:313 or 314.

In some embodiments, clone CH3C.35.23.1.1 may have a knob mutation (e.g., as referenced to T136W numbered by SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A numbered by SEQ ID NO: 1)), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered by SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:386 or 387. In some embodiments, clone CH3C.35.23.1.1, having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:386 or 387.

In some embodiments, clone ch3c.35.23.1.1 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1) and at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO: 315. In some embodiments, clone CH3C.35.23.1.1 with a hole mutation has the sequence of SEQ ID NO: 315.

In some embodiments, clone CH3C.35.23.1.1 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that modulates effector function (e.g., L4A, L5A, and/or P99G (e.g., L4A and L5A) as referenced to SEQ ID NO: 1), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:316 or 317. In some embodiments, clone CH3C.35.23.1.1, having a hole mutation and a mutation that modulates effector function, has the sequence of SEQ ID NO:316 or 317.

In some embodiments, clone ch3c.35.23.1.1 may have hole mutations (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), mutations that increase serum stability or serum half-life (e.g., M22Y, S24T, and T26E as referenced to SEQ ID NO: 1), and have at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 318. In some embodiments, clone CH3C.35.23.1.1, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 318.

In some embodiments, clone ch3c.35.23.1.1 may have a hole mutation (e.g., T136S, L138A, and Y177V as referenced to SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., N204S plus or minus M198L as referenced to SEQ ID NO: 1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO: 388. In some embodiments, clone CH3C.35.23.1.1, having a hole mutation and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO: 388.

In some embodiments, clone ch3c.35.23.1.1 may have a hole mutation (e.g., as referenced to T136S, L138A, and Y177V of SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) of SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to M22Y, S24T, and T26E of SEQ ID NO: 1), and has at least 85%, at least 90%, or at least 95% identity to the sequence of SEQ ID NO:319 or 320. In some embodiments, clone CH3C.35.23.1.1, having a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:319 or 320.

In some embodiments, clone ch3c.35.23.1.1 may have a hole mutation (e.g., as referenced to T136S, L138A, and Y177V numbered SEQ ID NO: 1), a mutation that modulates effector function (e.g., as referenced to L4A, L5A, and/or P99G (e.g., L4A and L5A) numbered SEQ ID NO: 1), a mutation that increases serum stability or serum half-life (e.g., as referenced to N204S numbered SEQ ID NO:1 plus or minus M198L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:389 or 390. In some embodiments, clone CH3C.35.23.1.1, having a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life, has the sequence of SEQ ID NO:389 or 390.

Forms of TfR binding proteins

In some embodiments, a modified TfR binding polypeptide as described herein is a subunit of a protein dimer. In some embodiments, the dimer is a heterodimer. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer comprises a single Fc polypeptide that binds to a TfR receptor, i.e., is monovalent for a TfR receptor binding. In some embodiments, the dimer comprises a second polypeptide that binds to a TfR receptor. The second polypeptide may comprise the same modified Fc polypeptide to provide a bivalent homodimeric protein, or the second modified Fc polypeptide described herein may provide a second TfR receptor binding site.

The TfR-binding polypeptides and dimeric or multimeric proteins comprising the polypeptides described herein can have a wide range of binding affinities, e.g., based on the form of the polypeptide. For example, in some embodiments, a polypeptide comprising a modified Fc polypeptide as described herein has an affinity for TfR that is any value in the range of 1pM to 10 μ Μ. In some embodiments, affinity can be measured in a monovalent format. In other embodiments, affinity may be measured in a bivalent form, e.g., as a protein dimer comprising a modified Fc polypeptide.

Methods for analyzing binding affinity, binding kinetics, and cross-reactivity to analyze binding to TfR are known in the art. These methods include, but are not limited to, solid phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore)^TM(GE Healthcare, Piscataway, NJ)), kinetic exclusion analysis (e.g.) Flow cytometry, Fluorescence Activated Cell Sorting (FACS), BioLayer interferometry (e.g.

(Forte Bio, Inc., Menlo Park, CA)) and Western blot analysis. In some casesIn embodiments, the binding affinity and/or cross-reactivity is determined using ELISA. Methods of performing ELISA assays are known in the art and are also described in the examples section below. In some embodiments, Surface Plasmon Resonance (SPR) is used to determine binding affinity, binding kinetics, and/or cross-reactivity. In some embodiments, a kinetic exclusion assay is used to determine binding affinity, binding kinetics, and/or cross-reactivity. In some embodiments, binding affinity, binding kinetics, and/or cross-reactivity are determined using BioLayer interferometry analysis. FcRn binding of TfR binding polypeptides can also be assessed using these types of assays. FcRn binding is typically assayed under acidic conditions, e.g., at a pH of about 5 to about 6.

