阅读说明：本技术 工程改造的转铁蛋白受体结合多肽 (Engineered TfR combination polypeptide ) 是由 陈晓诚 马克·S·丹尼斯 米哈利斯·卡里奥利斯 亚当·P·西尔弗曼 安基塔·斯利瓦斯塔瓦 于 2018-02-15 设计创作，主要内容包括：本文提供结合到转铁蛋白受体的多肽、产生此类多肽的方法和使用所述多肽以使组合物靶向转铁蛋白受体表达细胞的方法。(Provided herein is be integrated to the polypeptide of TfR, generate the method for such polypeptide and using the polypeptide so that the method for composition targeting Expression of Transferrin Receptor cell.)

1. a kind of polypeptide comprising being specifically bound to the CH3 structural domain of the modification of TfR, wherein the modification CH3 structural domain includes five at one group of amino acid position comprising 157,159,160,161,162,163,186,189 and 194 A, six, seven, eight or nine substitutions；And wherein the substitution and the position are the amino referring to SEQ ID NO:1 Sour 114-220 is determined.

2. polypeptide as described in claim 1, wherein the CH3 structural domain of the modification is also including 153,164,165 and 188 Include one, two, three or four substitution at position.

3. polypeptide as claimed in claim 1 or 2, wherein the CH3 structural domain of the modification is also including 187,197 and 199 Replace at position comprising one, two or three.

4. polypeptide as claimed any one in claims 1 to 3, wherein polypeptide is integrated to the top knot of the TfR Structure domain.

5. polypeptide as claimed in claim 4 wherein the polypeptide is integrated to the TfR, but does not inhibit to turn iron egg The combination of the white and described TfR.

6. polypeptide as described in claim 4 or 5, wherein the polypeptide is integrated to the ammonia comprising the TfR sequence The epitope of base acid 208.

7. such as polypeptide described in any one of claims 1 to 6, wherein the CH3 structural domain of the modification is included in 161 Trp。

8. the polypeptide as described in any one of claims 1 to 7, wherein the CH3 structural domain of the modification is included in 194 virtues Race's amino acid.

9. polypeptide as claimed in claim 8, wherein the aromatic amino acid at 194 is Trp or Phe.

10. such as polypeptide described in any one of claims 1 to 6, wherein the CH3 structural domain of the modification includes at least one choosing From position below: 157 are Leu, Tyr, Met or Val；159 are Leu, Thr, His or Pro；160 be Val, Pro or Acidic amino acid；161 are Trp；162 are Val, Ser or Ala；186 are Glu, Ala, Ser, Leu, Thr or Pro；189 Position is Thr or acidic amino acid；It is Trp, Tyr, His or Phe with 194.

11. polypeptide as claimed in claim 10, wherein the CH3 structural domain of the modification includes two selected from the following, three, Four, five, six, seven or eight positions: 157 are Leu, Tyr, Met or Val；159 be Leu, Thr, His or Pro；160 are Val, Pro or acidic amino acid；161 are Trp；162 are Val, Ser or Ala；186 be Glu, Ala, Ser, Leu, Thr or Pro；189 are Thr or acidic amino acid；It is Trp, Tyr, His or Phe with 194.

12. the polypeptide as described in any one of claims 1 to 11, wherein the CH3 structural domain of the modification is included in 157 Leu or Met；In 159 Leu, His or Pro；In 160 Val；In 161 Trp；In 162 Val or Ala；In 186 Pro；In 189 Thr；And/or in 194 Trp.

13. polypeptide as claimed in claim 12, wherein the CH3 structural domain of the modification be also included in 164 Ser, Thr, Gln or Phe.

14. polypeptide as described in claim 12 or 13, wherein the CH3 structural domain of the modification be also included in 153 Trp, Tyr, Leu or Gln.

15. the polypeptide as described in any one of claim 12 to 14, wherein the CH3 structural domain of the modification is also included in 165 Gln, Phe or His of position.

16. polypeptide as described in claim 12 or 13, wherein the CH3 structural domain of the modification is also included in 153 Trp And/or in 165 Gln.

17. the polypeptide as described in any one of claim 10 to 16, wherein the CH3 structural domain modified also includes one, two Or three positions selected from the following: 187 are Lys, Arg, Gly or Pro；197 are Ser, Thr, Glu or Lys；With 199 It is Ser, Trp or Gly.

18. the polypeptide as described in any one of claims 1 to 11, wherein the CH3 structural domain of the modification is included in 157 Tyr；In 159 Thr；In 160 Glu or Val；In 161 Trp；In 162 Ser；In 186 Ser or Thr；In 189 Glu；And/or in 194 Phe.

19. polypeptide as claimed in claim 18, wherein the CH3 structural domain of the modification be also included in 153 Trp, Tyr, Leu or Gln.

20. the polypeptide as described in claim 18 or 19, wherein the CH3 structural domain of the modification is also included in 188 Glu.

21. polypeptide as claimed in claim 18, wherein the CH3 structural domain of the modification be also included in 153 Trp and/or In 188 Glu.

22. the polypeptide as described in any one of claim 18 to 21, wherein the CH3 structural domain of the modification is included in 163 Asn.

23. the polypeptide as described in any one of claim 2 to 6, wherein the CH3 structural domain of the modification includes in following substitution One or more: in 153 Trp；In 159 Thr；In 161 Trp；In 162 Val；In 186 Ser Or Thr；In 188 Glu；And/or in 194 Phe.

24. the polypeptide as described in any one of claim 1 to 23, wherein the CH3 structural domain of the modification and SEQ ID NO: The amino acid 1 14-220 of any of 4-29,236-299 and 422-435 have at least 85% identity, at least 90% identity Or at least 95% identity.

25. the polypeptide as described in any one of claim 1 to 23, wherein the CH3 structural domain of the modification and SEQ ID NO:1 Amino acid 1 14-220 have at least 85% identity, restrictive condition be the homogeneity percentage do not include described group of position Set 157,159,160,161,162,163,186,189 and 194.

26. the polypeptide as described in any one of claim 1 to 25, wherein the CH3 structural domain of the modification includes SEQ ID The amino acid 1 57-163 and/or 186-194 of any of NO:4-29,236-299 and 422-435.

27. the polypeptide as described in any one of claim 2 to 6, wherein the CH3 structural domain of the modification includes at least one choosing From position below: 153 are Trp, Leu or Glu；157 are Tyr or Phe；159 are Thr；160 are Glu；161 It is Trp；162 are Ser, Ala, Val or Asn；163 are Ser or Asn；186 are Thr or Ser；188 be Glu or Ser；189 are Glu；It is Phe with 194.

28. polypeptide as claimed in claim 27, wherein the CH3 structural domain of the modification include it is selected from the following 2,3,4,5,6, 7,8,9,10 or 11 positions: 153 are Trp, Leu or Glu；157 are Tyr or Phe；159 are Thr；160 are Glu； 161 are Trp；162 are Ser, Ala, Val or Asn；163 are Ser or Asn；186 are Thr or Ser；188 are Glu Or Ser；189 are Glu；It is Phe with 194.

29. polypeptide as claimed in claim 28, wherein the CH3 structural domain of the modification includes following 11 positions: 153 are Trp, Leu or Glu；157 are Tyr or Phe；159 are Thr；160 are Glu；161 are Trp；162 be Ser, Ala, Val or Asn；163 are Ser or Asn；186 are Thr or Ser；188 are Glu or Ser；189 are Glu；It is with 194 Phe。

30. the polypeptide as described in claim 28 or 29, wherein the CH3 structural domain of the modification and SEQ ID NO:4-29, The amino acid 1 14-220 of any of 236-299 and 422-435 has at least 85% identity, at least 90% identity or extremely Few 95% identity.

31. polypeptide as claimed in claim 30, wherein appointing in corresponding to SEQ ID NO:4-29,236-299 and 422-435 One position 153,157,159,160,161,162,163,164,165,186,187,188,189,194,197 and 199 The residue at least 5,6,7,8,9,10,11,12,13,14,15 or 16 in position does not lack or replaces.

32. the polypeptide as described in any one of claims 1 to 31, wherein the CH3 structural domain of the modification also includes (i) In 139 Trp, or (ii) 139 Ser, in 141 Ala and in 180 Val, wherein the amino acid position is It is determined referring to SEQ ID NO:1.

33. the polypeptide as described in any one of claims 1 to 32, wherein the CH3 structural domain of the modification also includes (i) In 201 Leu and in 207 Ser, or (ii) in 207 Ser or Ala, wherein the amino acid position is referring to SEQ ID NO:1 is determined.

34. a kind of polypeptide comprising being specifically bound to the CH3 structural domain of the modification of TfR, wherein the modification CH3 structural domain is in one group of amino acid position comprising 153,157,159,160,162,163,186,188,189,194,197 and 199 Place is set to replace comprising one or more；And wherein the substitution and the position are that the sequence of reference SEQ ID NO:13 is true It is fixed.

35. polypeptide as claimed in claim 34, wherein the CH3 structural domain of the modification included in 153 Glu, Leu, Ser, Val, Trp or Tyr；Aromatic amino acid, Met, Pro or Val at 157；In 159 Thr, Asn or Val；160 Glu, Ile, Pro or Val of position；In 162 aliphatic amino acids, Ser or Thr；In 163 Ser, Asn, Arg or Thr； In 186 Thr, His or Ser；In 188 Glu, Ser, Asp, Gly, Thr, Pro, Gln or Arg；In 189 Glu Or Arg；In 194 Phe, His, Lys, Tyr or Trp；In 197 Ser, Thr or Trp；With 199 Ser, Cys, Pro, Met or Trp.

36. polypeptide as claimed in claim 35, wherein the aromatic amino acid at 157 is Tyr, Phe or Trp and described It is Ala, Ile or Val in 162 aliphatic amino acids.

37. the polypeptide as described in any one of claim 34 or 35, wherein the CH3 structural domain of the modification is included in 153 Glu, Leu or Trp；In 157 aromatic amino acids；In 159 Thr；In 160 Glu；In 162 aliphatic ammonia Base acid or Ser；In 163 Ser or Asn；In 186 Thr or Ser；In 188 Glu or Ser；In 189 Glu； In 194 Phe, His, Tyr or Trp；In 197 Ser；With in 199 Ser.

38. polypeptide as claimed in claim 37, wherein the aromatic amino acid at 157 be Tyr or Phe and it is described 162 aliphatic amino acids are Ala or Val.

39. the polypeptide as described in any one of claim 34 to 38, wherein the CH3 structural domain of the modification has SEQ ID The sequence of NO:556 or 559.

40. the polypeptide as described in any one of claim 34 to 38, wherein the CH3 structural domain of the modification comprising 153, 157, include a substitution at 159,160,162,163,186,188,189,194,197 and 199 one group of amino acid position.

41. polypeptide as claimed in claim 40, wherein the CH3 structural domain of the modification has in SEQ ID NO:563-574 The sequence of any one.

42. the polypeptide as described in any one of claim 34 or 35, wherein the CH3 structural domain of the modification is included in 153 Glu, Leu or Trp；In 157 Tyr or Phe；In 159 Thr；In 160 Glu；In 162 Ala, Val or Ser；In 163 Ser or Asn；In 186 Thr or Ser；In 188 Glu or Ser；In 189 Glu；194 The Phe of position；In 197 Ser；With in 199 Ser.

43. polypeptide as claimed in claim 42, wherein the CH3 structural domain of the modification has the sequence of SEQ ID NO:562.

44. a kind of polypeptide comprising being specifically bound to the CH3 structural domain of the modification of TfR, wherein the modification CH3 structural domain includes at one group of amino acid position comprising 153,157,159,160,162,163,164,186,189 and 194 One or more replaces；And wherein the substitution and the position are that the sequence of reference SEQ ID NO:9 determines.

45. polypeptide as claimed in claim 44, wherein the CH3 structural domain of the modification is included in 153 Glu or Trp；In 157 Val, Trp, Leu or Tyr；In 159 Leu, Pro, Phe, Thr or His；In 160 Pro, Val or Glu； In 162 Ala, Ser, Val or Gly；In 163 Leu, His, Gln, Gly, Val, Ala, Asn, Asp, Thr or Glu； In 164 Thr, Phe, Gln, Val or Tyr；In 186 Leu, Ser, Glu, Ala or Pro；189 Glu, Asp, Thr or Asn；With in 194 Trp, Tyr, Phe or His.

46. polypeptide as claimed in claim 45, wherein the CH3 structural domain of the modification is included in 153 Glu or Trp；In 157 Trp, Leu or Tyr；In 159 Thr or His；In 160 Val；In 162 Ala, Ser or Val；In 163 Val, Asn or Thr；In 164 Gln or Tyr；In 186 Pro；In 189 Thr or Asn；With 194 Trp, Tyr, Phe or His of position.

47. the polypeptide as described in any one of claim 44 to 46, wherein the CH3 structural domain of the modification has SEQ ID The sequence of NO:577 or 580.

48. a kind of polypeptide comprising being specifically bound to the CH3 structural domain of the modification of TfR, wherein the modification CH3 structural domain includes five, six at one group of amino acid position comprising 118,119,120,122,210,211,212 and 213 A, seven or eight substitutions；And wherein the substitution and the position are the amino acid 1 14-220 referring to SEQ ID NO:1 It determines.

49. polypeptide as claimed in claim 48, wherein the CH3 structural domain of the modification is included in 210 Gly；At 211 Phe；And/or in 213 Asp.

50. polypeptide as claimed in claim 48, wherein the CH3 structural domain of the modification includes at least one position selected from the following Set: 118 are Phe or Ile；119 are Asp, Glu, Gly, Ala or Lys；120 are Tyr, Met, Leu, Ile or Asp； 122 are Thr or Ala；210 are Gly；211 are Phe；212 are His, Tyr, Ser or Phe；It is Asp with 213.

51. polypeptide as claimed in claim 50, wherein the CH3 structural domain of the modification includes two selected from the following, three, Four, five, six, seven or eight positions: 118 are Phe or Ile；119 are Asp, Glu, Gly, Ala or Lys；120 Position is Tyr, Met, Leu, Ile or Asp；122 are Thr or Ala；210 are Gly；211 are Phe；212 be His, Tyr, Ser or Phe；It is Asp with 213.

52. the polypeptide as described in any one of claim 48 to 51, wherein the CH3 structural domain of the modification and SEQ ID NO: The amino acid 1 14-220 of any of 30-46 has at least 85% identity, at least 90% identity or at least 95% same Property.

53. the polypeptide as described in any one of claim 48 to 51, wherein the CH3 structural domain of the modification and SEQ ID NO: 1 amino acid 1 14-220 has at least 85% identity, and restrictive condition is that the homogeneity percentage does not include described group of position Set 118,119,120,122,210,211,212 and 213.

54. the polypeptide as described in any one of claim 48 to 53, wherein the CH3 structural domain of the modification includes SEQ ID The amino acid 1 18-122 and/or 210-213 of any of NO:30-46.

55. the polypeptide as described in any one of claim 34 to 54, wherein the CH3 structural domain of the modification also includes (i) In 139 Trp, or (ii) 139 Ser, in 141 Ala and in 180 Val, wherein the amino acid position is It is determined referring to SEQ ID NO:1.

56. the polypeptide as described in any one of claim 34 to 55, wherein the CH3 structural domain of the modification also includes (i) In 201 Leu and in 207 Ser, or (ii) in 207 Ser or Ala, wherein the amino acid position is referring to SEQ ID NO:1 is determined.

57. the polypeptide as described in any one of claim 1 to 56, wherein corresponding unmodified CH3 structural domain be human IgG 1, IgG2, IgG3 or IgG4 CH3 structural domain.

58. the polypeptide as described in any one of claim 1 to 57, wherein the polypeptide is joined to CH2 structural domain.

59. polypeptide as claimed in claim 58, wherein the CH2 structural domain contains the amino acid sequence with reference to SEQ ID NO:1 One or two of following each group modification of column:

(a) at 7 and in 8 Ala；With

(b) 25 Tyr, in 27 Thr and in 29 Glu.

60. polypeptide as claimed in claim 59, wherein (a) group is also included in 102 Gly.

61. the polypeptide as described in any one of claim 58 to 60, wherein the CH2 structural domain be human IgG 1, IgG2, IgG3 or IgG4 CH2 structural domain.

62. the polypeptide as described in any one of claim 58 to 61, wherein the polypeptide is further joined to Fab.

63. the polypeptide as described in any one of claim 58 to 62, wherein the polypeptide is the first polypeptide of dimer, thus The dimer is set to be combined into unit price about TfR.

64. the polypeptide as described in any one of claim 58 to 62, wherein the polypeptide is the first polypeptide, with the second polypeptide Dimer is formed, second polypeptide is integrated to the TfR and includes the CH3 structural domain of modification.

65. the polypeptide as described in claim 64, wherein the CH3 structural domain of the modification of second polypeptide and first polypeptide Modification CH3 structural domain it is identical.

66. a kind of polypeptide comprising being specifically bound to the CH2 structural domain of the modification of TfR, wherein the modification CH2 structural domain includes five, six, seven at one group of amino acid position comprising 47,49,56,58,59,60,61,62 and 63 A, eight or nine substitutions；And wherein the substitution and the position are that the amino acid 4-113 of reference SEQ ID NO:1 is true It is fixed.

67. the polypeptide as described in claim 66, wherein the CH2 structural domain of the modification is included in 60 Glu and/or 61 The Trp of position.

68. the polypeptide as described in claim 66, wherein the CH2 structural domain of the modification includes at least one position selected from the following Set: 47 are Glu, Gly, Gln, Ser, Ala, Asn, Tyr or Trp；49 are Ile, Val, Asp, Glu, Thr, Ala or Tyr； 56 are Asp, Pro, Met, Leu, Ala, Asn or Phe；58 are Arg, Ser, Ala or Gly；59 be Tyr, Trp, Arg or Val；60 are Glu；61 are Trp or Tyr；62 are Gln, Tyr, His, Ile, Phe, Val or Asp；With 63 be Leu, Trp, Arg, Asn, Tyr or Val.

69. polypeptide as recited in claim 68, wherein the CH2 structural domain of the modification include it is selected from the following at least two, Three, four, five, six, seven, eight or nine positions: 47 be Glu, Gly, Gln, Ser, Ala, Asn, Tyr or Trp；49 are Ile, Val, Asp, Glu, Thr, Ala or Tyr；56 are Asp, Pro, Met, Leu, Ala, Asn or Phe；58 Position is Arg, Ser, Ala or Gly；59 are Tyr, Trp, Arg or Val；60 are Glu；61 are Trp or Tyr；62 are Gln, Tyr, His, Ile, Phe, Val or Asp；It is Leu, Trp, Arg, Asn, Tyr or Val with 63.

70. the polypeptide as described in claim 68 or 69, wherein the CH2 structural domain of the modification included in 47 Glu, Gly, Gln, Ser, Ala, Asn or Tyr；In 49 Ile, Val, Asp, Glu, Thr, Ala or Tyr；56 Asp, Pro, Met, Leu, Ala or Asn；In 58 Arg, Ser or Ala；In 59 Tyr, Trp, Arg or Val；In 60 Glu；In 61 Trp；In 62 Gln, Tyr, His, Ile, Phe or Val；And/or in 63 Leu, Trp, Arg, Asn or Tyr.

71. the polypeptide as described in any one of claim 68 to 70, wherein the CH2 structural domain of the modification is included in 58 Arg；In 59 Tyr or Trp；In 60 Glu；In 61 Trp；And/or in 63 Arg or Trp.

72. the polypeptide as described in any one of claim 66 to 71, wherein the CH2 structural domain of the modification and SEQ ID NO: The amino acid 4-113 of any of 47-62 has at least 85% identity, at least 90% identity or at least 95% identity.

73. the polypeptide as described in any one of claim 66 to 71, wherein the CH2 structural domain of the modification and SEQ ID NO: 1 amino acid 4-113 has at least 85% identity, and restrictive condition is that the homogeneity percentage does not include described group of position 47,49,56,58,59,60,61,62 and 63.

74. the polypeptide as described in any one of claim 66 to 73, wherein the CH2 structural domain of the modification includes SEQ ID The amino acid 47-49 and/or 56-63 of any of NO:47-62.

75. a kind of polypeptide comprising being specifically bound to the CH2 structural domain of the modification of TfR, wherein the modification CH2 structural domain at one group of amino acid position comprising 39,40,41,42,43,44,68,70,71 and 72 comprising five, six, Seven, eight, nine or ten substitutions；And wherein the substitution and the position are the amino acid referring to SEQ ID NO:1 4-113 is determined.

76. the polypeptide as described in claim 75, wherein the CH2 structural domain of the modification is included in 43 Pro, at 68 Glu and/or in 70 Tyr.

77. the polypeptide as described in claim 75, wherein the CH2 structural domain of the modification includes at least one position selected from the following Set: 39 are Pro, Phe, Ala, Met or Asp；40 are Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp or Tyr； 41 are Thr, Ser, Gly, Met, Val, Phe, Trp or Leu；42 are Pro, Val, Ala, Thr or Asp；43 be Pro, Val or Phe；44 are Trp, Gln, Thr or Glu；68 are Glu, Val, Thr, Leu or Trp；70 are Tyr, His, Val Or Asp；71 are Thr, His, Gln, Arg, Asn or Val；It is Tyr, Asn, Asp, Ser or Pro with 72.

78. the polypeptide as described in claim 77, wherein the CH2 structural domain of the modification includes two selected from the following, three, Four, five, six, seven, eight, nine or ten positions: 39 are Pro, Phe, Ala, Met or Asp；40 be Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp or Tyr；41 are Thr, Ser, Gly, Met, Val, Phe, Trp or Leu； 42 are Pro, Val, Ala, Thr or Asp；43 are Pro, Val or Phe；44 are Trp, Gln, Thr or Glu；68 are Glu, Val, Thr, Leu or Trp；70 are Tyr, His, Val or Asp；71 are Thr, His, Gln, Arg, Asn or Val；With 72 are Tyr, Asn, Asp, Ser or Pro.

79. the polypeptide as described in claim 77 or 78, wherein the CH2 structural domain of the modification is included in 39 Pro, Phe Or Ala；In 40 Gln, Pro, Arg, Lys, Ala or Ile；In 41 Thr, Ser, Gly, Met, Val, Phe or Trp； In 42 Pro, Val or Ala；In 43 Pro；In 44 Trp or Gln；In 68 Glu；In 70 Tyr；In 71 Thr, His or Gln；And/or in 72 Tyr, Asn, Asp or Ser.

80. the polypeptide as described in claim 77 or 78, wherein the CH2 structural domain of the modification is included in 39 Met；40 The Leu or Glu of position；In 41 Trp；In 42 Pro；In 43 Val；In 44 Thr；In 68 Val or Thr；In 70 His；In 71 His, Arg or Asn；And/or in 72 Pro.

81. the polypeptide as described in claim 77 or 78, wherein the CH2 structural domain of the modification is included in 39 Asp；40 The Asp of position；In 41 Leu；In 42 Thr；In 43 Phe；In 44 Gln；In 68 Val or Leu；70 The Val of position；In 71 Thr；And/or in 72 Pro.

82. the polypeptide as described in any one of claim 75 to 81, wherein the CH2 structural domain of the modification and SEQ ID NO: The amino acid 4-113 of any of 63-85 has at least 85% identity, at least 90% identity or at least 95% identity.

83. the polypeptide as described in any one of claim 75 to 81, wherein the CH2 structural domain of the modification and SEQ ID NO: 1 amino acid 4-113 has at least 85% identity, and restrictive condition is that the homogeneity percentage does not include described group of position 39,40,41,42,43,44,68,70,71 and 72.

84. the polypeptide as described in any one of claim 75 to 83, wherein the CH2 structural domain of the modification includes SEQ ID The amino acid 39-44 and/or 68-72 of any of NO:63-85.

85. a kind of polypeptide comprising being specifically bound to the CH2 structural domain of the modification of TfR, wherein the modification CH2 structural domain at one group of amino acid position comprising 41,42,43,44,45,65,66,67,69 and 73 comprising five, six, Seven, eight, nine or ten substitutions；And wherein the substitution and the position are the amino acid referring to SEQ ID NO:1 4-113 is determined.

86. the polypeptide as described in claim 85, wherein the CH2 structural domain of the modification includes at least one position selected from the following Set: 41 are Val or Asp；42 are Pro, Met or Asp；43 are Pro or Trp；44 are Arg, Trp, Glu or Thr；45 Position is Met, Tyr or Trp；65 are Leu or Trp；66 are Thr, Val, Ile or Lys；67 be Ser, Lys, Ala or Leu；69 are His, Leu or Pro；It is Val or Trp with 73.

87. the polypeptide as described in claim 86, wherein the CH2 structural domain of the modification includes two selected from the following, three, Four, five, six, seven, eight, nine or ten positions: 41 are Val or Asp；42 are Pro, Met or Asp；43 Position is Pro or Trp；44 are Arg, Trp, Glu or Thr；45 are Met, Tyr or Trp；65 are Leu or Trp；66 are Thr, Val, Ile or Lys；67 are Ser, Lys, Ala or Leu；69 are His, Leu or Pro；It is Val or Trp with 73.

88. the polypeptide as described in claim 86 or 87, wherein the CH2 structural domain of the modification is included in 41 Val；42 The Pro of position；In 43 Pro；In 44 Arg or Trp；In 45 Met；In 65 Leu；In 66 Thr；67 The Ser of position；In 69 His；And/or in 73 Val.

89. the polypeptide as described in claim 86 or 87, wherein the CH2 structural domain of the modification is included in 41 Asp；42 The Met or Asp of position；In 43 Trp；In 44 Glu or Thr；In 45 Tyr or Trp；In 65 Trp；At 66 Val, Ile or Lys；In 67 Lys, Ala or Leu；In 69 Leu or Pro；And/or in 73 Trp.

90. the polypeptide as described in any one of claim 85 to 89, wherein the CH2 structural domain of the modification and SEQ ID NO: The amino acid 4-113 of any of 86-90 has at least 85% identity, at least 90% identity or at least 95% identity.

91. the polypeptide as described in any one of claim 85 to 89, wherein the CH2 structural domain of the modification and SEQ ID NO: 1 amino acid 4-113 has at least 85% identity, and restrictive condition is that the homogeneity percentage does not include described group of position 41,42,43,44,45,65,66,67,69 and 73.

92. the polypeptide as described in any one of claim 85 to 91, wherein the CH2 structural domain of the modification includes SEQ ID The amino acid 41-45 and/or 65-73 of any of NO:86-90.

93. a kind of polypeptide comprising being specifically bound to the CH2 structural domain of the modification of TfR, wherein the modification CH2 structural domain at one group of amino acid position comprising 45,47,49,95,97,99,102,103 and 104 comprising five, six, Seven, eight or nine substitutions；And wherein the substitution and the position are the amino acid 4-113 referring to SEQ ID NO:1 It determines.

94. the polypeptide as described in claim 93, wherein the CH2 structural domain of the modification is included in 103 Trp.

95. the polypeptide as described in claim 93 or 94, wherein the CH2 structural domain of the modification includes at least one selected from following Position: 45 are Trp, Val, Ile or Ala；47 are Trp or Gly；49 are Tyr, Arg or Glu；95 are Ser, Arg Or Gln；97 are Val, Ser or Phe；99 are Ile, Ser or Trp；102 are Trp, Thr, Ser, Arg or Asp；103 It is Trp；It is Ser, Lys, Arg or Val with 104.

96. the polypeptide as described in claim 95, wherein the CH2 structural domain of the modification includes two selected from the following, three, Four, five, six, seven, eight or nine positions: 45 are Trp, Val, Ile or Ala；47 are Trp or Gly；49 It is Tyr, Arg or Glu；95 are Ser, Arg or Gln；97 are Val, Ser or Phe；99 are Ile, Ser or Trp；102 It is Trp, Thr, Ser, Arg or Asp；103 are Trp；It is Ser, Lys, Arg or Val with 104.

97. the polypeptide as described in claim 93 or 94, wherein the CH2 structural domain of the modification included in 45 Val or Ile；In 47 Gly；In 49 Arg；In 95 Arg；In 97 Ser；In 99 Ser；102 Thr, Ser or Arg；In 103 Trp；And/or in 104 Lys or Arg.

98. the polypeptide as described in any one of claim 93 to 97, wherein the CH2 structural domain of the modification and SEQ ID NO: The amino acid 4-113 of any of 91-95 has at least 85% identity, at least 90% identity or at least 95% identity.

99. the polypeptide as described in any one of claim 93 to 97, wherein the CH2 structural domain of the modification and SEQ ID NO: 1 amino acid 4-113 has at least 85% identity, and restrictive condition is that the homogeneity percentage does not include described group of position 45,47,49,95,97,99,102,103 and 104.

100. the polypeptide as described in any one of claim 93 to 99, wherein the CH2 structural domain of the modification includes SEQ ID The amino acid 45-49 and/or 95-104 of any of NO:91-95.

101. the polypeptide as described in any one of claim 66 to 100, wherein corresponding unmodified CH2 structural domain is the mankind IgG1, IgG2, IgG3 or IgG4 CH2 structural domain.

102. the polypeptide as described in any one of claim 66 to 101, wherein the CH2 structural domain of the modification contain with reference to One or two of following each group modification of the amino acid sequence of SEQ ID NO:1:

(a) at 7 and in 8 Ala；With

(b) 25 Tyr, in 27 Thr and in 29 Glu.

103. the polypeptide as described in claim 102, wherein (a) group is also included in 102 Gly.

104. the polypeptide as described in any one of claim 66 to 103, wherein the polypeptide is joined to CH3 structural domain.

105. the polypeptide as described in claim 104, wherein the CH3 structural domain includes the Trp of (i) at 139, or (ii) In 139 Ser, in 141 Ala and in 180 Val, wherein the amino acid position is true referring to SEQ ID NO:1 It is fixed.

106. the polypeptide as described in claim 104 or 105, wherein the CH3 structural domain include (i) 201 Leu and 207 Ser, or (ii) in 207 Ser or Ala, wherein the amino acid position is determined referring to SEQ ID NO:1.

107. the polypeptide as described in any one of claim 104 to 106, wherein the polypeptide is further joined to Fab.

108. the polypeptide as described in any one of claim 104 to 107, wherein the polypeptide is the first polypeptide of dimer, Thus the dimer is made to be combined into unit price about TfR.

109. the polypeptide as described in any one of claim 104 to 107, wherein the polypeptide is the first polypeptide, with second Polypeptide forms dimer, and second polypeptide is integrated to the TfR and includes the CH2 structural domain of modification.

110. the polypeptide as described in claim 109, wherein the CH2 structural domain of the modification of second polypeptide and described the The CH2 structural domain of the modification of one polypeptide is identical.

111. a kind of polypeptide for being specifically bound to TfR, it includes:

The amino acid 1 57-194 of any of SEQ ID NO:4-29,236-299 and 422-435,

The amino acid 1 18-213 of any of SEQ ID NO:30-46,

The amino acid 47-63 of any of SEQ ID NO:47-62,

The amino acid 39-72 of any of SEQ ID NO:63-85,

The amino acid 41-73 of any of SEQ ID NO:86-90, or

The amino acid 45-104 of any of SEQ ID NO:91-95, wherein the amino acid position is referring to SEQ ID NO:1 It determines.

112. a kind of polypeptide for being specifically bound to TfR, it includes section mutation and with SEQ ID NO:349,361, 373, any of 385,397,409,436,448,460 and 472 sequence has at least 85% identity, at least 90% same Property or at least 95% identity sequence, wherein as numbered referring to SEQ ID NO:1, the section mutation is T139W.

113. a kind of polypeptide for being specifically bound to TfR, it includes section mutation, the mutation of mediating effect+6 function and Have at least with the sequence of any of SEQ ID NO:350,362,374,386,398,410,437,449,461 and 473 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, Section mutation is that the mutation of T139W and the mediating effect+6 function is L7A and L8A.

114. a kind of polypeptide for being specifically bound to TfR, it includes section mutation, the mutation of mediating effect+6 function and Have at least with the sequence of any of SEQ ID NO:351,363,375,387,399,411,438,450,462 and 474 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, Section mutation is that the mutation of T139W and the mediating effect+6 function is L7A, L8A and P102G.

115. a kind of polypeptide for being specifically bound to TfR, it includes section mutation, the mutation of increase serum stability Have at least with the sequence of any of SEQ ID NO:352,364,376,388,400,412,439,451,463 and 475 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, The section mutation is T139W and the mutation for increasing serum stability is M25Y, S27T and T29E.

116. a kind of polypeptide for being specifically bound to TfR, it includes section mutation, the mutation of increase serum stability Have at least with the sequence of any of SEQ ID NO:485,492,499,506,513,520,527,534,541 and 548 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, The section mutation is T139W and the mutation for increasing serum stability is M201L and N207S.

117. a kind of polypeptide for being specifically bound to TfR it includes section mutation, the mutation of mediating effect+6 function, increases The mutation of increase serum stability and in SEQ ID NO:353,365,377,389,401,413,440,452,464 and 476 appoint One sequence has at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein such as reference SEQ ID NO:1 is numbered, and the section mutation is T139W, and the mutation of the mediating effect+6 function is L7A and L8A and the increasing The mutation of increase serum stability is M25Y, S27T and T29E.

118. a kind of polypeptide for being specifically bound to TfR it includes section mutation, the mutation of mediating effect+6 function, increases The mutation of increase serum stability and in SEQ ID NO:486,493,500,507,514,521,528,535,542 and 549 appoint One sequence has at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein such as reference SEQ ID NO:1 is numbered, and the section mutation is T139W, and the mutation of the mediating effect+6 function is L7A and L8A and the increasing The mutation of increase serum stability is M201L and N207S.

119. a kind of polypeptide for being specifically bound to TfR it includes section mutation, the mutation of mediating effect+6 function, increases The mutation of increase serum stability and in SEQ ID NO:354,366,378,390,402,414,441,453,465 and 477 appoint One sequence has at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein such as reference SEQ ID NO:1 is numbered, and section mutation is T139W, the mutation of the mediating effect+6 function be L7A, L8A and P102G and The mutation for increasing serum stability is M25Y, S27T and T29E.

120. a kind of polypeptide for being specifically bound to TfR it includes section mutation, the mutation of mediating effect+6 function, increases The mutation of increase serum stability and in SEQ ID NO:487,494,501,508,515,522,529,536,543 and 550 appoint One sequence has at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein such as reference SEQ ID NO:1 is numbered, and section mutation is T139W, the mutation of the mediating effect+6 function be L7A, L8A and P102G and The mutation for increasing serum stability is M201L and N207S.

121. a kind of polypeptide for being specifically bound to TfR, it includes hole mutation and with SEQ ID NO:355,367, 379, any of 391,403,415,442,454,466 and 478 sequence has at least 85% identity, at least 90% same Property or at least 95% identity sequence, wherein as numbered referring to SEQ ID NO:1, the hole mutation is T139S, L141A And Y180V.

122. a kind of polypeptide for being specifically bound to TfR, it includes hole mutation, the mutation of mediating effect+6 function and Have at least with the sequence of any of SEQ ID NO:356,368,380,392,404,416,443,455,467 and 479 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, Hole mutation is that the mutation of T139S, L141A and Y180V and the mediating effect+6 function is L7A and L8A.

123. a kind of polypeptide for being specifically bound to TfR, it includes hole mutation, the mutation of mediating effect+6 function and Have at least with the sequence of any of SEQ ID NO:357,369,381,393,405,417,444,456,468 and 480 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, Hole mutation is that the mutation of T139S, L141A and Y180V and the mediating effect+6 function is L7A, L8A and P102G.

124. a kind of polypeptide for being specifically bound to TfR, it includes hole mutation, the mutation of increase serum stability Have at least with the sequence of any of SEQ ID NO:358,370,382,394,406,418,445,457,469 and 481 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, The hole mutation is T139S, L141A and Y180V and the mutation for increasing serum stability is M25Y, S27T and T29E.

125. a kind of polypeptide for being specifically bound to TfR, it includes hole mutation, the mutation of increase serum stability Have at least with the sequence of any of SEQ ID NO:488,495,502,509,516,523,530,537,544 and 551 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, The hole mutation is T139S, L141A and Y180V and the mutation for increasing serum stability is M201L and N207S.

126. a kind of polypeptide for being specifically bound to TfR it includes hole mutation, the mutation of mediating effect+6 function, increases The mutation of increase serum stability and in SEQ ID NO:359,371,383,395,407,419,446,458,470 and 482 appoint One sequence has at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein such as reference SEQ ID NO:1 is numbered, and the hole mutation is T139S, L141A and Y180V, and the mutation of the mediating effect+6 function is L7A With L8A and the mutation for increasing serum stability is M25Y, S27T and T29E.

127. a kind of polypeptide for being specifically bound to TfR it includes hole mutation, the mutation of mediating effect+6 function, increases The mutation of increase serum stability and in SEQ ID NO:489,496,503,510,517,524,531,538,545 and 552 appoint One sequence has at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein such as reference SEQ ID NO:1 is numbered, and the hole mutation is T139S, L141A and Y180V, and the mutation of the mediating effect+6 function is L7A With L8A and the mutation for increasing serum stability is M201L and N207S.

128. a kind of polypeptide for being specifically bound to TfR it includes hole mutation, the mutation of mediating effect+6 function, increases The mutation of increase serum stability and in SEQ ID NO:360,372,384,396,408,420,447,459,471 and 483 appoint One sequence has at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein such as reference SEQ ID NO:1 is numbered, and hole mutation is T139S, L141A and Y180V, the mutation of the mediating effect+6 function be L7A, The mutation of L8A and P102G and the increase serum stability is M25Y, S27T and T29E.

129. a kind of polypeptide for being specifically bound to TfR it includes hole mutation, the mutation of mediating effect+6 function, increases The mutation of increase serum stability and in SEQ ID NO:490,497,504,511,518,525,532,539,546 and 553 appoint One sequence has at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein such as reference SEQ ID NO:1 is numbered, and hole mutation is T139S, L141A and Y180V, the mutation of the mediating effect+6 function be L7A, The mutation of L8A and P102G and the increase serum stability is M201L and N207S.

130. a kind of polypeptide for being specifically bound to TfR, it includes SEQ ID NO:116-233,303-345 and The sequence of any of 581-608.

131. a kind of polypeptide for being specifically bound to TfR, it includes any of SEQ ID NO:116-130's First ray and the second sequence independently selected from the group being made of SEQ ID NO:131-139.

132. the polypeptide as described in claim 131, it includes appoint in SEQ ID NO:121,116,122,123 or 126-130 One First ray and the second sequence independently selected from the group being made of SEQ ID NO:136,137 and 139.

133. the polypeptide as described in claim 131, it includes the first sequences of any of SEQ ID NO:120 or 124-126 Column and the second sequence independently selected from the group being made of SEQ ID NO:135,138 and 139.

134. the polypeptide as described in claim 131, it includes:

A) SEQ ID NO:116 and SEQ ID NO:131,

B) SEQ ID NO:116 and SEQ ID NO:136,

C) SEQ ID NO:117 and SEQ ID NO:132,

D) SEQ ID NO:118 and SEQ ID NO:133,

E) SEQ ID NO:119 and SEQ ID NO:134,

F) SEQ ID NO:120 and SEQ ID NO:135,

G) SEQ ID NO:121 and SEQ ID NO:136,

H) SEQ ID NO:122 and SEQ ID NO:137,

I) SEQ ID NO:123 and SEQ ID NO:136,

J) SEQ ID NO:124 and SEQ ID NO:138,

K) SEQ ID NO:125 and SEQ ID NO:135,

L) SEQ ID NO:126 and SEQ ID NO:139,

M) SEQ ID NO:127 and SEQ ID NO:136,

N) SEQ ID NO:128 and SEQ ID NO:136,

O) SEQ ID NO:129 and SEQ ID NO:136, or

P) SEQ ID NO:130 and SEQ ID NO:136.

135. a kind of polypeptide for being specifically bound to TfR, it includes any of SEQ ID NO:303-339's First ray and the second sequence independently selected from the group being made of SEQ ID NO:136,138 and 340-345.