TfR binding protein conjugates

In some embodiments, a modified polypeptide that binds TfR and initiates transport across the BBB comprises a modified Fc polypeptide as described herein and further comprises a portion or all of a hinge region. The hinge region may be from any immunoglobulin subclass or isotype. An illustrative immunoglobulin hinge is an IgG hinge region, such as an IgG1 hinge region, for example the human IgG1 hinge amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 62). In other embodiments, a polypeptide that may comprise a hinge or a portion of a hinge region is further fused to another portion, such as an immunoglobulin variable region, thereby producing a TfR binding polypeptide-variable region fusion polypeptide. The variable region may bind to any antigen of interest, such as a therapeutic neural target, or a diagnostic neural target.

In some embodiments, a TfR-binding polypeptide (e.g., a modified Fc polypeptide) is fused to a variable region via a linker. As indicated in the preceding paragraph, a TfR-binding polypeptide (e.g., a modified Fc polypeptide) can be fused to a variable region via a hinge region. In some embodiments, a TfR-binding polypeptide (e.g., a modified Fc polypeptide) can be fused to a variable region by a peptide linker. The peptide linker can be configured such that it allows the variable region and the TfR-binding polypeptide to rotate relative to each other; and/or resistance to digestion by proteases. In some embodiments, the linker may be a flexible linker, e.g., containing amino acids such as: gly, Asn, Ser, Thr, Ala, etc. Such joints are designed using known parameters. For example, the linker may have repeats, such as Gly-Ser repeats.

The variable region may be in any antibody format, for example a Fab or scFv format. In some embodiments, the antibody variable region sequence comprises two antibody variable region heavy chains and two antibody variable region light chains, or fragments thereof.

A TfR binding polypeptide (e.g., a modified Fc polypeptide) can also be fused to a polypeptide that is not an immunoglobulin variable region that targets an antigen of interest. In some embodiments, such polypeptides are fused to a TfR-binding polypeptide using a peptide linker (e.g., a flexible linker as described above).

In some embodiments, a TfR-binding polypeptide may be fused to a polypeptide, e.g., a therapeutic polypeptide, in need of targeting a cell expressing a TfR-binding polypeptide. In some embodiments, the TfR-binding polypeptide is fused to a biologically active polypeptide for transport across the BBB, e.g., a soluble protein, e.g., an extracellular domain of a receptor or a growth factor, cytokine, or enzyme.

In other embodiments, the TfR-binding polypeptide may be fused to a peptide or protein suitable for protein purification, such as polyhistidine, an epitope tag (e.g., FLAG, c-Myc, hemagglutinin tag, etc.), Glutathione S Transferase (GST), thioredoxin, protein a, protein G, or Maltose Binding Protein (MBP). In some cases, a peptide or protein fused to a TfR-binding polypeptide may comprise a protease cleavage site, such as that of factor Xa or thrombin. In certain embodiments, the linkage may be cleaved by an enzyme present in the central nervous system.

A non-polypeptide agent can also be attached to the TfR-binding polypeptide. Such agents include cytotoxic agents, imaging agents, DNA or RNA molecules or compounds. In some embodiments, the agent may be a therapeutic agent or an imaging compound. In some embodiments, the agent is a small molecule, such as less than 1000Da, less than 750Da, or less than 500 Da.

A polypeptide or non-polypeptide agent may be attached to the N-or C-terminal region of a TfR-binding polypeptide, or to any region of the polypeptide, so long as the agent does not interfere with the binding of the TfR-binding polypeptide to the TfR.

In various embodiments, conjugates can be generated using well-known chemical crosslinking reagents and protocols. For example, there are a large number of chemical cross-linking agents known to those skilled in the art and suitable for cross-linking polypeptides with an agent of interest. For example, the crosslinker is a heterobifunctional crosslinker that can be used to stepwise bond molecules. Heterobifunctional cross-linkers provide the ability to design more specific coupling methods for conjugating proteins, thereby reducing the occurrence of undesirable side reactions (such as homologous protein polymers).

The agent of interest may be a therapeutic agent, including cytotoxic agents and the like, or a chemical moiety. In some embodiments, the agent may be a peptide or small molecule therapeutic or imaging agent.