136. the polypeptide as described in claim 135, it includes:

A) SEQ ID NO:303 and SEQ ID NO:340,

B) SEQ ID NO:304 and SEQ ID NO:340,

C) SEQ ID NO:305 and SEQ ID NO:340,

D) SEQ ID NO:306 and SEQ ID NO:341,

E) SEQ ID NO:307 and SEQ ID NO:340,

F) SEQ ID NO:308 and SEQ ID NO:340,

G) SEQ ID NO:309 and SEQ ID NO:340,

H) SEQ ID NO:310 and SEQ ID NO:340,

I) SEQ ID NO:311 and SEQ ID NO:340,

J) SEQ ID NO:312 and SEQ ID NO:341,

K) SEQ ID NO:313 and SEQ ID NO:340,

L) SEQ ID NO:314 and SEQ ID NO:340,

M) SEQ ID NO:315 and SEQ ID NO:340,

N) SEQ ID NO:316 and SEQ ID NO:340,

O) SEQ ID NO:317 and SEQ ID NO:340,

P) SEQ ID NO:318 and SEQ ID NO:341,

Q) SEQ ID NO:319 and SEQ ID NO:340,

R) SEQ ID NO:320 and SEQ ID NO:340,

S) SEQ ID NO:321 and SEQ ID NO:340,

T) SEQ ID NO:322 and SEQ ID NO:340,

U) SEQ ID NO:323 and SEQ ID NO:340,

V) SEQ ID NO:324 and SEQ ID NO:341,

W) SEQ ID NO:325 and SEQ ID NO:340,

X) SEQ ID NO:326 and SEQ ID NO:340,

Y) SEQ ID NO:327 and SEQ ID NO:340,

Z) SEQ ID NO:328 and SEQ ID NO:340,

Aa) SEQ ID NO:329 and SEQ ID NO:340,

Ab) SEQ ID NO:330 and SEQ ID NO:341,

Ac) SEQ ID NO:331 and SEQ ID NO:340,

Ad) SEQ ID NO:332 and SEQ ID NO:340,

Ae) SEQ ID NO:306 and SEQ ID NO:340,

Af) SEQ ID NO:312 and SEQ ID NO:340,

Ag) SEQ ID NO:324 and SEQ ID NO:138,

Ah) SEQ ID NO:318 and SEQ ID NO:340,

Ai) SEQ ID NO:324 and SEQ ID NO:340,

Aj) SEQ ID NO:330 and SEQ ID NO:340,

Ak) SEQ ID NO:318 and SEQ ID NO:138,

Al) SEQ ID NO:333 and SEQ ID NO:136,

Am) SEQ ID NO:334 and SEQ ID NO:136,

An) SEQ ID NO:312 and SEQ ID NO:138,

Ao) SEQ ID NO:333 and SEQ ID NO:342,

Ap) SEQ ID NO:335 and SEQ ID NO:342,

Aq) SEQ ID NO:336 and SEQ ID NO:342,

Ar) SEQ ID NO:334 and SEQ ID NO:342,

As) SEQ ID NO:330 and SEQ ID NO:138,

At) SEQ ID NO:330 and SEQ ID NO:343,

Au) SEQ ID NO:330 and SEQ ID NO:345,

Av) SEQ ID NO:337 and SEQ ID NO:136,

Aw) SEQ ID NO:338 and SEQ ID NO:136,

Ax) SEQ ID NO:339 and SEQ ID NO:136,

Ay) SEQ ID NO:330 and SEQ ID NO:344,

Az) SEQ ID NO:312 and SEQ ID NO:343, or

Ba) SEQ ID NO:312 and SEQ ID NO:345.

137. a kind of polypeptide for being specifically bound to TfR, it includes SEQ ID NO:581,583,585,587, 589, any of 591 and 593 First ray and independently selected from by SEQ ID NO:582,884,586,588,590,592 With the second sequence of the group of 594 compositions.

138. the polypeptide as described in claim 137, it includes:

A) SEQ ID NO:581 and SEQ ID NO:582,

B) SEQ ID NO:583 and SEQ ID NO:584,

C) SEQ ID NO:585 and SEQ ID NO:586,

D) SEQ ID NO:587 and SEQ ID NO:588,

E) SEQ ID NO:589 and SEQ ID NO:590,

F) SEQ ID NO:591 and SEQ ID NO:592, or

G) SEQ ID NO:593 and SEQ ID NO:594.

139. a kind of polypeptide for being specifically bound to TfR, it includes any in SEQ ID NO:554,557 and 560 A First ray and the second sequence independently selected from the group being made of SEQ ID NO:555,558 and 561.

140. the polypeptide as described in claim 139, it includes:

A) SEQ ID NO:554 and SEQ ID NO:555,

B) SEQ ID NO:557 and SEQ ID NO:558, or

C) SEQ ID NO:560 and SEQ ID NO:561.

A kind of 141. polypeptides for being specifically bound to TfR, it includes any in SEQ ID NO:554,557 and 560 A First ray and independently selected from being made of SEQ ID NO:340-345,582,584,586,588,590,592 and 594 Group the second sequence.

A kind of 142. polypeptides for being specifically bound to TfR, it includes SEQ ID NO:303-339,581,583, 585, any of 587,589,591 and 593 First ray and independently selected from by SEQ ID NO:555,558 and 561 groups At group the second sequence.

A kind of 143. polypeptides for being specifically bound to TfR, it includes any of SEQ ID NO:609-614's First ray and the second sequence independently selected from the group being made of SEQ ID NO:615-620.

A kind of 144. polypeptides for being specifically bound to TfR, it includes SEQ ID NO:303,312,315-318 and Any of 328 First ray and the second sequence independently selected from the group being made of SEQ ID NO:135,340 and 341.

145. polypeptide as described in claim 144, it includes:

A) SEQ ID NO:303 and SEQ ID NO:340,

B) SEQ ID NO:316 and SEQ ID NO:340,

C) SEQ ID NO:317 and SEQ ID NO:340,

D) SEQ ID NO:318 and SEQ ID NO:340,

E) SEQ ID NO:328 and SEQ ID NO:341,

F) SEQ ID NO:318 and SEQ ID NO:340,

G) SEQ ID NO:312 and SEQ ID NO:340,

H) SEQ ID NO:303 and SEQ ID NO:341,

I) SEQ ID NO:316 and SEQ ID NO:135, or

J) SEQ ID NO:315 and SEQ ID NO:341.

A kind of 146. polypeptides for being specifically bound to TfR, it includes SEQ ID NO:595,597,599,601, 603, any of 605 and 607 First ray and independently selected from by SEQ ID NO:596,598,600,602,604,606 With the second sequence of the group of 608 compositions.

147. polypeptide as described in claim 146, it includes:

A) SEQ ID NO:595 and SEQ ID NO:596,

B) SEQ ID NO:597 and SEQ ID NO:598,

C) SEQ ID NO:599 and SEQ ID NO:600,

D) SEQ ID NO:601 and SEQ ID NO:602,

E) SEQ ID NO:603 and SEQ ID NO:604,

F) SEQ ID NO:605 and SEQ ID NO:606, or

G) SEQ ID NO:607 and SEQ ID NO:608.

A kind of 148. polypeptides for being specifically bound to TfR, it includes any of SEQ ID NO:575's and 578 First ray and the second sequence independently selected from the group being made of SEQ ID NO:576 and 579.

149. polypeptide as described in claim 148, it includes:

A) SEQ ID NO:575 and SEQ ID NO:576, or

B) SEQ ID NO:578 and SEQ ID NO:579.

A kind of 150. polypeptides for being specifically bound to TfR, it includes any of SEQ ID NO:575's and 578 First ray and the second sequence independently selected from the group being made of SEQ ID NO:136,138 and 340-345.

A kind of 151. polypeptides for being specifically bound to TfR, it includes any of SEQ ID NO:303-339's First ray and the second sequence independently selected from the group being made of SEQ ID NO:576 and 579.

A kind of 152. polypeptides for being specifically bound to TfR, it includes any of SEQ ID NO:140-153's First ray and the second sequence independently selected from the group being made of SEQ ID NO:154-157.

153. polypeptide as described in claim 152, it includes:

A) SEQ ID NO:140 and SEQ ID NO:154,

B) SEQ ID NO:141 and SEQ ID NO:154,

C) SEQ ID NO:142 and SEQ ID NO:154,

D) SEQ ID NO:143 and SEQ ID NO:154,

E) SEQ ID NO:144 and SEQ ID NO:154,

F) SEQ ID NO:145 and SEQ ID NO:154,

G) SEQ ID NO:146 and SEQ ID NO:154,

H) SEQ ID NO:147 and SEQ ID NO:154,

I) SEQ ID NO:148 and SEQ ID NO:155,

J) SEQ ID NO:149 and SEQ ID NO:154,

K) SEQ ID NO:140 and SEQ ID NO:156,

L) SEQ ID NO:150 and SEQ ID NO:156,

M) SEQ ID NO:151 and SEQ ID NO:157,

N) SEQ ID NO:152 and SEQ ID NO:155, or

O) SEQ ID NO:153 and SEQ ID NO:154.

A kind of 154. polypeptides for being specifically bound to TfR, it includes any of SEQ ID NO:158-171's First ray and the second sequence independently selected from the group being made of SEQ ID NO:172-186.

155. polypeptide as described in claim 154, it includes:

A) SEQ ID NO:158 and SEQ ID NO:172,

B) SEQ ID NO:158 and SEQ ID NO:179,

C) SEQ ID NO:159 and SEQ ID NO:173,

D) SEQ ID NO:159 and SEQ ID NO:181,

E) SEQ ID NO:160 and SEQ ID NO:174,

F) SEQ ID NO:161 and SEQ ID NO:175,

G) SEQ ID NO:162 and SEQ ID NO:176,

H) SEQ ID NO:163 and SEQ ID NO:177,

I) SEQ ID NO:164 and SEQ ID NO:178,

J) SEQ ID NO:165 and SEQ ID NO:180,

K) SEQ ID NO:166 and SEQ ID NO:182,

L) SEQ ID NO:167 and SEQ ID NO:183,

M) SEQ ID NO:168 and SEQ ID NO:184,

N) SEQ ID NO:169 and SEQ ID NO:185,

O) SEQ ID NO:170 and SEQ ID NO:174, or

P) SEQ ID NO:171 and SEQ ID NO:186.

A kind of 156. polypeptides for specifically binding TfR, it includes the of any of SEQ ID NO:187-204 One sequence and the second sequence independently selected from the group being made of SEQ ID NO:205-215.

157. polypeptide as described in claim 156, it includes:

A) SEQ ID NO:187 and SEQ ID NO:205,

B) SEQ ID NO:187 and SEQ ID NO:206,

C) SEQ ID NO:188 and SEQ ID NO:206,

D) SEQ ID NO:189 and SEQ ID NO:207,

E) SEQ ID NO:190 and SEQ ID NO:206,

F) SEQ ID NO:191 and SEQ ID NO:205,

G) SEQ ID NO:192 and SEQ ID NO:206,

H) SEQ ID NO:193 and SEQ ID NO:208,

I) SEQ ID NO:194 and SEQ ID NO:206,

J) SEQ ID NO:195 and SEQ ID NO:209,

K) SEQ ID NO:196 and SEQ ID NO:206,

L) SEQ ID NO:197 and SEQ ID NO:205,

M) SEQ ID NO:198 and SEQ ID NO:206,

N) SEQ ID NO:199 and SEQ ID NO:208,

O) SEQ ID NO:200 and SEQ ID NO:206,

P) SEQ ID NO:201 and SEQ ID NO:210,

Q) SEQ ID NO:201 and SEQ ID NO:211,

R) SEQ ID NO:201 and SEQ ID NO:212,

S) SEQ ID NO:202 and SEQ ID NO:212,

T) SEQ ID NO:203 and SEQ ID NO:213,

U) SEQ ID NO:203 and SEQ ID NO:214, or

V) SEQ ID NO:204 and SEQ ID NO:215.

A kind of 158. polypeptides for being specifically bound to TfR, it includes any of SEQ ID NO:216-220's First ray and the second sequence independently selected from the group being made of SEQ ID NO:221-224.

159. polypeptide as described in claim 158, it includes:

A) SEQ ID NO:216 and SEQ ID NO:221,

B) SEQ ID NO:217 and SEQ ID NO:221,

C) SEQ ID NO:218 and SEQ ID NO:222,

D) SEQ ID NO:219 and SEQ ID NO:223, or

E) SEQ ID NO:220 and SEQ ID NO:224.

A kind of 160. polypeptides for being specifically bound to TfR, it includes any of SEQ ID NO:225-228's First ray and the second sequence independently selected from the group being made of SEQ ID NO:229-233.

161. polypeptide as described in claim 160, it includes:

A) SEQ ID NO:225 and SEQ ID NO:229,

B) SEQ ID NO:226 and SEQ ID NO:230,

C) SEQ ID NO:226 and SEQ ID NO:231,

D) SEQ ID NO:227 and SEQ ID NO:232, or

E) SEQ ID NO:228 and SEQ ID NO:233.

A kind of 162. polynucleotides, it includes the nucleic acid sequences of polypeptide of the coding as described in any one of claim 1 to 161.

A kind of 163. carriers, it includes the polynucleotides as described in claim 162.

A kind of 164. host cells, it includes the polynucleotides as described in claim 162.

A kind of 165. methods of the polypeptide for the CH2 structural domain for generating the CH3 structural domain comprising modification or modification comprising expressing Host cell is cultivated under conditions of the polypeptide encoded as the polynucleotide as described in claim 162.

A kind of 166. pharmaceutical compositions, it includes polypeptides as described in any one of claim 1 to 161 and pharmaceutically acceptable Carrier.

A kind of 167. methods for wearing born of the same parents' transport compositions across endothelium, the method includes wanting the endothelium with comprising right The composition of any one of 1 to 161 polypeptide is asked to contact.

168. method as described in claim 167, wherein the endothelium is blood-brain barrier (BBB).

169. is a kind of engineered at the method for being specifically bound to TfR by CH3 structural domain, which comprises

(a) being modified into the polynucleotide for encoding the CH3 structural domain has extremely at comprising one group of amino acid position below Few five amino acid replaces:

(i) 157,159,160,161,162,163,186,189 and 194；Or

(ii) 118,119,120,122,210,211,212 and 213,

Wherein the substitution and position are that the amino acid 1 14-220 of reference SEQ ID NO:1 is determined；

(b) polypeptide of CH3 structural domain of the expression comprising the modification；With

(c) determine whether the CH3 structural domain of the modification is integrated to the TfR.

170. method as described in claim 169, wherein it is described expression comprising the modification CH3 structural domain polypeptide and really The step of whether the CH3 structural domain of the fixed modification is integrated to the TfR is carried out using display systems.

171. method as described in claim 170, wherein the display systems are cell surface display system, viral display system System, mRNA display systems, polysome display systems or ribosome display system.

172. method as described in claim 169, wherein the polypeptide of the CH3 structural domain comprising the modification is to be expressed as Soluble protein form.

173. is a kind of engineered at the method for being specifically bound to TfR by CH2 structural domain, which comprises

(a) being modified into the polynucleotide for encoding the CH2 structural domain has extremely at comprising one group of amino acid position below Few five amino acid replaces:

(i) 47,49,56,58,59,60,61,62 and 63；

(ii) 39,40,41,42,43,44,68,70,71 and 72；

(iii) 41,42,43,44,45,65,66,67,69 and 73；Or

(iv) 45,47,49,95,97,99,102,103 and 104；

Wherein the substitution and the position are that the amino acid 4-113 of reference SEQ ID NO:1 is determined；

(b) polypeptide of CH2 structural domain of the expression comprising the modification；With

(c) determine whether the CH2 structural domain of the modification is integrated to the TfR.

174. method as described in claim 173, wherein it is described expression comprising the modification CH2 structural domain polypeptide and really The step of whether the CH2 structural domain of the fixed modification is integrated to the TfR is carried out using display systems.

175. method as described in claim 174, wherein the display systems are cell surface display system, viral display system System, mRNA display systems, polysome display systems or ribosome display system.

176. method as described in claim 173, wherein the polypeptide of the CH2 structural domain comprising the modification is to be expressed as Soluble protein form.

177. is a kind of engineered at the method for being specifically bound to TfR by CH3 structural domain, which comprises

(a) by the polynucleotide for encoding the CH3 structural domain be modified into at least five as claim 1 to 23,27 to 29, Amino acid substitution described in any one of 32 to 33,48 to 51 and 55 to 56；With

(b) express and recycle the polypeptide of the CH3 structural domain comprising the modification.

178. is a kind of engineered at the method for being specifically bound to TfR by CH2 structural domain, which comprises

(a) by the polynucleotide for encoding the CH2 structural domain be modified into at least five as claim 66 to 71,75 to 81, amino acid substitution described in any one of 85 to 89 and 93 to 97；With

(b) express and recycle the polypeptide of the CH2 structural domain comprising the modification.

A kind of 179. methods of the combination of the Fc polypeptide and target of modification of the enhancing comprising non-natural binding site, the method packet It includes:

(a) in the non-natural binding siteInterior one or more positions introduce one or more replace；With

(b) combination of the Fc polypeptide and the target of the modification is tested.

180. method as described in claim 179, wherein one or more in following position of the non-natural binding site A place includes to replace: 157,159,160,161,162,163,186,189 and 194.

181. method as described in claim 179 or 180, wherein referring to SEQ ID NO:1, in the non-natural binding site 'sOne or more of substitutions of interior one or more positions are selected from the group being made up of: K21, R28, Q115、R117、E118、Q120、T132、K133、N134、Q135、S137、K143、E153、E155、S156、G158、Y164、 K165、T166、D172、S173、D174、S176、K182、L183、T184、V185、K187、S188、Q191、Q192、G193、 V195, F196, S197, S199, Q211, S213, S215, L216, S217, P218, G219 and K220.

182. method as described in any one of claim 179 to 181, wherein the target is TfR.

Background of invention

The various technologies that protein engineering is transformed into and is integrated to its usual uncombined target are developed.For example, It is engineered at the protein with required combination or enzymatic activity to screen to can produce library.

TfR is the carrier protein of transferrins, and transferrins is to input iron in cell and other function institutes Need and regulated and controled in response to intracellular iron concentration.TfR table on the endothelium including blood-brain barrier endothelial It reaches, and the expression quantity in various cancer cells and inflammatory cell is higher.It is that homoreceptor is mediated to wear born of the same parents across blood-brain barrier One of receptor of transport.Therefore, TfR, which can be used as, is introduced into medicament in cell with endocytosis in cell or across thin Born of the same parents wear the ideal target of born of the same parents' transport.

Summary of the invention

The polypeptide including CH2 the and CH3 structural domain of transferrins (TfR) receptor can be combined is developed.These are more Peptide is by engineered at the substitution in CH2 or CH3 structural domain with generation novelty TfR binding site.TfR is in blood-brain barrier (BBB) great expression on, and TfR natively moves to transferrins in brain from blood.Due to these polypeptides combination TfR, therefore It also across BBB transhipment and can be also used for therapeutic agent that across BBB transhipment connected (such as therapeutical peptide, antibody variable region Such as Fab and small molecule).The method can generally improved treatment agent brain absorb and be therefore particularly suitable for treatment benefit In the illness and disease of brain delivery.

In an aspect, the polypeptide of the CH3 structural domain of the modification comprising being specifically bound to TfR is provided, Wherein the CH3 structural domain of the modification is in one group of amino acid comprising 157,159,160,161,162,163,186,189 and 194 Include four, five, six, seven, eight or nine substitutions at position；And wherein the substitution and the position are references The amino acid 1 14-220 of SEQ ID NO:1 is determined.In some embodiments, the CH3 structural domain of the modification also comprising 153, include one, two, three or four substitution at 164,165 and 188 position.In some embodiments, described more Peptide is integrated to the apical domains of TfR.In some embodiments, the polypeptide is integrated to TfR, The combination of transferrins and TfR is not inhibited simultaneously.In some embodiments, the polypeptide is integrated to comprising turning The epitope of the amino acid 208 of Human Placental Ferritin Receptor sequence.

In some embodiments, the CH3 structural domain of modification is included in 161 Trp.In some embodiments, it repairs The CH3 structural domain of decorations is included in 194 aromatic amino acids.In some embodiments, 194 aromatic amino acids are Trp Or Phe.

In some embodiments, the CH3 structural domain of the modification includes at least one position selected from the following: 157 It is Leu, Tyr, Met or Val；159 are Leu, Thr, His or Pro；160 are Val, Pro or acidic amino acid；161 are Trp or Gly；162 are Val, Ser or Ala；186 are Glu, Ala, Ser, Leu, Thr or Pro；189 are Thr or acidity Amino acid；It is Trp, Tyr, His or Phe with 194.In some embodiments, the CH3 structural domain of modification includes selected from following Two, three, four, five, six, seven or eight positions: 157 are Leu, Tyr, Met or Val；159 be Leu, Thr, His or Pro；160 are Val, Pro or acidic amino acid；161 are Trp or Gly；162 are Val, Ser or Ala； 186 are Glu, Ala, Ser, Leu, Thr or Pro；189 are Thr or acidic amino acid；With 194 be Trp, Tyr, His or Phe.In some embodiments, the CH3 structural domain of modification is included in 157 Leu or Met；In 159 Leu, His or Pro；In 160 Val；In 161 Trp or Gly；In 162 Val or Ala；In 186 Pro；At 189 Thr；And/or in 194 Trp.

In some embodiments, the CH3 structural domain of the modification includes at least one position selected from the following: 157 It is Leu, Tyr, Met or Val；159 are Leu, Thr, His or Pro；160 are Val, Pro or acidic amino acid；161 are Trp；162 are Val, Ser or Ala；186 are Glu, Ala, Ser, Leu, Thr or Pro (such as Thr or Pro)；189 are Thr or acidic amino acid；It is Trp, Tyr, His or Phe with 194.In some embodiments, the CH3 structural domain packet of modification Containing two selected from the following, three, four, five, six, seven or eight positions: 157 are Leu, Tyr, Met or Val； 159 are Leu, Thr, His or Pro；160 are Val, Pro or acidic amino acid；161 are Trp；162 are Val, Ser Or Ala；186 are Glu, Ala, Ser, Leu, Thr or Pro (such as Thr or Pro)；189 are Thr or acidic amino acid；With 194 are Trp, Tyr, His or Phe.In some embodiments, the CH3 structural domain of modification included in 157 Leu or Met；In 159 Leu, His or Pro；In 160 Val；In 161 Trp；In 162 Val or Ala；At 186 Pro；In 189 Thr；And/or in 194 Trp.

In some embodiments, the CH3 structural domain of modification is also included in 164 Ser, Thr, Gln or Phe.One In a little embodiments, the CH3 structural domain of modification is also included in 153 Trp, Tyr, Leu or Gln.In some embodiments, The CH3 structural domain of modification is also included in 165 Gln, Phe or His.In some embodiments, the CH3 structural domain of modification is also Included in 153 Trp and/or in 165 Gln.In some embodiments, the CH3 structural domain of modification is also included in 188 The Glu of position.

In some embodiments, the CH3 structural domain of modification is included in 157 Tyr；In 159 Thr；At 160 Glu or Val；In 161 Trp；In 162 Ser；In 186 Ser or Thr；In 189 Glu；And/or 194 Phe.In some embodiments, the CH3 structural domain of modification is also included in 153 Trp, Tyr, Leu or Gln.In In some embodiments, the CH3 structural domain of modification is also included in 188 Glu.In some embodiments, the CH3 knot of modification Structure domain is also included in 153 Trp and/or in 188 Glu.In some embodiments, the CH3 structural domain of modification also wraps It is contained in 153 Leu and/or in 188 Glu.In some embodiments, the CH3 structural domain of modification is included in 163 Asn。

In some embodiments, the CH3 structural domain of modification includes one or more of following substitution: at 153 Trp；In 159 Thr；In 161 Trp；In 162 Val；In 186 Ser or Thr；In 188 Glu；With/ Or in 194 Phe.

In some embodiments, the CH3 structural domain of the modification also includes one, two or three position selected from the following Set: 187 are Lys, Arg, Gly or Pro；197 are Ser, Thr, Glu or Lys；It is Ser, Trp or Gly with 199.

In some embodiments, the CH3 structural domain Yu SEQ ID NO:4-29,236-299 and 422-435 of the modification Any of amino acid 1 14-220 have at least 85% identity, at least 90% identity or at least 95% identity.One In a little embodiments, the CH3 structural domain of the modification and the amino acid 1 14-220 of SEQ ID NO:1 are same at least 85% Property, restrictive condition be the homogeneity percentage do not include described group of position 157,159,160,161,162,163,186, 189 and 194.In some embodiments, the CH3 structural domain of the modification includes SEQ ID NO:4-29,236-299 and 422- Any of 435 amino acid 1 57-163 and/or 186-194.

In some embodiments, the CH3 structural domain of the modification includes at least one position selected from the following: 153 It is Trp, Leu or Glu；157 are Tyr or Phe；159 are Thr；160 are Glu；161 are Trp；162 be Ser, Ala, Val or Asn；163 are Ser or Asn；186 are Thr or Ser；188 are Glu or Ser；189 are Glu；With 194 Position is Phe.In some embodiments, the CH3 structural domain of the modification includes selected from the following 2,3,4,5,6,7,8,9,10 Or 11 positions (such as 11 positions): 153 are Trp, Leu or Glu (such as Trp or Leu)；157 are Tyr or Phe； 159 are Thr；160 are Glu；161 are Trp；162 are Ser, Ala, Val or Asn；163 are Ser or Asn；186 Position is Thr or Ser；188 are Glu or Ser；189 are Glu；It is Phe with 194.In some embodiments, described to repair Any of CH3 structural domain and SEQ ID NO:236-299 and 422-435 of decorations have at least 85% identity, at least 90% identity or at least 95% identity.

In some embodiments, the CH3 structural domain of the modification include following amino acid: 153 be Trp, Leu or Glu (such as Trp or Leu)；157 are Tyr or Phe；159 are Thr；160 are Glu；161 are Trp；162 are Ser, Ala, Val or Asn；163 are Ser or Asn；186 are Thr or Ser；188 are Glu or Ser；189 are Glu； It is Phe with 194.In another embodiment, the CH3 structural domain Yu SEQ ID NO:236-299 and 422-435 of the modification Any of have at least 85% identity, at least 90% identity or at least 95% identity.

In some embodiments, the CH3 structural domain of the modification includes in SEQ ID NO:236-299 and 422-435 The amino acid 1 53-163 and/or 186-194 of any one.

In some embodiments, provided herein is a kind of CH3 comprising being specifically bound to the modification of TfR The polypeptide of structural domain, wherein any of the CH3 structural domain of the modification and SEQ ID NO:4-29,236-299 and 422-435 Amino acid 1 14-220 have at least 85% identity, at least 90% identity or at least 95% identity.In certain embodiment party In case, correspond to the position 153 determined referring to SEQ ID NO:1,157,159,160,161,162,163,164,165, 186, at least 5,6,7,8,9,10,11,12,13,14,15 or 16 in 187,188,189,194,197 and 199 position Residue at (such as 11 to 16) does not lack or replaces in SEQ ID NO:4-29 or 236-299.

In either one or two of in embodiments above, the CH3 structural domain of the modification also includes the Trp of (i) at 139 (T139W), or (ii) 139 Ser (T139S), in 141 Ala (L141A) and in 180 Val (Y180V), Described in amino acid position be referring to SEQ ID NO:1 determine.In either one or two of in embodiments above, the modification CH3 structural domain also includes the Leu (M201L) of (i) at the 201 and Ser (N207S) at 207, or (ii) in 207 Ser Or Ala (N207S or N207A), wherein the amino acid position is determined referring to SEQ ID NO:1.

In another aspect, the present invention is characterized in that it is a kind of comprising being specifically bound to the modification of TfR The polypeptide of CH3 structural domain, wherein the CH3 structural domain of the modification comprising 153,157,159,160,162,163,186,188, 189, replace at 194,197 and 199 one group of amino acid position comprising one or more；And the wherein substitution and institute's rheme Set be referring to SEQ ID NO:13 sequence determine.In some embodiments, the CH3 structural domain of the modification is included in 153 Glu, Leu, Ser, Val, Trp or Tyr of position；Aromatic amino acid, Met, Pro or Val at 157；159 Thr, Asn or Val；In 160 Glu, Ile, Pro or Val；In 162 aliphatic amino acids, Ser or Thr；163 Ser, Asn, Arg or Thr；In 186 Thr, His or Ser；In 188 Glu, Ser, Asp, Gly, Thr, Pro, Gln or Arg； In 189 Glu or Arg；In 194 Phe, His, Lys, Tyr or Trp；In 197 Ser, Thr or Trp；With 199 Ser, Cys, Pro, Met or Trp of position.In specific embodiments, the aromatic amino acid at 157 be Tyr, Phe or The Trp and aliphatic amino acid at 162 is Ala, Ile or Val.

In some embodiments, the CH3 structural domain of the modification is included in 153 Glu, Leu or Trp；At 157 Aromatic amino acid；In 159 Thr；In 160 Glu；In 162 aliphatic amino acids or Ser；In 163 Ser Or Asn；In 186 Thr or Ser；In 188 Glu or Ser；In 189 Glu；In 194 Phe, His, Tyr or Trp；In 197 Ser；With in 199 Ser.In specific embodiments, the aromatic amino acid at 157 is Tyr Or Phe and the aliphatic amino acid at 162 are Ala or Val.

In some embodiments, the CH3 structural domain of the modification has the sequence of SEQ ID NO:556 or 559.

In some embodiments, the CH3 structural domain of the modification comprising 153,157,159,160,162,163, 186, include a substitution at 188,189,194,197 and 199 one group of amino acid position.In specific embodiments, described The CH3 structural domain of modification has the sequence of any of SEQ ID NO:563-574.

In some embodiments, the CH3 structural domain of the modification is included in 153 Glu, Leu or Trp；At 157 Tyr or Phe；In 159 Thr；In 160 Glu；In 162 Ala, Val or Ser；In 163 Ser or Asn； In 186 Thr or Ser；In 188 Glu or Ser；In 189 Glu；In 194 Phe；In 197 Ser；With In 199 Ser.In specific embodiments, the CH3 structural domain of the modification has the sequence of SEQ ID NO:562.

In another aspect, the present invention is characterized in that it is a kind of comprising being specifically bound to the modification of TfR The polypeptide of CH3 structural domain, wherein the CH3 structural domain of the modification comprising 153,157,159,160,162,163,164,186, Replace at 189 and 194 one group of amino acid position comprising one or more；And wherein the substitution and the position are references The sequence of SEQ ID NO:9 determines.In some embodiments, the CH3 structural domain of the modification included in 153 Glu or Trp；In 157 Val, Trp, Leu or Tyr；In 159 Leu, Pro, Phe, Thr or His；In 160 Pro, Val Or Glu；In 162 Ala, Ser, Val or Gly；In 163 Leu, His, Gln, Gly, Val, Ala, Asn, Asp, Thr Or Glu；In 164 Thr, Phe, Gln, Val or Tyr；In 186 Leu, Ser, Glu, Ala or Pro；At 189 Glu, Asp, Thr or Asn；With in 194 Trp, Tyr, Phe or His.

In some embodiments, the CH3 structural domain of the modification is included in 153 Glu or Trp；At 157 Trp, Leu or Tyr；In 159 Thr or His；In 160 Val；In 162 Ala, Ser or Val；At 163 Val, Asn or Thr；In 164 Gln or Tyr；In 186 Pro；In 189 Thr or Asn；With 194 Trp, Tyr, Phe or His.In specific embodiments, the CH3 structural domain of the modification has the sequence of SEQ ID NO:577 or 580 Column.

In another aspect, it provides a kind of comprising being specifically bound to the CH3 structural domain of the modification of TfR Polypeptide, wherein the CH3 structural domain of the modification is in one group of amino comprising 118,119,120,122,210,211,212 and 213 Include four, five, six, seven or eight substitutions at sour position；And wherein the substitution and the position are referring to SEQ The amino acid 1 14-220 of ID NO:1 is determined.In some embodiments, the CH3 structural domain of modification is included in 210 Gly； In 211 Phe；And/or in 213 Asp.In some embodiments, the CH3 structural domain of the modification includes at least one A position selected from the following: 118 are Phe or Ile；119 are Asp, Glu, Gly, Ala or Lys；120 be Tyr, Met, Leu, Ile or Asp；122 are Thr or Ala；210 are Gly；211 are Phe；212 are His, Tyr, Ser or Phe；With 213 are Asp.In some embodiments, the CH3 structural domain of modification include two selected from the following, three, four, five, Six, seven or eight positions: 118 are Phe or Ile；119 are Asp, Glu, Gly, Ala or Lys；120 be Tyr, Met, Leu, Ile or Asp；122 are Thr or Ala；210 are Gly；211 are Phe；212 be His, Tyr, Ser or Phe；It is Asp with 213.In some embodiments, the CH3 structural domain of the modification with it is any in SEQ ID NO:30-46 A amino acid 1 14-220 has at least 85% identity, at least 90% identity or at least 95% identity.In some implementations In scheme, the CH3 structural domain of the modification and the amino acid 1 14-220 of SEQ ID NO:1 have at least 85% identity, limit Condition processed is that the homogeneity percentage does not include described group of position 118,119,120,122,210,211,212 and 213.One In a little embodiments, the CH3 structural domain of the modification include any of SEQ ID NO:30-46 amino acid 1 18-122 and/ Or 210-213.In some embodiments, the CH3 structural domain of the modification also includes the Trp (T139W) of (i) at 139, or (ii) 139 Ser (T139S), in 141 Ala (L141A) and in 180 Val (Y180V), wherein the amino Sour position is determined referring to SEQ ID NO:1.In some embodiments, the CH3 structural domain of the modification also includes (i) In 201 Leu (M201L) and in 207 Ser (N207S), or (ii) in 207 Ser or Ala (N207S or N207A), Wherein the amino acid position is determined referring to SEQ ID NO:1.

In some embodiments, corresponding unmodified CH3 structural domain is human IgG 1, IgG2, IgG3 or IgG4 CH3 knot Structure domain.In some embodiments, the polypeptide be joined to CH2 structural domain (such as IgG1, IgG2, IgG3 or IgG4 CH2 knot Structure domain).In some embodiments, the CH2 structural domain contains the following each group of the amino acid sequence with reference to SEQ ID NO:1 One or two of modification: (a) at 7 and in 8 Ala (L7A and L8A)；(b) 25 Tyr (M25Y), 27 The Thr (S27T) of position and in 29 Glu (T29E).In some embodiments, (a) group is also included in 102 Gly (P102G).In some embodiments, the polypeptide is further joined to Fab via hinge area.In some embodiments, Fab is integrated to tau protein (such as mankind's tau protein) or its segment.The tau protein can be phosphorylation tau protein, unphosphorylated τ Albumen, the truncated tau protein of montage isoform, N-terminal of tau protein, the truncated tau protein of C-terminal and/or its segment.In some realities It applies in scheme, Fab is integrated to beta-secretase 1 (BACE1) albumen (such as mankind BACE1 albumen) or its segment.The BACE1 egg White can be the montage isoform or its segment of BACE1 albumen.In some embodiments, Fab is integrated on bone marrow cell Triggering receptor 2 (TREM2) albumen (such as mankind TREM2 albumen) of expression or its segment.In some embodiments, Fab It is integrated to alpha-synapse nucleoprotein (such as mankind's alpha-synapse nucleoprotein) or its segment.The alpha-synapse nucleoprotein can be monomer α- Synapse nucleoprotein, oligomeric alpha-synapse nucleoprotein, alpha-synapse nucleoprotein fibrinogen, soluble alpha-synapse nucleoprotein and/or its segment. In some embodiments, the polypeptide is the first polypeptide of dimer, thus makes the dimer about TfR It is combined into unit price.In some embodiments, the polypeptide is the first polypeptide, forms dimer with the second polypeptide, described Second polypeptide is integrated to TfR and includes the CH3 structural domain of modification.In some embodiments, second polypeptide Modification CH3 structural domain it is identical as the CH3 structural domain of modification of first polypeptide.

In another aspect, the polypeptide of the CH2 structural domain of the modification comprising being specifically bound to TfR is provided, Wherein the CH2 structural domain of the modification wraps at one group of amino acid position comprising 47,49,56,58,59,60,61,62 and 63 Replace containing four, five, six, seven, eight or nine；And wherein the substitution and the position are referring to SEQ ID The amino acid 4-113 of NO:1 is determined.In some embodiments, the CH2 structural domain of the modification included in 60 Glu and/ Or in 61 Trp.In some embodiments, the CH2 structural domain of the modification includes at least one position selected from the following: 47 are Glu, Gly, Gln, Ser, Ala, Asn, Tyr or Trp；49 are Ile, Val, Asp, Glu, Thr, Ala or Tyr；56 Position is Asp, Pro, Met, Leu, Ala, Asn or Phe；58 are Arg, Ser, Ala or Gly；59 be Tyr, Trp, Arg or Val；60 are Glu；61 are Trp or Tyr；62 are Gln, Tyr, His, Ile, Phe, Val or Asp；With 63 be Leu, Trp, Arg, Asn, Tyr or Val.In some embodiments, the CH2 structural domain of the modification includes selected from the following at least two A, three, four, five, six, seven, eight or nine positions: 47 are Glu, Gly, Gln, Ser, Ala, Asn, Tyr Or Trp；49 are Ile, Val, Asp, Glu, Thr, Ala or Tyr；56 are Asp, Pro, Met, Leu, Ala, Asn or Phe； 58 are Arg, Ser, Ala or Gly；59 are Tyr, Trp, Arg or Val；60 are Glu；61 are Trp or Tyr；62 are Gln, Tyr, His, Ile, Phe, Val or Asp；It is Leu, Trp, Arg, Asn, Tyr or Val with 63.In some embodiments In, the CH2 structural domain of the modification is included in 47 Glu, Gly, Gln, Ser, Ala, Asn or Tyr；49 Ile, Val, Asp, Glu, Thr, Ala or Tyr；In 56 Asp, Pro, Met, Leu, Ala or Asn；In 58 Arg, Ser or Ala；In 59 Tyr, Trp, Arg or Val；In 60 Glu；In 61 Trp；62 Gln, Tyr, His, Ile, Phe or Val；And/or in 63 Leu, Trp, Arg, Asn or Tyr.

In some embodiments, the CH2 structural domain of the modification is included in 58 Arg；In 59 Tyr or Trp； In 60 Glu；In 61 Trp；And/or in 63 Arg or Trp.In some embodiments, the CH2 of the modification The amino acid 4-113 of any of structural domain and SEQ ID NO:47-62 have at least 85% identity, at least 90% identity Or at least 95% identity.In some embodiments, the amino acid 4- of the CH2 structural domain of the modification and SEQ ID NO:1 113 have at least 85% identity, restrictive condition be the homogeneity percentage do not include described group of position 47,49,56, 58,59,60,61,62 and 63.In some embodiments, the CH2 structural domain of the modification includes in SEQ ID NO:47-62 The amino acid 47-49 and/or 56-63 of any one.

In another aspect, the polypeptide of the CH2 structural domain of the modification comprising being specifically bound to TfR is provided, Wherein the CH2 structural domain of the modification is at one group of amino acid position comprising 39,40,41,42,43,44,68,70,71 and 72 Replace comprising four, five, six, seven, eight, nine or ten；And wherein the substitution and the position are references The amino acid 4-113 of SEQ ID NO:1 is determined.In some embodiments, the CH2 structural domain of the modification is included in 43 Pro, in 68 Glu and/or in 70 Tyr.In some embodiments, the CH2 structural domain of the modification includes at least One position selected from the following: 39 are Pro, Phe, Ala, Met or Asp；40 be Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp or Tyr；41 are Thr, Ser, Gly, Met, Val, Phe, Trp or Leu；42 are Pro, Val, Ala, Thr Or Asp；43 are Pro, Val or Phe；44 are Trp, Gln, Thr or Glu；68 are Glu, Val, Thr, Leu or Trp；70 Position is Tyr, His, Val or Asp；71 are Thr, His, Gln, Arg, Asn or Val；With 72 be Tyr, Asn, Asp, Ser or Pro.In some embodiments, the CH2 structural domain of modification includes two selected from the following, three, four, five, six, seven A, eight, nine or ten positions: 39 are Pro, Phe, Ala, Met or Asp；40 be Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp or Tyr；41 are Thr, Ser, Gly, Met, Val, Phe, Trp or Leu；42 be Pro, Val, Ala, Thr or Asp；43 are Pro, Val or Phe；44 are Trp, Gln, Thr or Glu；68 are Glu, Val, Thr, Leu Or Trp；70 are Tyr, His, Val or Asp；71 are Thr, His, Gln, Arg, Asn or Val；With 72 be Tyr, Asn, Asp, Ser or Pro.