Method for increasing effector function

For some applications, it is desirable to introduce modifications into the modified Fc polypeptides or modified Fc polypeptide dimers described herein to increase effector function (e.g., ADCC). A method for increasing effector function involves preparing a non-fucosylated or fucose-deficient modified Fc polypeptide or modified Fc polypeptide dimer.

One method for producing a fucose-deficient modified Fc polypeptide or modified Fc polypeptide dimer is the use of fucose analogs, such as 2-fluoro fucose (2-FF). Fucose analogs can consume or reduce the availability of GDP-fucose, a substrate required by fucosyltransferases to incorporate fucose into proteins.

An alternative method for large scale production of commonly used modified Fc polypeptides or modified Fc polypeptide dimers that produce fucose-deficient properties is to knock out the cell line using a-1, 6 fucosyltransferase (FUT8) to express the modified Fc polypeptide or modified Fc polypeptide dimer. A non-limiting example of a suitable FUT8 knock-out cell line is the Chinese Hamster Ovary (CHO) FUT8 knock-out cell line available from Lonza Biologics. In addition, FUT8 small interfering RNA (siRNA) can be used to transform CHO cell lines (e.g., by constitutive expression of FUT8 siRNA) for the preparation of fucose-deficient proteins, as described in Mori et al (Biotechnol. Bioeng. (2004)88: 901-908; incorporated herein by reference in its entirety).

XI nucleic acids, vectors and host cells

Recombinant methods are typically used to prepare modified TfR binding polypeptides as described herein. Thus, an isolated nucleic acid comprising a nucleic acid sequence encoding any of the polypeptides comprising a modified Fc polypeptide as described herein, and a host cell into which the nucleic acid is introduced for replication of the nucleic acid encoding the polypeptide and/or expression of the polypeptide. In some embodiments, the host cell is a eukaryotic cell, such as a human cell.

In another aspect, polynucleotides comprising a nucleotide sequence encoding a polypeptide described herein are provided. The polynucleotide may be single-stranded or double-stranded. In some embodiments, the polynucleotide is DNA. In a particular embodiment, the polynucleotide is a cDNA. In some embodiments, the polynucleotide is RNA.

In some embodiments, the polynucleotide is included in a nucleic acid construct. In some embodiments, the construct is a replicable vector. In some embodiments, the vector is selected from the group consisting of a plasmid, a viral vector, a phagemid, a yeast chromosomal vector, and a non-episomal mammalian vector.

In some embodiments, the polynucleotide is operably linked to one or more regulatory nucleotide sequences in the expression construct. In one series of embodiments, the nucleic acid expression construct is suitable for use as a surface expression library. In some embodiments, the library is suitable for surface expression in yeast. In some embodiments, the library is suitable for surface expression in a bacteriophage. In another series of embodiments, the nucleic acid expression construct is adapted to express the polypeptide in a system that allows isolation of the polypeptide in milligram or gram quantities. In some embodiments, the system is a mammalian cell expression system. In some embodiments, the system is a yeast cell expression system.

Expression vehicles for the preparation of recombinant polypeptides include plasmids and other vectors. Suitable vectors include, for example, the following types of plasmids: pBR 322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotic cells such as e. pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Alternatively, viral derivatives such as bovine papilloma virus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of the polypeptide in eukaryotic cells. In some embodiments, it may be desirable to express the recombinant polypeptide by using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors. Additional expression systems include adenovirus, adeno-associated virus, and other viral expression systems.

The vector may be transformed into any suitable host cell. In some embodiments, host cells (e.g., bacterial or yeast cells) may be suitable for use as surface expression libraries. In some cells, the vector is expressed in a host cell to express a relatively large amount of the polypeptide. Such host cells include mammalian cells, yeast cells, insect cells, and prokaryotic cells. In some embodiments, the cell is a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, Baby Hamster Kidney (BHK) cell, NS0 cell, Y0 cell, HEK293 cell, COS cell, Vero cell (Vero cell), or hela cell (HeLacell).

Host cells transfected with an expression vector encoding a TfR-binding polypeptide can be cultured under appropriate conditions to allow expression of the polypeptide to occur. The polypeptide can be secreted and isolated from the cell mixture and the culture medium containing the polypeptide. Alternatively, the polypeptide may be retained in the cytoplasm or membrane fraction and the cells harvested, lysed, and the polypeptide isolated using a desired method.

XII example

The following examples are included to demonstrate particular embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques which function well in the practice of the present disclosure, and thus can be considered to constitute specific modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

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