In some embodiments, the CH2 structural domain of the modification is included in 39 Pro, Phe or Ala；At 40 Gln, Pro, Arg, Lys, Ala or Ile；In 41 Thr, Ser, Gly, Met, Val, Phe or Trp；In 42 Pro, Val Or Ala；In 43 Pro；In 44 Trp or Gln；In 68 Glu；In 70 Tyr；In 71 Thr, His or Gln；And/or in 72 Tyr, Asn, Asp or Ser.In some embodiments, the CH2 structural domain of the modification is included in 39 Met；In 40 Leu or Glu；In 41 Trp；In 42 Pro；In 43 Val；In 44 Thr；In 68 Val or Thr；In 70 His；In 71 His, Arg or Asn；And/or in 72 Pro.In some embodiment party In case, the CH2 structural domain of the modification is included in 39 Asp；In 40 Asp；In 41 Leu；In 42 Thr； In 43 Phe；In 44 Gln；In 68 Val or Leu；In 70 Val；In 71 Thr；And/or at 72 Pro.

In some embodiments, the amino acid of any of the CH2 structural domain of the modification and SEQ ID NO:63-85 4-113 has at least 85% identity, at least 90% identity or at least 95% identity.In some embodiments, described The CH2 structural domain of modification and the amino acid 4-113 of SEQ ID NO:1 have at least 85% identity, and restrictive condition is described Homogeneity percentage does not include described group of position 39,40,41,42,43,44,68,70,71 and 72.In some embodiments, The CH2 structural domain of the modification includes the amino acid 39-44 and/or 68-72 of any of SEQ ID NO:63-85.

In another aspect, the polypeptide of the CH2 structural domain of the modification comprising being specifically bound to TfR is provided, Wherein the CH2 structural domain of the modification is at one group of amino acid position comprising 41,42,43,44,45,65,66,67,69 and 73 Replace comprising four, five, six, seven, eight, nine or ten；And wherein the substitution and the position are references The amino acid 4-113 of SEQ ID NO:1 is determined.In some embodiments, the CH2 structural domain of the modification includes at least one Position selected from the following: 41 are Val or Asp；42 are Pro, Met or Asp；43 are Pro or Trp；44 be Arg, Trp, Glu or Thr；45 are Met, Tyr or Trp；65 are Leu or Trp；66 are Thr, Val, Ile or Lys；67 are Ser, Lys, Ala or Leu；69 are His, Leu or Pro；It is Val or Trp with 73.In some embodiments, modification CH2 structural domain includes two selected from the following, three, four, five, six, seven, eight, nine or ten positions: 41 It is Val or Asp；42 are Pro, Met or Asp；43 are Pro or Trp；44 are Arg, Trp, Glu or Thr；45 are Met, Tyr or Trp；65 are Leu or Trp；66 are Thr, Val, Ile or Lys；67 are Ser, Lys, Ala or Leu；69 Position is His, Leu or Pro；It is Val or Trp with 73.

In some embodiments, the CH2 structural domain of modification is included in 41 Val；In 42 Pro；At 43 Pro；In 44 Arg or Trp；In 45 Met；In 65 Leu；In 66 Thr；In 67 Ser；At 69 His；And/or in 73 Val.In some embodiments, the CH2 structural domain of modification is included in 41 Asp；At 42 Met or Asp；In 43 Trp；In 44 Glu or Thr；In 45 Tyr or Trp；In 65 Trp；At 66 Val, Ile or Lys；In 67 Lys, Ala or Leu；In 69 Leu or Pro；And/or in 73 Trp.

In some embodiments, the amino acid of any of the CH2 structural domain of the modification and SEQ ID NO:86-90 4-113 has at least 85% identity, at least 90% identity or at least 95% identity.In some embodiments, described The CH2 structural domain of modification and the amino acid 4-113 of SEQ ID NO:1 have at least 85% identity, and restrictive condition is described Homogeneity percentage does not include described group of position 41,42,43,44,45,65,66,67,69 and 73.In some embodiments, The CH2 structural domain of the modification includes the amino acid 41-45 and/or 65-73 of any of SEQ ID NO:86-90.

In another aspect, the polypeptide of the CH2 structural domain of the modification comprising being specifically bound to TfR is provided, Wherein the CH2 structural domain of the modification is at one group of amino acid position comprising 45,47,49,95,97,99,102,103 and 104 Replace comprising four, five, six, seven, eight or nine；And wherein the substitution and the position are referring to SEQ ID The amino acid 4-113 of NO:1 is determined.In some embodiments, the CH2 structural domain of modification is included in 103 Trp.Some In embodiment, the CH2 structural domain of the modification includes at least one position selected from the following: 45 be Trp, Val, Ile or Ala；47 are Trp or Gly；49 are Tyr, Arg or Glu；95 are Ser, Arg or Gln；97 are Val, Ser or Phe； 99 are Ile, Ser or Trp；102 are Trp, Thr, Ser, Arg or Asp；103 are Trp；With 104 be Ser, Lys, Arg or Val.In some embodiments, the CH2 structural domain of modification includes two selected from the following, three, four, five, six A, seven, eight or nine positions: 45 are Trp, Val, Ile or Ala；47 are Trp or Gly；49 be Tyr, Arg or Glu；95 are Ser, Arg or Gln；97 are Val, Ser or Phe；99 are Ile, Ser or Trp；102 be Trp, Thr, Ser, Arg or Asp；103 are Trp；It is Ser, Lys, Arg or Val with 104.

In some embodiments, the CH2 structural domain of modification is included in 45 Val or Ile；In 47 Gly；49 The Arg of position；In 95 Arg；In 97 Ser；In 99 Ser；In 102 Thr, Ser or Arg；At 103 Trp；And/or in 104 Lys or Arg.

In some embodiments, the amino acid of any of the CH2 structural domain of the modification and SEQ ID NO:91-95 4-113 has at least 85% identity, at least 90% identity or at least 95% identity.In some embodiments, described The CH2 structural domain of modification and the amino acid 4-113 of SEQ ID NO:1 have at least 85% identity, and restrictive condition is described Homogeneity percentage does not include described group of position 45,47,49,95,97,99,102,103 and 104.In some embodiments, The CH2 structural domain of the modification includes the amino acid 45-49 and/or 95-104 of any of SEQ ID NO:91-95.

In some embodiments, corresponding unmodified CH2 structural domain is human IgG 1, IgG2, IgG3 or IgG4 CH2 knot Structure domain.In some embodiments, the CH2 structural domain of the modification contain with reference to SEQ ID NO:1 amino acid sequence with One or two of lower each group modification: (a) at 7 and in 8 Ala (L7A and L8A)；(b) in 25 Tyr (M25Y), the Thr (S27T) at the 27 and Glu (T29E) at 29.In some embodiments, (a) group is also included in 102 The Gly (P102G) of position.In some embodiments, the polypeptide is joined to CH3 structural domain.In some embodiments, described CH3 structural domain include the Trp (T139W) of (i) at 139, or (ii) 139 Ser (T139S), in 141 Ala (L141A) and in 180 Val (Y180V), wherein the amino acid position is the amino acid sequence referring to SEQ ID NO:1 It determines.In some embodiments, the CH3 structural domain includes the Leu (M201L) of (i) at the 201 and Ser at 207 (N207S), or (ii) Ser or Ala (N207S or N207A) at 207, wherein the amino acid position is referring to SEQ ID NO:1 is determined.In some embodiments, the polypeptide is further joined to Fab.In some embodiments, Fab is integrated to τ Albumen (such as mankind's tau protein) or its segment.The tau protein can be phosphorylation tau protein, unphosphorylated tau protein, tau protein Montage isoform, the truncated tau protein of N-terminal, the truncated tau protein of C-terminal and/or its segment.In some embodiments, Fab is integrated to beta-secretase 1 (BACE1) albumen (such as mankind BACE1 albumen) or its segment.The BACE1 albumen can be The montage isoform or its segment of BACE1 albumen.In some embodiments, Fab is integrated to the touching expressed on bone marrow cell Hair property receptor 2 (TREM2) albumen (such as mankind TREM2 albumen) or its segment.In some embodiments, Fab is integrated to α- Synapse nucleoprotein (such as mankind's alpha-synapse nucleoprotein) or its segment.The alpha-synapse nucleoprotein can be monomer alpha-synapse core egg White, oligomeric alpha-synapse nucleoprotein, alpha-synapse nucleoprotein fibrinogen, soluble alpha-synapse nucleoprotein and/or its segment.

In some embodiments, the polypeptide includes the CH2 structural domain of modification or the CH3 structural domain of modification, with this Any of described polypeptide of text, such as any of SEQ ID NO:4-95,236-299,302 and 347-553 competition knot Close TfR.In some embodiments, the polypeptide includes the CH2 structural domain of modification or the CH3 structure of modification Domain, any of with polypeptide described herein, such as any in SEQ ID NO:4-95,236-299,302 and 347-553 A same epitope being integrated on TfR.

In some embodiments, the polypeptide is the first polypeptide of dimer, thus makes the dimer about turning iron Protein receptor binding is monovalent.In some embodiments, the polypeptide is the first polypeptide, forms dimerization with the second polypeptide Body, second polypeptide are integrated to TfR and include the CH2 structural domain of modification.In some embodiments, described The CH2 structural domain of the modification of second polypeptide is identical as the CH2 of modification of first polypeptide.

In another aspect, the polypeptide for being specifically bound to TfR is provided, it includes SEQ ID NO:4-29, The amino acid 1 57-194 of any of 236-299 and 422-435, or amino acid 1 53-194 or amino in some embodiments Sour 153-199；The amino acid 1 18-213 of any of SEQ ID NO:30-46；The ammonia of any of SEQ ID NO:47-62 Base acid 47-63；The amino acid 39-72 of any of SEQ ID NO:63-85；The amino of any of SEQ ID NO:86-90 Sour 41-73；Or the amino acid 45-104 of any of SEQ ID NO:91-95.

In another aspect, provided herein is a kind of polypeptide, it includes section mutation and with SEQ ID NO:4-95,236-299 There is at least 85% identity, at least 90% identity or at least 95% identity with the sequence of any of 422-435, wherein As numbered referring to SEQ ID NO:1, the section mutation is T139W.

In some embodiments, the polypeptide include section mutation and with SEQ ID NO:349,361,373,385,397, 409, any of 436,448,460 and 472 sequence has at least 85% identity, at least 90% identity or at least 95% The sequence of identity, wherein the section mutation is T139W as numbered referring to SEQ ID NO:1.In specific embodiments, The polypeptide includes the sequence of any of SEQ ID NO:349,361,373,385,397,409,436,448,460 and 472.

In another aspect, provided herein is a kind of polypeptides, and it includes section mutation, the mutation of mediating effect+6 function and and SEQ The sequence of any of ID NO:4-95,236-299 and 422-435 have at least 85% identity, at least 90% identity or At least 95% identity, wherein the section mutation is T139W and the mediating effect+6 function as numbered referring to SEQ ID NO:1 The mutation of energy is L7A, L8A and/or P102G (such as L7A and L8A).

In some embodiments, the polypeptide include section mutation, mediating effect+6 function mutation and with SEQ ID NO: 350, any of 362,374,386,398,410,437,449,461 and 473 sequence has at least 85% identity, at least The sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, the section mutation is The mutation of T139W and the mediating effect+6 function is L7A and L8A.In specific embodiments, the polypeptide includes SEQ ID The sequence of any of NO:350,362,374,386,398,410,437,449,461 and 473.

In some embodiments, the polypeptide include section mutation, mediating effect+6 function mutation and with SEQ ID NO: 351, any of 363,375,387,399,411,438,450,462 and 474 sequence has at least 85% identity, at least The sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, the section mutation is The mutation of T139W and the mediating effect+6 function is L7A, L8A and P102G.In specific embodiments, the polypeptide includes The sequence of any of SEQ ID NO:351,363,375,387,399,411,438,450,462 and 474.

In another aspect, provided herein is a kind of polypeptide, have section mutation, increase serum stability mutation and with The sequence of any of SEQ ID NO:4-95,236-299 and 422-435 have at least 85% identity, at least 90% same Property or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, the section mutation is T139W and the increase blood The mutation of clear stability is (i) such as M25Y, S27T and the T29E numbered referring to SEQ ID NO:1, or (ii) such as reference SEQ N207S that ID NO:1 is numbered and presence or absence of M201L.

In some embodiments, the polypeptide include section mutation, increase serum stability mutation and with SEQ ID The sequence of any of NO:352,364,376,388,400,412,439,451,463 and 475 have at least 85% identity, At least sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, the section mutation is The mutation of T139W and the increase serum stability is M25Y, S27T and T29E.In specific embodiments, the polypeptide packet The sequence of any of ID containing SEQ NO:352,364,376,388,400 and 412.

In some embodiments, the polypeptide include section mutation, increase serum stability mutation and with SEQ ID The sequence of any of NO:485,492,499,506,513,520,527,534,541 and 548 have at least 85% identity, At least sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, the section mutation is T139W and the mutation for increasing serum stability are N207S and the existence or non-existence as numbered referring to SEQ ID NO:1 M201L.In specific embodiments, the polypeptide include SEQ ID NO:485,492,499,506,513,520,527,534, Any of 541 and 548 sequence.

In another aspect, provided herein is a kind of polypeptides, have section mutation, the mutation of mediating effect+6 function, increase blood The mutation of clear stability and have with the sequence of any of SEQ ID NO:4-95,236-299 and 422-435 at least 85% same One property, at least 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, the section mutation is T139W, the mutation of the mediating effect+6 function are L7A, L8A and/or P102G (such as L7A and L8A), and the increase blood The mutation of clear stability is (i) such as M25Y, S27T and the T29E numbered referring to SEQ ID NO:1, or (ii) such as reference SEQ N207S that ID NO:1 is numbered and presence or absence of M201L.

In some embodiments, the polypeptide includes section mutation, the mutation of mediating effect+6 function, increases serum stability Mutation and have with the sequences of any of SEQ ID NO:353,365,377,389,401,413,440,452,464 and 476 There are at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as referring to SEQ ID NO:1 institute Number, the section mutation is T139W, and the mutation of the mediating effect+6 function is L7A and L8A and the increase serum stability Mutation is M25Y, S27T and T29E.In specific embodiments, the polypeptide include SEQ ID NO:353,365,377,389, 401, any of 413,440,452,464 and 476 sequence.

In some embodiments, the polypeptide includes section mutation, the mutation of mediating effect+6 function, increases serum stability Mutation and have with the sequences of any of SEQ ID NO:486,493,500,507,514,521,528,535,542 and 549 There are at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as referring to SEQ ID NO:1 institute Number, the section mutation is T139W, and the mutation of the mediating effect+6 function is L7A and L8A and the increase serum stability Mutation is the N207S as numbered referring to SEQ ID NO:1 and presence or absence of M201L.In specific embodiments, described Polypeptide includes the sequence of any of SEQ ID NO:486,493,500,507,514,521,528,535,542 and 549.

In some embodiments, the polypeptide includes section mutation, the mutation of mediating effect+6 function, increases serum stability Mutation and have with the sequences of any of SEQ ID NO:354,366,378,390,402,414,441,453,465 and 477 There are at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as referring to SEQ ID NO:1 institute Number, the section mutation is T139W, and the mutation of the mediating effect+6 function is L7A, L8A and P102G and the increase serum is steady Qualitatively mutation is M25Y, S27T and T29E.In specific embodiments, the polypeptide include SEQ ID NO:354,366, 378, any of 390,402,414,441,453,465 and 477 sequence.

In some embodiments, the polypeptide includes section mutation, the mutation of mediating effect+6 function, increases serum stability Mutation and have with the sequences of any of SEQ ID NO:487,494,501,508,515,522,529,536,543 and 550 There are at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as referring to SEQ ID NO:1 institute Number, the section mutation is T139W, and the mutation of the mediating effect+6 function is L7A, L8A and P102G and the increase serum is steady Qualitatively mutation is N207S as numbered referring to SEQ ID NO:1 and presence or absence of M201L.In specific embodiment In, the polypeptide includes the sequence of any of SEQ ID NO:487,494,501,508,515,522,529,536,543 and 550 Column.

In another aspect, provided herein is a kind of polypeptide, it includes hole mutation and with SEQ ID NO:4-95,236-299 There is at least 85% identity, at least 90% identity or at least 95% identity with the sequence of any of 422-435, wherein As numbered referring to SEQ ID NO:1, the hole mutation is T139S, L141A and Y180V.

In some embodiments, the polypeptide include hole be mutated and with SEQ ID NO:355,367,379,391,403, 415, any of 442,454,466 and 478 sequence has at least 85% identity, at least 90% identity or at least 95% The sequence of identity, wherein the hole mutation is T139S, L141A and Y180V as numbered referring to SEQ ID NO:1.In spy Determine in embodiment, the polypeptide includes in SEQ ID NO:355,367,379,391,403,415,442,454,466 and 478 The sequence of any one.

In another aspect, provided herein is a kind of polypeptides, and it includes hole mutation, the mutation of mediating effect+6 function and and SEQ The sequence of any of ID NO:4-95,236-299 and 422-435 have at least 85% identity, at least 90% identity or At least 95% identity, wherein the hole mutation is T139S, L141A and Y180V and institute as numbered referring to SEQ ID NO:1 The mutation for stating mediating effect+6 function is L7A, L8A and/or P102G (such as L7A and L8A).

In some embodiments, the polypeptide include hole mutation, mediating effect+6 function mutation and with SEQ ID NO: 356, any of 368,380,392,404,416,443,455,467 and 479 sequence has at least 85% identity, at least The sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, the hole mutation is The mutation of T139S, L141A and Y180V and the mediating effect+6 function is L7A and L8A.In specific embodiments, described more Peptide includes the sequence of any of SEQ ID NO:356,368,380,392,404,416,443,455,467 and 479.

In some embodiments, the polypeptide include hole mutation, mediating effect+6 function mutation and with SEQ ID NO: 357, any of 369,381,393,405,417,444,456,468 and 480 sequence has at least 85% identity, at least The sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, the hole mutation is The mutation of T139S, L141A and Y180V and the mediating effect+6 function is L7A, L8A and P102G.In specific embodiments, The polypeptide includes the sequence of any of SEQ ID NO:357,369,381,393,405,417,444,456,468 and 480.

In another aspect, provided herein is a kind of polypeptide, with hole mutation, increase serum stability mutation and with The sequence of any of SEQ ID NO:4-95,236-299 and 422-435 have at least 85% identity, at least 90% same Property or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, the hole mutation is T139S, L141A and Y180V And the mutation for increasing serum stability is (i) such as M25Y, S27T and T29E for being numbered referring to SEQ ID NO:1, or (ii) such as the N207S that is numbered referring to SEQ ID NO:1 and presence or absence of M201L.

In some embodiments, the polypeptide include hole mutation, increase serum stability mutation and with SEQ ID The sequence of any of NO:358,370,382,394,406,418,445,457,469 and 481 have at least 85% identity, At least sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, the hole mutation is The mutation of T139S, L141A and Y180V and the increase serum stability is M25Y, S27T and T29E.In specific embodiment In, the polypeptide includes the sequence of any of SEQ ID NO:358,370,382,394,406,418,445,457,469 and 481 Column.

In some embodiments, the polypeptide include hole mutation, increase serum stability mutation and with SEQ ID The sequence of any of NO:488,495,502,509,516,523,530,537,544 and 551 have at least 85% identity, At least sequence of 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, the hole mutation is T139S, L141A and Y180V and the mutation for increasing serum stability are the N207S as numbered referring to SEQ ID NO:1 And presence or absence of M201L.In specific embodiments, the polypeptide include SEQ ID NO:488,495,502,509, 516, any of 523,530,537,544 and 551 sequence.

In another aspect, provided herein is a kind of polypeptides, with hole mutation, the mutation of mediating effect+6 function, increase blood The mutation of clear stability and have with the sequence of any of SEQ ID NO:4-95,236-299 and 422-435 at least 85% same One property, at least 90% identity or at least 95% identity, wherein as numbered referring to SEQ ID NO:1, the hole mutation is T139S, L141A and Y180V, the mutation of the mediating effect+6 function are L7A, L8A and/or P102G (such as L7A and L8A), and And the mutation for increasing serum stability is (i) such as M25Y, S27T and T29E for being numbered referring to SEQ ID NO:1, or (ii) such as the N207S that is numbered referring to SEQ ID NO:1 and presence or absence of M201L.

In some embodiments, the polypeptide includes hole mutation, the mutation of mediating effect+6 function, increases serum stability Mutation and have with the sequences of any of SEQ ID NO:359,371,383,395,407,419,446,458,470 and 482 There are at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as referring to SEQ ID NO:1 institute Number, the hole mutation is T139S, L141A and Y180V, and the mutation of the mediating effect+6 function is L7A and L8A and the increasing The mutation of increase serum stability is M25Y, S27T and T29E.In specific embodiments, the polypeptide includes SEQ ID NO: 359, any of 371,383,395,407,419,446,458,470 and 482 sequence.

In some embodiments, the polypeptide includes hole mutation, the mutation of mediating effect+6 function, increases serum stability Mutation and have with the sequences of any of SEQ ID NO:489,496,503,510,517,524,531,538,545 and 552 There are at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as referring to SEQ ID NO:1 institute Number, the hole mutation is T139S, L141A and Y180V, and the mutation of the mediating effect+6 function is L7A and L8A and the increasing The mutation of increase serum stability is the N207S as numbered referring to SEQ ID NO:1 and presence or absence of M201L.Specific In embodiment, the polypeptide includes to appoint in SEQ ID NO:489,496,503,510,517,524,531,538,545 and 552 One sequence.

In some embodiments, the polypeptide includes hole mutation, the mutation of mediating effect+6 function, increases serum stability Mutation and have with the sequences of any of SEQ ID NO:360,372,384,396,408,420,447,459,471 and 483 There are at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as referring to SEQ ID NO:1 institute Number, hole mutation are T139S, L141A and Y180V, the mutation of the mediating effect+6 function be L7A, L8A and P102G and The mutation for increasing serum stability is M25Y, S27T and T29E.In specific embodiments, the polypeptide includes SEQ The sequence of any of IDNO:360,372,384,396,408,420,447,459,471 and 483.

In some embodiments, the polypeptide includes hole mutation, the mutation of mediating effect+6 function, increases serum stability Mutation and have with the sequences of any of SEQ ID NO:490,497,504,511,518,525,532,539,546 and 553 There are at least 85% identity, at least sequence of 90% identity or at least 95% identity, wherein as referring to SEQ ID NO:1 institute Number, hole mutation are T139S, L141A and Y180V, the mutation of the mediating effect+6 function be L7A, L8A and P102G and The mutation for increasing serum stability is N207S as numbered referring to SEQ ID NO:1 and presence or absence of M201L. In specific embodiments, the polypeptide includes the and of SEQ ID NO:490,497,504,511,518,525,532,539,546 Any of 553 sequence.

In another aspect, the polypeptide for being specifically bound to TfR is provided, it includes SEQ ID NO:116- 233, the sequence of any of 303-345 and 581-608.

In some embodiments, the polypeptide for being specifically bound to TfR is provided, it includes SEQ ID NO: The First ray of any of 116-130 and the second sequence independently selected from the group being made of SEQ ID NO:131-139.In In some embodiments, the polypeptide includes the first sequence of any of SEQ ID NO:121,116,122,123 or 126-130 Column and the second sequence independently selected from the group being made of SEQ ID NO:136,137 and 139.In some embodiments, institute State First ray of the polypeptide comprising any of SEQ ID NO:120 or 124-126 and independently selected from by SEQ ID NO: 135, the second sequence of the group of 138 and 139 compositions.

In some embodiments, the polypeptide includes SEQ ID NO:116 and SEQ ID NO:131, SEQ ID NO: 116 and SEQ ID NO:136, SEQ ID NO:117 and SEQ ID NO:132, SEQ ID NO:118 and SEQ ID NO:133, SEQ ID NO:119 and SEQ ID NO:134, SEQ ID NO:120 and SEQ ID NO:135, SEQ ID NO:121 and SEQ ID NO:136, SEQ ID NO:122 and SEQ ID NO:137, SEQ ID NO:123 and SEQ ID NO:136, SEQ ID NO:124 and SEQ ID NO:138, SEQ ID NO:125 and SEQ ID NO:135, SEQ ID NO:126 and SEQ ID NO: 139, SEQ ID NO:127 and SEQ ID NO:136, SEQ ID NO:128 and SEQ ID NO:136, SEQ ID NO:129 and SEQ ID NO:136 or SEQ ID NO:130 and SEQ ID NO:136.

In some embodiments, the polypeptide for being specifically bound to TfR is provided, it includes SEQ ID NO: The First ray of any of 303-339 and independently selected from the group being made of SEQ ID NO:136,138 and 340-345 Two sequences.In some embodiments, the polypeptide includes SEQ ID NO:303 and SEQ ID NO:340, SEQ ID NO: 304 and SEQ ID NO:340, SEQ ID NO:305 and SEQ ID NO:340, SEQ ID NO:306 and SEQ ID NO:341, SEQ ID NO:307 and SEQ ID NO:340, SEQ ID NO:308 and SEQ ID NO:340, SEQ ID NO:309 and SEQ ID NO:340, SEQ ID NO:310 and SEQ ID NO:340, SEQ ID NO:311 and SEQ ID NO:340, SEQ ID NO:312 and SEQ ID NO:341, SEQ ID NO:313 and SEQ ID NO:340, SEQ ID NO:314 and SEQ ID NO: 340, SEQ ID NO:315 and SEQ ID NO:340, SEQ ID NO:316 and SEQ ID NO:340, SEQ ID NO:317 and SEQ ID NO:340, SEQ ID NO:318 and SEQ ID NO:341, SEQ ID NO:319 and SEQ ID NO:340, SEQ ID NO:320 and SEQ ID NO:340, SEQ ID NO:321 and SEQ ID NO:340, SEQ ID NO:322 and SEQ ID NO:340, SEQ ID NO:323 and SEQ ID NO:340, SEQ ID NO:324 and SEQ ID NO:341, SEQ ID NO: 325 and SEQ ID NO:340, SEQ ID NO:326 and SEQ ID NO:340, SEQ ID NO:327 and SEQ ID NO:340, SEQ ID NO:328 and SEQ ID NO:340, SEQ ID NO:329 and SEQ ID NO:340, SEQ ID NO:330 and SEQ ID NO:341, SEQ ID NO:331 and SEQ ID NO:340, SEQ ID NO:332 and SEQ ID NO:340, SEQ ID NO:306 and SEQ ID NO:340, SEQ ID NO:312 and SEQ ID NO:340, SEQ ID NO:324 and SEQ ID NO: 138, SEQ ID NO:318 and SEQ ID NO:340, SEQ ID NO:324 and SEQ ID NO:340, SEQ ID NO:330 and SEQ ID NO:340, SEQ ID NO:318 and SEQ ID NO:138, SEQ ID NO:333 and SEQ ID NO:136, SEQ ID NO:334 and SEQ ID NO:136, SEQ ID NO:312 and SEQ ID NO:138, SEQ ID NO:333 and SEQ ID NO:342, SEQ ID NO:335 and SEQ ID NO:342, SEQ ID NO:336 and SEQ ID NO:342, SEQ ID NO: 334 and SEQ ID NO:342, SEQ ID NO:330 and SEQ ID NO:138, SEQ ID NO:330 and SEQ ID NO:343, SEQ ID NO:330 and SEQ ID NO:345, SEQ ID NO:337 and SEQ ID NO:136, SEQ ID NO:338 and SEQ ID NO:136, SEQ ID NO:339 and SEQ ID NO:136, SEQ ID NO:330 and SEQ ID NO:344, SEQ ID NO:312 and SEQ ID NO:343 or SEQ ID NO:312 and SEQ ID NO:345.

In another aspect, the present invention is characterized in that a kind of polypeptide for being specifically bound to TfR, packet The First ray of any of ID containing SEQ NO:581,583,585,587,589,591 and 593 and independently selected from by SEQ Second sequence of the group of the composition of ID NO:582,884,586,588,590,592 and 594.In specific embodiments, described more Peptide includes SEQ ID NO:581 and SEQ ID NO:582, SEQ ID NO:583 and SEQ ID NO:584, SEQ ID NO:585 With SEQ ID NO:586, SEQ ID NO:587 and SEQ ID NO:588, SEQ ID NO:589 and SEQ ID NO:590, SEQ ID NO:591 and SEQ ID NO:592 or SEQ ID NO:593 and SEQ ID NO:594.

In another aspect, the present invention is characterized in that a kind of polypeptide for being specifically bound to TfR, packet The First ray of any of ID containing SEQ NO:554,557 and 560 and independently selected from by SEQ ID NO:555,558 and Second sequence of the group of 561 compositions.In specific embodiments, the polypeptide includes SEQ ID NO:554 and SEQ ID NO: 555, SEQ ID NO:557 and SEQ ID NO:558 or SEQ ID NO:560 and SEQ ID NO:561.

In another aspect, the present invention is characterized in that a kind of polypeptide for being specifically bound to TfR, packet The First ray of any of ID containing SEQ NO:554,557 and 560 and independently selected from by SEQ ID NO:340-345, 582, the second sequence of the group of 584,586,588,590,592 and 594 compositions.In specific embodiments, the polypeptide includes SEQ ID NO:554 and SEQ ID NO:340, SEQ ID NO:554 and SEQ ID NO:341, SEQ ID NO:554 and SEQ ID NO:342, SEQ ID NO:554 and SEQ ID NO:343, SEQ ID NO:554 and SEQ ID NO:344, SEQ ID NO:554 and SEQ ID NO:345, SEQ ID NO:554 and SEQ ID NO:582, SEQ ID NO:554 and SEQ ID NO: 584, SEQ ID NO:554 and SEQ ID NO:586, SEQ ID NO:554 and SEQ ID NO:588, SEQ ID NO:554 and SEQ ID NO:590, SEQ ID NO:554 and SEQ ID NO:592, SEQ ID NO:554 and SEQ ID NO:594, SEQ ID NO:557 and SEQ ID NO:340, SEQ ID NO:557 and SEQ ID NO:341, SEQ ID NO:557 and SEQ ID NO:342, SEQ ID NO:557 and SEQ ID NO:343, SEQ ID NO:557 and SEQ ID NO:344, SEQ ID NO: 557 and SEQ ID NO:345, SEQ ID NO:557 and SEQ ID NO:582, SEQ ID NO:557 and SEQ ID NO:584, SEQ ID NO:557 and SEQ ID NO:586, SEQ ID NO:557 and SEQ ID NO:588, SEQ ID NO:557 and SEQ ID NO:590, SEQ ID NO:557 and SEQ ID NO:592, SEQ ID NO:557 and SEQ ID NO:594, SEQ ID NO:560 and SEQ ID NO:340, SEQ ID NO:560 and SEQ ID NO:341, SEQ ID NO:560 and SEQ ID NO: 342, SEQ ID NO:560 and SEQ ID NO:343, SEQ ID NO:560 and SEQ ID NO:344 or SEQ ID NO:560 With SEQ ID NO:345, SEQ ID NO:560 and SEQ ID NO:582, SEQ ID NO:560 and SEQ ID NO:584, SEQ ID NO:560 and SEQ ID NO:586, SEQ ID NO:560 and SEQ ID NO:588, SEQ ID NO:560 and SEQ ID NO:590, SEQ ID NO:560 and SEQ ID NO:592 or SEQ ID NO:560 and SEQ ID NO:594.

In another aspect, the present invention is characterized in that a kind of polypeptide for being specifically bound to TfR, packet It the First ray of any of ID containing SEQ NO:303-339,581,583,585,587,589,591 and 593 and independently selects Second sequence of the group of the composition of free SEQ ID NO:555,558 and 561.In specific embodiments, the polypeptide includes SEQ ID NO:303 and SEQ ID NO:555, SEQ ID NO:303 and SEQ ID NO:558, SEQ ID NO:303 and SEQ ID NO:561, SEQ ID NO:304 and SEQ ID NO:555, SEQ ID NO:304 and SEQ ID NO:558, SEQ ID NO: 304 and SEQ ID NO:561, SEQ ID NO:305 and SEQ ID NO:555, SEQ ID NO:305 and SEQ ID NO:558, SEQ ID NO:305 and SEQ ID NO:561, SEQ ID NO:306 and SEQ ID NO:555, SEQ ID NO:306 and SEQ ID NO:558, SEQ ID NO:306 and SEQ ID NO:561, SEQ ID NO:307 and SEQ ID NO:555, SEQ ID NO:307 and SEQ ID NO:558, SEQ ID NO:307 and SEQ ID NO:561, SEQ ID NO:308 and SEQ ID NO: 555, SEQ ID NO:308 and SEQ ID NO:558, SEQ ID NO:308 and SEQ ID NO:561, SEQ ID NO:309 and SEQ ID NO:555, SEQ ID NO:309 and SEQ ID NO:558, SEQ ID NO:309 and SEQ ID NO:561, SEQ ID NO:310 and SEQ ID NO:555, SEQ ID NO:310 and SEQ ID NO:558, SEQ ID NO:310 and SEQ ID NO:561, SEQ ID NO:311 and SEQ ID NO:555, SEQ ID NO:311 and SEQ ID NO:558, SEQ ID NO: 311 and SEQ ID NO:561, SEQ ID NO:312 and SEQ ID NO:555, SEQ ID NO:312 and SEQ ID NO:558, SEQ ID NO:312 and SEQ ID NO:561, SEQ ID NO:313 and SEQ ID NO:555, SEQ ID NO:313 and SEQ ID NO:558, SEQ ID NO:313 and SEQ ID NO:561, SEQ ID NO:314 and SEQ ID NO:555, SEQ ID NO:314 and SEQ ID NO:558, SEQ ID NO:314 and SEQ ID NO:561, SEQ ID NO:315 and SEQ ID NO: 555, SEQ ID NO:315 and SEQ ID NO:558, SEQ ID NO:315 and SEQ ID NO:561, SEQ ID NO:316 and SEQ ID NO:555, SEQ ID NO:316 and SEQ ID NO:558, SEQ ID NO:316 and SEQ ID NO:561, SEQ ID NO:317 and SEQ ID NO:555, SEQ ID NO:317 and SEQ ID NO:558, SEQ ID NO:317 and SEQ ID NO:561, SEQ ID NO:318 and SEQ ID NO:555, SEQ ID NO:318 and SEQ ID NO:558, SEQ ID NO: 318 and SEQ ID NO:561, SEQ ID NO:319 and SEQ ID NO:555, SEQ ID NO:319 and SEQ ID NO:558, SEQ ID NO:319 and SEQ ID NO:561, SEQ ID NO:320 and SEQ ID NO:555, SEQ ID NO:320 and SEQ ID NO:558, SEQ ID NO:320 and SEQ ID NO:561, SEQ ID NO:321 and SEQ ID NO:555, SEQ ID NO:321 and SEQ ID NO:558, SEQ ID NO:321 and SEQ ID NO:561, SEQ ID NO:322 and SEQ ID NO: 555, SEQ ID NO:322 and SEQ ID NO:558, SEQ ID NO:322 and SEQ ID NO:561, SEQ ID NO:323 and SEQ ID NO:555, SEQ ID NO:323 and SEQ ID NO:558, SEQ ID NO:323 and SEQ ID NO:561, SEQ ID NO:324 and SEQ ID NO:555, SEQ ID NO:324 and SEQ ID NO:558, SEQ ID NO:324 and SEQ ID NO:561, SEQ ID NO:325 and SEQ ID NO:555, SEQ ID NO:325 and SEQ ID NO:558, SEQ ID NO: 325 and SEQ ID NO:561, SEQ ID NO:326 and SEQ ID NO:555, SEQ ID NO:326 and SEQ ID NO:558, SEQ ID NO:326 and SEQ ID NO:561, SEQ ID NO:327 and SEQ ID NO:555, SEQ ID NO:327 and SEQ ID NO:558, SEQ ID NO:327 and SEQ ID NO:561, SEQ ID NO:328 and SEQ ID NO:555, SEQ ID NO:328 and SEQ ID NO:558, SEQ ID NO:328 and SEQ ID NO:561, SEQ ID NO:329 and SEQ ID NO: 555, SEQ ID NO:329 and SEQ ID NO:558, SEQ ID NO:329 and SEQ ID NO:561, SEQ ID NO:330 and SEQ ID NO:555, SEQ ID NO:330 and SEQ ID NO:558, SEQ ID NO:330 and SEQ ID NO:561, SEQ ID NO:331 and SEQ ID NO:555, SEQ ID NO:331 and SEQ ID NO:558, SEQ ID NO:331 and SEQ ID NO:561, SEQ ID NO:332 and SEQ ID NO:555, SEQ ID NO:332 and SEQ ID NO:558, SEQ ID NO: 332 and SEQ ID NO:561, SEQ ID NO:333 and SEQ ID NO:555, SEQ ID NO:333 and SEQ ID NO:558, SEQ ID NO:333 and SEQ ID NO:561, SEQ ID NO:334 and SEQ ID NO:555, SEQ ID NO:334 and SEQ ID NO:558, SEQ ID NO:334 and SEQ ID NO:561, SEQ ID NO:335 and SEQ ID NO:555, SEQ ID NO:335 and SEQ ID NO:558, SEQ ID NO:335 and SEQ ID NO:561, SEQ ID NO:336 and SEQ ID NO: 555, SEQ ID NO:336 and SEQ ID NO:558, SEQ ID NO:336 and SEQ ID NO:561, SEQ ID NO:337 and SEQ ID NO:555, SEQ ID NO:337 and SEQ ID NO:558, SEQ ID NO:337 and SEQ ID NO:561, SEQ ID NO:338 and SEQ ID NO:555, SEQ ID NO:338 and SEQ ID NO:558, SEQ ID NO:338 and SEQ ID NO:561, SEQ ID NO:339 and SEQ ID NO:555, SEQ ID NO:339 and SEQ ID NO:558, SEQ ID NO: 339 and SEQ ID NO:561, SEQ ID NO:581 and SEQ ID NO:555, SEQ ID NO:581 and SEQ ID NO:558, SEQ ID NO:581 and SEQ ID NO:561, SEQ ID NO:583 and SEQ ID NO:555, SEQ ID NO:583 and SEQ ID NO:558, SEQ ID NO:583 and SEQ ID NO:561, SEQ ID NO:585 and SEQ ID NO:555, SEQ ID NO:585 and SEQ ID NO:558, SEQ ID NO:585 and SEQ ID NO:561, SEQ ID NO:587 and SEQ ID NO: 555, SEQ ID NO:587 and SEQ ID NO:558, SEQ ID NO:587 and SEQ ID NO:561, SEQ ID NO:589 and SEQ ID NO:555, SEQ ID NO:589 and SEQ ID NO:558, SEQ ID NO:589 and SEQ ID NO:561, SEQ ID NO:591 and SEQ ID NO:555, SEQ ID NO:591 and SEQ ID NO:558, SEQ ID NO:591 and SEQ ID NO:561, SEQ ID NO:593 and SEQ ID NO:555, SEQ ID NO:593 and SEQ ID NO:558 or SEQ ID NO: 593 and SEQ ID NO:561.

In another aspect, the present invention is characterized in that a kind of polypeptide for being specifically bound to TfR, packet The First ray of any of the NO:609-614 of ID containing SEQ and independently selected from the group being made of SEQ ID NO:615-620 The second sequence.

In another aspect, the present invention is characterized in that a kind of polypeptide for being specifically bound to TfR, packet The First ray of any of ID containing SEQ NO:303,312,315-318 and 328 and independently selected from by SEQ ID NO: 135, the second sequence of the group of 340 and 341 compositions.In specific embodiments, the polypeptide include SEQ ID NO:303 and SEQ ID NO:340, SEQ ID NO:316 and SEQ ID NO:340, SEQ ID NO:317 and SEQ ID NO:340, SEQ ID NO:318 and SEQ ID NO:340, SEQ ID NO:328 and SEQ ID NO:341, SEQ ID NO:318 and SEQ ID NO:340, SEQ ID NO:312 and SEQ ID NO:340, SEQ ID NO:303 and SEQ ID NO:341, SEQ ID NO: 316 and SEQ ID NO:135 or SEQ ID NO:315 and SEQ ID NO:341.

In another aspect, the present invention is characterized in that a kind of polypeptide for being specifically bound to TfR, packet The First ray of any of ID containing SEQ NO:595,597,599,601,603,605 and 607 and independently selected from by SEQ Second sequence of the group of the composition of ID NO:596,598,600,602,604,606 and 608.In specific embodiments, described more Peptide includes SEQ ID NO:595 and SEQ ID NO:596, SEQ ID NO:597 and SEQ ID NO:598, SEQ ID NO:599 With SEQ ID NO:600, SEQ ID NO:601 and SEQ ID NO:602, SEQ ID NO:603 and SEQ ID NO:604, SEQ ID NO:605 and SEQ ID NO:606 or SEQ ID NO:607 and SEQ ID NO:608.

In another aspect, the present invention is characterized in that a kind of polypeptide for being specifically bound to TfR, packet The First ray of any of the NO:575 and 578 of ID containing SEQ and independently selected from being made of SEQ ID NO:576 and 579 Second sequence of group.In specific embodiments, the polypeptide includes SEQ ID NO:575 and SEQ ID NO:576 or SEQ ID NO:578 and SEQ ID NO:579.

In another aspect, the present invention is characterized in that a kind of polypeptide for being specifically bound to TfR, packet The First ray of any of the NO:575 and 578 of ID containing SEQ and independently selected from by SEQ ID NO:136,138 and 340- Second sequence of the group of 345 compositions.

In another aspect, the present invention is characterized in that a kind of polypeptide for being specifically bound to TfR, packet The First ray of any of the NO:303-339 of ID containing SEQ and independently selected from the group being made of SEQ ID NO:576 and 579 The second sequence.

In some embodiments, the polypeptide for being specifically bound to TfR is provided, it includes SEQ ID NO: The First ray of any of 140-153 and the second sequence independently selected from the group being made of SEQ ID NO:154-157.In In some embodiments, the polypeptide includes SEQ ID NO:140 and SEQ ID NO:154, SEQ ID NO:141 and SEQ ID NO:154, SEQ ID NO:142 and SEQ ID NO:154, SEQ ID NO:143 and SEQ ID NO:154, SEQ ID NO: 144 and SEQ ID NO:154, SEQ ID NO:145 and SEQ ID NO:154, SEQ ID NO:146 and SEQ ID NO:154, SEQ ID NO:147 and SEQ ID NO:154, SEQ ID NO:148 and SEQ ID NO:155, SEQ ID NO:149 and SEQ ID NO:154, SEQ ID NO:140 and SEQ ID NO:156, SEQ ID NO:150 and SEQ ID NO:156, SEQ ID NO:151 and SEQ ID NO:157, SEQ ID NO:152 and SEQ ID NO:155 or SEQ ID NO:153 and SEQ ID NO:154。

In some embodiments, the polypeptide for being specifically bound to TfR is provided, it includes SEQ ID NO: The First ray of any of 158-171 and the second sequence independently selected from the group being made of SEQ ID NO:172-186.In In some embodiments, the polypeptide includes SEQ ID NO:158 and SEQ ID NO:172, SEQ ID NO:158 and SEQ ID NO:179, SEQ ID NO:159 and SEQ ID NO:173, SEQ ID NO:159 and SEQ ID NO:181, SEQ ID NO: 160 and SEQ ID NO:174, SEQ ID NO:161 and SEQ ID NO:175, SEQ ID NO:162 and SEQ ID NO:176, SEQ ID NO:163 and SEQ ID NO:177, SEQ ID NO:164 and SEQ ID NO:178, SEQ ID NO:165 and SEQ ID NO:180, SEQ ID NO:166 and SEQ ID NO:182, SEQ ID NO:167 and SEQ ID NO:183, SEQ ID NO:168 and SEQ ID NO:184, SEQ ID NO:169 and SEQ ID NO:185, SEQ ID NO:170 and SEQ ID NO: 174 or SEQ ID NO:171 and SEQ ID NO:186.

In some embodiments, the polypeptide of specific binding TfR is provided, it includes SEQ ID NO: The First ray of any of 187-204 and the second sequence independently selected from the group being made of SEQ ID NO:205-215.In In some embodiments, the polypeptide includes SEQ ID NO:187 and SEQ ID NO:205, SEQ ID NO:187 and SEQ ID NO:206, SEQ ID NO:188 and SEQ ID NO:206, SEQ ID NO:189 and SEQ ID NO:207, SEQ ID NO: 190 and SEQ ID NO:206, SEQ ID NO:191 and SEQ ID NO:205, SEQ ID NO:192 and SEQ ID NO:206, SEQ ID NO:193 and SEQ ID NO:208, SEQ ID NO:194 and SEQ ID NO:206, SEQ ID NO:195 and SEQ ID NO:209, SEQ ID NO:196 and SEQ ID NO:206, SEQ ID NO:197 and SEQ ID NO:205, SEQ ID NO:198 and SEQ ID NO:206, SEQ ID NO:199 and SEQ ID NO:208, SEQ ID NO:200 and SEQ ID NO: 206, SEQ ID NO:201 and SEQ ID NO:210, SEQ ID NO:201 and SEQ ID NO:211, SEQ ID NO:201 and SEQ ID NO:212, SEQ ID NO:202 and SEQ ID NO:212, SEQ ID NO:203 and SEQ ID NO:213, SEQ ID NO:203 and SEQ ID NO:214 or SEQ ID NO:204 and SEQ ID NO:215.

In some embodiments, the polypeptide for being specifically bound to TfR is provided, it includes SEQ ID NO: The First ray of any of 216-220 and the second sequence independently selected from the group being made of SEQ ID NO:221-224.In In some embodiments, the polypeptide includes SEQ ID NO:216 and SEQ ID NO:221, SEQ ID NO:217 and SEQ ID NO:221, SEQ ID NO:218 and SEQ ID NO:222, SEQ ID NO:219 and SEQ ID NO:223 or SEQ ID NO: 220 and SEQ ID NO:224.

In some embodiments, the polypeptide for being specifically bound to TfR is provided, it includes SEQ ID NO: The First ray of any of 225-228 and the second sequence independently selected from the group being made of SEQ ID NO:229-233.In In some embodiments, the polypeptide include SEQ ID NO:225 and 229, SEQ ID NO:226 and SEQ ID NO:230, SEQ ID NO:226 and SEQ ID NO:231, SEQ ID NO:227 and SEQ ID NO:232 or SEQ ID NO:228 and SEQ ID NO:233。

In either one or two of in embodiments above, the Leu that the polypeptide can be additionally included in 7 and 8 becomes taking for Ala Generation (L7A and L8A).In some embodiments, the Ala at 7 and 8 replace (L7A and L8A) to be become with the Pro at 102 It is combined for the substitution (P102G) of Gly.

In another aspect, polynucleotide is provided, it includes codings to be specifically bound to transferrins as described herein The nucleic acid sequence of the polypeptide of receptor.

In another aspect, carrier is provided comprising containing coding as described herein be specifically bound to transferrins by The polynucleotide of the nucleic acid sequence of the polypeptide of body.

In another aspect, host cell is provided comprising be specifically bound to as described herein containing coding and turn iron egg The polynucleotide of the nucleic acid sequence of the polypeptide of polymeric immunoglobulin receptor.

In another aspect, the polypeptide of the CH2 structural domain for generating the CH3 structural domain comprising modification or modification is provided Method comprising cultivate host cell under conditions of the polypeptide that expression is encoded by polynucleotide as described herein.

In another aspect, pharmaceutical composition is provided, it includes as described herein be specifically bound to transferrins by The polypeptide and pharmaceutically acceptable carrier of body.

In another aspect, the method for wearing born of the same parents' transport compositions across endothelium is provided, the method includes make endothelium with Composition contact comprising being specifically bound to the polypeptide of TfR as described herein.In some embodiments, The endothelium is blood-brain barrier (BBB).

In another aspect, it provides for CH3 structural domain is engineered at being specifically bound to TfR Method, which comprises

(a) polynucleotide for encoding the CH3 structural domain is modified into comprising having at one group of amino acid position below There are at least five amino acid substitutions:

(i) 157,159,160,161,162,163,186,189 and 194；Or

(ii) 118,119,120,122,210,211,212 and 213,

Wherein the substitution and position are that the amino acid 1 14-220 of reference SEQ ID NO:1 is determined；

(b) polypeptide of CH3 structural domain of the expression comprising the modification；With

(c) determine whether the CH3 structural domain of the modification is integrated to the TfR.

In another aspect, provided herein is one kind for turning iron egg at being specifically bound to for CH3 structural domain is engineered The method of polymeric immunoglobulin receptor, the method includes (a) to be modified into the polynucleotide for encoding CH3 structural domain in the CH3 structural domain Amino acid substitution described in any of previous paragraph replaced at least five such as description CH3 structural domain；(b) Express and recycle the polypeptide of the CH3 structural domain comprising the modification.

In some embodiments, the polypeptide of the CH3 structural domain of the expression comprising the modification and the determining modification CH3 structural domain the step of whether being integrated to the TfR be to be carried out using display systems.In some embodiments In, the display systems are cell surface display system, viral display system, mRNA display systems, polysome display systems Or ribosome display system.In some embodiments, the polypeptide of the CH3 structural domain comprising modification is to be expressed as soluble protein The form of matter.

In another aspect, it provides for CH2 structural domain is engineered at being specifically bound to TfR Method, which comprises

(a) polynucleotide for encoding the CH2 structural domain is modified into comprising having at one group of amino acid position below There are at least five amino acid substitutions:

(i) 47,49,56,58,59,60,61,62 and 63；

(ii) 39,40,41,42,43,44,68,70,71 and 72；

(iii) 41,42,43,44,45,65,66,67,69 and 73；Or

(iv) 45,47,49,95,97,99,102,103 and 104；

Wherein the substitution and the position are that the amino acid 4-113 of reference SEQ ID NO:1 is determined；

(b) polypeptide of CH2 structural domain of the expression comprising the modification；With

(c) determine whether the CH2 structural domain of the modification is integrated to the TfR.

In another aspect, provided herein is one kind for turning iron egg at being specifically bound to for CH2 structural domain is engineered The method of polymeric immunoglobulin receptor, the method includes (a) to be modified into the polynucleotide for encoding CH2 structural domain in the CH2 structural domain Amino acid substitution described in any of previous paragraph replaced at least five such as description CH2 structural domain；(b) Express and recycle the polypeptide of the CH2 structural domain comprising the modification.

In some embodiments, the polypeptide of the CH2 structural domain of the expression comprising the modification and the determining modification CH2 structural domain the step of whether being integrated to the TfR be to be carried out using display systems.In some embodiments In, the display systems are cell surface display system, viral display system, mRNA display systems, polysome display systems Or ribosome display system.In some embodiments, the polypeptide of the CH2 structural domain comprising modification is to be expressed as soluble protein The form of matter.

In another aspect, the present invention is characterized in that it is a kind of for enhancing the Fc of the modification comprising non-natural binding site The method of the combination of polypeptide and target (such as TfR), which comprises (a) is in the non-natural binding siteInterior (such as OrIt is interior) one or more One or more replace is introduced at a position；(b) combination of the Fc polypeptide and the target of the modification is tested.

In some embodiments, the non-natural binding site is at the one or more in following position comprising taking Generation: 157,159,160,161,162,163,186,189 and 194.

In some embodiments, referring to SEQ ID NO:1, in the non-natural binding siteInterior one Or one or more of substitutions at multiple positions are selected from the group that is made up of: K21, R28, Q115, R117, E118, Q120、T132、K133、N134、Q135、S137、K143、E153、E155、S156、G158、Y164、K165、T166、D172、 S173、D174、S176、K182、L183、T184、V185、K187、S188、Q191、Q192、G193、V195、F196、S197、 S199, Q211, S213, S215, L216, S217, P218, G219 and K220.

Detailed description of the invention

Figure 1A-Fig. 1 D shows the Phage-ELISA result of four CH2A2 clone.CH2A2Fc variant is in phage surface Upper expression and test it and the anti-c-Myc antibody 9E10 (expression compare), the negative control, mankind's transferrins receptor that are coated on plate (TfR) and the combination of Macaca inus (cyno) TfR.X-axis shows the OD of phage solution₂₆₈, it is the measurement of phagocytosis bulk concentration. The ELISA result of Figure 1A display clone CH2A2.5.The ELISA result of Figure 1B display clone CH2A2.1.Fig. 1 C display clone The ELISA result of CH2A2.4.The ELISA result of Fig. 1 D display clone CH2A2.16.

Fig. 2A and Fig. 2 B shows the Phage-ELISA result for being integrated to the CH2A2 clone of mankind TfR.By bacteriophage with close Like in conjunction with EC₅₀It is added on the elisa plate for being coated with TfR, and adds the soluble iron saturation Tf or soluble T fR of various concentration. Data show the TfR being coated on CH2A2 clone and soluble T fR competitive binding to plate, but do not compete with iron saturation Tf.Fig. 2A is aobvious Show the result of the experiment of addition soluble iron saturation Tf.The result of the experiment of Fig. 2 B display addition soluble T fR.

Fig. 3 A- Fig. 3 D is shown in the combination presence or absence of CH2C clone and TfR under iron saturation Tf.Fig. 3 A shows phagocytosis Body ELISA's as a result, wherein TfR is coated on elisa plate and presence or absence of a large amount of excessive iron saturations Tf (5 μM) It is lower to add the clone CH2C.23 being showed on bacteriophage.Fig. 3 B, which is shown, to be cloned and is coated in the CH2C of Fc-Fab fusions form The combination of the elisa plate of the mankind or Macaca inus TfR.Fig. 3 C show Phage-ELISA as a result, wherein by mankind TfR, food crab Macaque TfR, iron saturation Tf, anti-Myc or streptavidin are coated on elisa plate and are being not present or there are iron to be saturated Tf Under, add the clone CH2C.17 and CH2C.22 of the phage display of various dilutions.These data show, these clones not with Iron is saturated Tf competitive binding to TfR.Fig. 3 D is shown in the presence of 5 μM of iron saturation Tf and subtracts independent iron saturation Tf combination background It is integrated to the clone CH2C.7's of the TfR- biotin on anti-streptavidin sensor(that is, biological membranous layer Interferometry) dynamics trace, show not with Tf competitive binding.

Fig. 4 A and Fig. 4 B are shown in the combination presence or absence of CH3B clone and TfR under iron saturation Tf.Fig. 4 A, which is shown, to be bitten Thallus ELISA's as a result, being wherein coated with mankind TfR, Macaca inus TfR, iron saturation Tf, anti-Myc or streptavidin In on elisa plate and in the case where being not present or being saturated Tf there are iron, the clone CH3B.11 of the phage display of various dilutions is added And CH3B.12.These data show that these clones are not saturated Tf competitive binding to TfR with iron.Fig. 4 B shows that CH3B clone combines The elisa plate being coated with to the mankind or Macaca inus TfR.By the area Fc comprising CH3B cloned sequence and Fab segment composition and with two Dimer form is measured.

Fig. 5 shows the mature library NNK patch (patch) of related CH3B clone.Each band shows the master of CH3 structural domain Chain, wherein dark colored surface indicates original CH3B positioning area (register) and light-colored surface patch indicates the pedigree of extension.

Fig. 6 is shown in the FACS figure of CH3C Immune Clone Selection on yeast, is shown in the enrichment that group is combined after 3 wheels sort.The 1st In wheel and the 2nd wheel sorting, biotinylation TfR is pre-loaded in streptavidin-AlexaOn 647, then It is cultivated together with yeast.In the 3rd wheel sorting, first biotinylation TfR is cultivated together with yeast, and add avidin chain bacterium Element-Alexa647 carry out secondary detecting.In all number wheel sortings, using for the C in yeast display construct The anti-c-Myc antibody of the chicken of terminal M yc label (being obtained from Thermo Fisher) monitoring expression.

Fig. 7 A- Fig. 7 C is shown in the combination presence or absence of CH3C clone and TfR under iron saturation Tf.Each clone be with The measurement of Fc-Fab fusions form.It uses Ab204 as positive control in this measurement, is to have to be integrated to the variable of TfR The standard antibody in area.Fig. 7 A shows the combination of CH3C variant and the mankind TfR being coated on elisa plate.Fig. 7 B is shown in 5 μM of iron It is saturated in the presence of Tf, the combination of CH3C variant and the mankind TfR being coated on elisa plate.Fig. 7 C shows CH3C variant and coating In the combination of the Macaca inus TfR on elisa plate.

Fig. 8 shows the combination of the 293F cell of CH3C clone and endogenous expression mankind TfR.Cell is allocated in 96 hole V On the culture plate of shape bottom, and add the CH3C clone in Fc-Fab fusion binding protein form of various concentration.1 is cultivated at 4 DEG C Hour after, each culture plate is centrifuged and is washed, and then with Goat anti-Human's class IgG-Alexa647 secondary antibodies one It rises and is cultivated 30 minutes at 4 DEG C.After washing cell again, in FACSCanto^TMCulture plate is read on II flow cytometer, and is usedSoftware determines the median fluorescence angle value in the channel APC (647nm).

Fig. 9 A and Fig. 9 B are shown in the internalization situation of CH3C.3 in the HEK293 cell of endogenous expression mankind TfR.37 DEG C and 8%CO₂Under concentration, the CH3C.3 or reference material of 1 μM of concentration are added, is kept for 30 minutes, is washed out cell, makes its infiltration Change and uses anti-human IgG-AlexaThe dyeing of 488 secondary antibodies.After washing again, cell is obtained using fluorescence microscope The quantity of image and quantitative dot.Fig. 9 A shows microexamination data.Fig. 9 B shows the figure of the quantity of every hole spot.

Figure 10 shows the selection scheme of the soft library CH3C (soft library).Using MACS for the mankind (H) or food crab Macaque (C) TfR sorts initial libraries.Then gained yeast is collected in object separation and such as first round FACS sorting generally directed to people Class or Macaca inus TfR are sorted respectively.Gained is collected object separation again to sort to carry out another wheel FACS.Finally, will HHH and CCC collects object and keeps separating and finally collecting while having collecting object there are two the other of target kind.

Figure 11 A and Figure 11 B show the CH3C clone identified from the first soft libraries of random mutants and the mankind and Macaca inus TfR Combination.Positive control is the anti-TfR antibody A b204 of high-affinity and the anti-TfR antibody A b084 of low-affinity.Figure 11 A is shown and people The combination of class TfR.Figure 11 B is shown and the combination of Macaca inus TfR.

Figure 12 A and Figure 12 B are shown in presence or absence of under iron saturation Tf, are identified from the first soft libraries of random mutants The combination of CH3C clone and mankind TfR.Each clone is in Fc-Fab fusions form.It is anti-using high-affinity in this measurement B204 is as positive control for TfR antibody A.Figure 12 A shows the combination of CH3C variant and the mankind TfR being coated on elisa plate.Figure 12B is shown in the presence of 5 μM of iron saturation Tf, the combination of CH3C variant and the mankind TfR being coated on elisa plate.

Figure 13 shows the combination of the CH3C clone and 293F cell that identify from the first soft libraries of random mutants.Cell is distributed In on 96 hole V-Bottom culture plates, and the CH3C in Fc-Fab fusion protein form for adding various concentration is cloned.It is trained at 4 DEG C After educating 1 hour, each culture plate is centrifuged and is washed, and then with Goat anti-Human's class IgG-Alexa647 second levels are anti- Body is cultivated 30 minutes at 4 DEG C together.After washing cell again, in FACSCanto^TMCulture plate is read on II flow cytometer, and It usesSoftware determines the median fluorescence angle value in the channel APC (647nm).

Figure 14 A- Figure 14 C shows the combination of the CH3C clone and CHO-K1 cell that identify from the first soft libraries of random mutants. Cell is allocated on 96 hole V-Bottom culture plates, and the CH3C in Fc-Fab fusions form for adding various concentration is cloned.In After being cultivated 1 hour at 4 DEG C, each culture plate is centrifuged and is washed, and then with Goat anti-Human's class IgG-Alexa647 Secondary antibody is cultivated 30 minutes at 4 DEG C together.After washing cell again, in FACSCanto^TMCulture is read on II flow cytometer Plate, and useSoftware determines the median fluorescence angle value in the channel APC (647nm).Figure 14 A display overexpression mankind The CHO-K1 cell of TfR.The CHO-K1 cell of Figure 14 B display overexpression Macaca inus TfR.Figure 14 C, which is shown, does not express the mankind The CHO-K1 parental cell of TfR.

Figure 15 A and Figure 15 B show TfR apical domains.Figure 15 A shows apical domains on mankind's TfR protein Position.Illustration shows the close-up view of seven residues different between the mankind and Macaca inus TfR.Figure 15 B is shown containing the mankind The sequence alignment of seven different residues between (SEQ ID NO:107) and Macaca inus (SEQ ID NO:108) TfR.Jointly Sequence is SEQ ID NO:622.

Figure 16 A- Figure 16 E shows the combination of CH3C clone and the apical domains being showed on bacteriophage.Figure 16 A is shown respectively The Myc expression of kind of TfR apical domains mutant shows that the expression quantity of the mutant is similar and through normalization.Figure 16 B is shown CH3C.18 and wild type and the combination for being mutated mankind TfR apical domains, display and the combination of R208G mutant are reduced.Figure 16C shows CH3C.35 and wild type and the combination for being mutated mankind TfR apical domains, and display and the combination of R208G mutant subtract It is few.Figure 16 D shows that CH3C.18 and Wild type human and Macaca inus TfR apical domains and G208R are mutated Macaca inus top The combination of terminal domains, display and the combination of the mutant restore.Figure 16 E shows CH3C.35 and Wild type human and food crab The combination of macaque TfR apical domains and G208R mutation Macaca inus apical domains, the combination of display and the mutant Restore.

Figure 17 A- Figure 17 D is shown to be determined by the paratope for the CH3C variant that mutated site is recovered to wildtype residues progress Position.Figure 17 A shows that the paratope of the revertant using the ELISA CH3C.35 for being integrated to mankind TfR carried out positions.Figure 17B shows that the paratope of the revertant using the ELISA CH3C.35 for being integrated to Macaca inus TfR carried out positions.Figure 17C shows that the paratope of the revertant using the ELISA CH3C.18 for being integrated to mankind TfR carried out positions.Figure 17 D is aobvious Show that the paratope of the revertant using the ELISA CH3C.18 for being integrated to Macaca inus TfR carried out positions.

Figure 18 A- Figure 18 D shows the design in the mature library jointly CH3C.Figure 18 A shows being total to based on CH3C.35 sample sequence Same library.Figure 18 B shows the common library based on CH3C.18 sample sequence.Figure 18 C shows the sky based on CH3C.18 and CH3C.35 Position library.Figure 18 D is shown based on CH3C.18 aromatic compounds library.

Figure 19 A- Figure 19 E is shown from the common mature CH3C variant in library and the combination of the mankind or Macaca inus TfR ELISA.Neomorph (that is, CH3C.3.2-1, CH3C.3.2-5 and CH3C.3.2-19) has Macaca inus and mankind TfR It is similar to combine EC₅₀Value, and parental clone CH3C.18 and CH3C.35 is to the EC of mankind TfR₅₀Value is substantially better than to Macaca inus The EC of TfR₅₀Value.Figure 19 A shows the data of CH3C.3.2-1.Figure 19 B shows the data of CH3C.3.2-19.Figure 19 C is shown The data of CH3C.3.2-5.Figure 19 D shows the data of CH3C.18.Figure 19 E shows the data of CH3C.35.

Figure 20 is shown in the CH3C variant from common mature library in the mankind (HEK293) and monkey (LLC-MK2) cell It is internalized by situation.Compared to and the combination of mankind TfR preferably clone CH3C.35, have the similar mankind and Macaca inus TfR affine Absorption of the clone CH3C.3.2-5 and CH3C3.2-19 of power in monkey cells obviously improves.Made using anti-BACE1 antibody A b107 For negative control.(BACE1 is not expressed on HEK293 or MK2 cell).Use anti-TfR antibody A b204 as positive control.

Figure 21 shows that being depicted in CH3 structure (adapts from the map of the NNK walking residue on PDB 4W4O).Black surface is aobvious Show original CH3C positioning area, gray face shows 44 residues being incorporated in NNK walking configuration, and band shows wild type master Chain.

Figure 22 is shown in the yeast group being enriched with after three-wheel sorting NNK walking library.Yeast is dyed with anti-c-Myc to monitor Express (x-axis) and and TfR apical domains (200nM Macaca inus or the 200nM mankind) combination (y-axis).The number presented herein Enhance according to display and the combination of two kinds of TfR apical domains ortholog things.

Figure 23 A and Figure 23 B show the FACS data of CH3C.35.21 mutant.Yeast is dyed with anti-c-Myc to monitor table Up to (x-axis) and and mankind TfR apical domains (200nM) combination (y-axis).The FACS of Figure 23 A display clone CH3C.35.21 Data.Figure 23 B shows the FACS data of mutant, wherein 11 position back mutations in clone CH3C.35.21 are at wild type The upper surface of (FACS figure one column) or the library the NNK (column below FACS figure, in any sorting for being expressed as all 20 amino acid Before).

Figure 24 A- Figure 24 D shows the ELISA ratio of divalent and the combination of unit price CH3C polypeptide and the mankind and Macaca inus TfR Compared with.Figure 24 A shows the combination of divalent CH3C polypeptide and mankind TfR.Figure 24 B shows divalent CH3C polypeptide and Macaca inus TfR's In conjunction with.Figure 24 C shows the combination of unit price CH3C polypeptide and mankind TfR.Figure 24 D shows unit price CH3C polypeptide and Macaca inus TfR Combination.

Figure 25 A-25E shows the cell combination of unit price CH3C polypeptide.Figure 25 A shows 293F cell.Figure 25 B is shown and figure The amplification of the combination of discribed 293F cell in 25A.Figure 25 C shows the CHO-K1 cell through mankind's TfR stable transfection.Figure 25D show and Figure 25 C in the discribed CHO-K1 cell through mankind's TfR stable transfection combination amplification.Figure 25 E display warp The CHO-K1 cell of Macaca inus TfR stable transfection.

Figure 26 is shown in the internalization situation of unit price and divalent CH3C polypeptide in HEK293 cell.

Figure 27 A- Figure 27 H shows the binding kinetics of CH3C polypeptide.Figure 27 A shows CH3C.35.N163's and mankind TfR In conjunction with data.Figure 27 B shows the data of the combination of CHC3.35 and mankind TfR.Figure 27 C shows CHC3.35.N163 and the mankind The data that TfR unit price combines.Figure 27 D shows data of the CHC3.35 in conjunction with mankind's TfR unit price.Figure 27 E is shown The data of the combination of CH3C.35.N163 and Macaca inus TfR.Figure 27 F shows the number of the combination of CHC3.35 and Macaca inus TfR According to.Figure 27 G shows data of the CHC3.35.N163 in conjunction with Macaca inus TfR unit price.Figure 27 H shows CHC3.35 and food crab Mi The data that monkey TfR unit price combines.

Figure 28 A- Figure 28 F shows the binding kinetics of CH3C polypeptide.Figure 28 A shows the knot of CH3C.3.2-1 and mankind TfR The data of conjunction.Figure 28 B shows the data of the combination of CH3C.3.2-5 and mankind TfR.Figure 28 C shows CH3C.3.2-19 and the mankind The data of the combination of TfR.Figure 28 D shows the data of the combination of CH3C.3.2-1 and Macaca inus TfR.Figure 28 E is shown The data of the combination of CH3C.3.2-5 and Macaca inus TfR.Figure 28 F shows the combination of CH3C.3.2-19 and Macaca inus TfR Data.

Figure 29 A- Figure 29 E is shown in presence (bottom trace) or there is no (top trace) mankind TfR extracellular domains Under, the combination of polypeptide-Fab fusions and FcRn at pH 5.5.

Figure 29 A shows the data of CH3C.35.Figure 29 B shows the data of CH3C.35.19.Figure 29 C shows CH3C.35.20 Data.Figure 29 D shows the data of CH3C.35.21.Figure 29 E shows the data of CH3C.35.24.

Figure 30 is shown in pharmacokinetics (PK) measurement of CH3C polypeptide in wild-type mice.Except CH3C.3.2-5 has Have outside very fast clearance rate, all polypeptide-Fab fusions all have with wild type Fc-Fab fusions (that is, anti-rsv antibodies Ab122 With the comparable clearance rate of anti-BACE1 antibody A b153).

Figure 31 is shown in the pharmacokinetics of the brain in Mice brain tissues/pharmacodynamics (PK/PD) data.To chimeric HuTfR chimeric mice (n=4/group) intravenous administration 42mg/kg Ab153 or unit price CH3C.35.N163 (is designated as " CH3C.35.N163_mono "), and wild-type mice (n=3) intravenous administration 50mg/kg control human IgG 1 (is designated as "huIgG1").Bar chart indicates the +/- SD of average value.

Figure 32 A and 32B are shown in 24 hours after 50mg/kg polypeptide therapeutic, hTfR^{Apical domains+/+}Found in mouse The concentration of IgG.Figure 32 A shows the IgG concentration in blood plasma.Figure 32 B shows the IgG concentration in brain tissue.

Figure 33 A and Figure 33 B shows as measured by being reduced by amyloid p-protein 40 (A β 40), hTfR after 24 hours^{Apical domains+/+}The target for the polypeptide given in mouse engages.Figure 33 A shows 40 concentration of A β in blood plasma.Figure 33 B is shown in brain tissue 40 concentration of A β.

Figure 34 shows that the SDS-PAGE of the different size part of CH3C.18Fc and TfR apical domains (AD) compound is solidifying Glue.Swimming lane 1: molecular weight marker.Swimming lane 2: the CH3C.18Fc-AD compound of the reduction after size exclusion chromatography.

Figure 35 A and Figure 35 B describe the combination between polypeptide and TfR of the present invention.Figure 35 A describes clone Combination interface between CH3C.18 and TfR apical domains.Figure 35 B shows discribed combination circle in Figure 35 A The enlarged drawing in face.

Figure 36 A and Figure 36 B describe the interaction between CH3C.18 and TfR apical domains.Figure 36 A describes the top TfR The mating surface of the structure framework (top figure) and TfR apical domains and CH3C.18Fc of structural domain and CH3C.18Fc is (at 5 angstroms It is interior) (base map).Altogether composite structure withResolution ratio parsing.The structure is disclosed on TfR apical domains and is integrated to The epitope of CH3C.18.In detail, the apical domains N-terminal area participate in CH3C Fc combine, and the structure with CH3C.18Fc with TfR apical domains mutagenesis data are consistent.In addition, the library CH3C.18 side chain also contacts TfR (In It is interior).The library CH3C.18 residue: L157, H159, V160, W161, A162, V163, P186, T189 and W194.Non- library residue: F196 and S156.The key interactions of Figure 36 B description CH3C.18Fc and TfR apical domains.W161 on CH3C.18Fc Cation-π interaction between the R208 on apical domains is important binding interactions.CH3C.18W388 or Apical domains R208 is mutated the combination that can destroy CH3C.18Fc and apical domains.It is consistent with this, from the mankind to Macaca inus R208G mutation explain Macaca inus affinity reduce the reason of.In addition, the non-conservative residue in mankind's apical domains (N292 and E294 (K292 and D294 in Macaca inus)) is neighbouring.Therefore, the Q192 in CH3C.18 can be mutated with phase It combines selectivity to improve Macaca inus the mankind to combine.

Figure 37 A and Figure 37 B describe the combination between polypeptide and TfR of the present invention.Figure 37 A describes clone Hydrogen bond between residue and TfR (D chain) apical domains and non-bond in CH3C.18 (A chain) contact.Figure 37 B Describe the hydrogen bond between residue and TfR (C chain) apical domains in clone's CH3C.18 (B chain) and non-bond connects Touching.

Figure 38 shows the comparison of human IgG 1, IgG2, IgG3 and IgG4 amino acid sequence (SEQ ID NO:623-626).

Figure 39 A- Figure 39 C describes the combination between polypeptide and TfR of the present invention.Figure 39 A describes the top TfR knot Mating surface (the In of the structure framework (top figure) and TfR apical domains and CH3C.35Fc of structure domain and CH3C.35Fc It is interior) (base map).Altogether composite structure withResolution ratio parsing.The structure is disclosed on TfR apical domains and is integrated to The epitope of CH3C.35.The library CH3C.35 side chain contacts TfR (InIt is interior).The library CH3C.35 residue: Y157, T159, E160, W161, S162, T186, E189 and W194.Non- library residue: F196, S156, Q192.Figure 39 B and 39C show Figure 39 A In it is discribed clone CH3C.35 and TfR apical domains between combination interface enlarged drawing.

Figure 40 A describes the covering between CH3C.35Fc and TfR-AD compound and CH3C.18Fc and TfR-AD compound Structure.

The enlarged drawing of overlay structure in Figure 40 B depiction 40A.

Figure 41 A and Figure 41 B describe the combination between polypeptide and TfR of the present invention.Figure 41 A describes clone Hydrogen bond between residue and TfR (D chain) apical domains and non-bond in CH3C.35 (A chain) contact.Figure 41 B Describe the hydrogen bond between residue and TfR (C chain) apical domains in clone's CH3C.35 (B chain) and non-bond connects Touching.

The Fc-Fab that Figure 42 A and Figure 42 B describe in Macaca inus comprising CH3C variant fusion to Ab153Fab structural domain melts Blood plasma PK and the A β 40 for closing polypeptide is reduced.Figure 42 A shows, Ab210 and CH3C.35.9:Ab153 due to the removing that TfR is mediated and Show the clearance rate faster than control IgG (Ab122) and Ab153.Figure 42 B shows, be respectively self-bonded and inhibit BACE1 Ab153, Ab210 and CH3C.35.9:Ab153 shows the reduction of the significant A β 40 in blood plasma.

Figure 43 A and Figure 43 B are depicted in Macaca inus using comprising CH3C variant fusion to Ab153 Fab structural domain Fc-Fab fused polypeptide substantially reduces celiolymph (CSF) A β and sAPP β/sAPP α.Figure 43 A shows, give Ab210 and The animal of CH3C.35.9:Ab153 shows that CSF A β 40 reduces about 70% compared to Ab153 and control IgG (Ab122).Figure 43 B It has been shown that, the animal for giving Ab210 and CH3C.35.9:Ab153 show sAPP β/sAPP α ratio compared to Ab153 and control IgG (Ab122) about 75% is reduced.N=4/group.Line chart indicates average value ± SEM.

Figure 44 A and Figure 44 B be depicted in the anti-BACE1_Ab153, CH3C35.21:Ab153 of single systemic injection 50mg/kg, HTfR apical domains after CH3C35.20:Ab153 or CH3C35:Ab153 Polypeptide fusions^+/+Gene knock-in (KI) mouse HuIgG1 concentration (± SEM, n=5/group of average value) in blood plasma (Figure 44 A) and brain lysate (Figure 44 B).

Figure 44 C be depicted in the anti-BACE1_Ab153, CH3C35.21:Ab153 of single systemic injection 50mg/kg, HTfR apical domains after CH3C35.20:Ab153 or CH3C35:Ab153 Polypeptide fusions^+/+The brain lysate of KI mouse In endogenous mouse A β concentration (average value ± SEM, n=5 only/group).

Figure 44 D be depicted in the anti-BACE1_Ab153, CH3C35.21:Ab153 of single systemic injection 50mg/kg, HTfR apical domains after CH3C35.20:Ab153 or CH3C35:Ab153 Polypeptide fusions^+/+The brain lysate of KI mouse In for the Western blot (Western blot) of the normalized brain TfR protein of actin quantify (average value ± SEM, n =5/group).

Figure 45 A and Figure 45 B be depicted in single systemic injection 50mg/kg anti-BACE1_Ab153, CH3C.35.23:Ab153 or HTfR after CH3C.35.23.3:Ab153 Polypeptide fusions^{Apical domains+/+}Blood plasma (Figure 45 A) and brain the lysate (figure of KI mouse HuIgG1 concentration (± SEM, n=5/group of average value) in 45B).

Figure 45 C be depicted in single systemic injection 50mg/kg anti-BACE1_Ab153, CH3C.35.23:Ab153 or HTfR after CH3C.35.23.3:Ab153 Polypeptide fusions^{Apical domains+/+}Endogenous mouse A β in the brain lysate of KI mouse Concentration (± SEM, n=5/group of average value).

Figure 45 D be depicted in single systemic injection 50mg/kg anti-BACE1_Ab153, CH3C.35.23:Ab153 or HTfR after CH3C.35.23.3:Ab153 Polypeptide fusions^{Apical domains+/+}In the brain lysate of KI mouse just for actin The Western standard measure (± SEM, n=4/group of average value) of the brain TfR protein of ruleization.

Figure 46 A- Figure 46 D is depicted in give the specified protein of single dose 30mg/kg after carried out in Macaca inus 28 Its PKPD research.Figure 46 A and Figure 46 B describe the plasma Ap concentration in serum huIgG1 and blood plasma in serum, and display is given Compound around exposure and thus caused by the influence to plasma A β content at any time.Figure 46 C and Figure 46 D are depicted in administration A β and sAPP β/sAPP α in the CSF of Macaca inus (± SEM, n=4-5/group of average value) afterwards.

Figure 47 A- Figure 47 C be depicted in single dose 30mg/kg specify protein after Macaca inus peripheral blood in blood Netted red blood cell is relative to amount (Figure 47 A), absolute serum iron content (Figure 47 B) and the absolute erythrocyte counts before administration (Figure 47 C) (± SEM, n=4-5/group of average value).

After the single dose 10mg/kg that Figure 48 A and Figure 48 B are depicted in 14 days is injected intravenously in hFcRn gene knock-in mouse The surrounding PK of specified protein measures (blood plasma huIgG1 concentration (Figure 48 A) and clearance rate value (Figure 48 B)) (average value ± SEM, n =3/group).

The median fluorescence intensity of Figure 49 description TfR combination CH3C.18 variant.

Specific embodiment

I. introduction

The polypeptide in conjunction with TfR (TfR) is described herein.The present invention is partly based on certain in the discovery area Fc Amino acid can be modified to generate the novel binding site for having specificity to TfR in Fc polypeptide.Using TfR in blood-brain barrier (BBB) transferrins is natively moved to the fact in brain from blood by great expression and TfR on, these polypeptides can be used for across BBB transports therapeutic agent (such as therapeutical peptide, antibody variable region such as Fab and small molecule).The method can be improved generally The brain of therapeutic agent absorbs and is therefore particularly suitable for the illness and disease that treatment has benefited from brain delivery.

In an aspect, several groups of amino acid that the present invention is partly based in discovery CH3 or CH2 Domain Polypeptide can be with It is substituted to generate the polypeptide for combining TfR.Therefore, in an aspect, provided herein is TfR combinations Polypeptide, such as referring to one group of amino acid (i) 157,159,160,161,162,163,186,189 of SEQ ID NO:1 number With 194；Or there are multiple substitutions at (ii) 118,119,120,122,210,211,212 and 213.In some embodiments, TfR combination polypeptide of the invention such as referring to one group of amino acid (iii) 47 of SEQ ID NO:1 number, 49,56, 58,59,60,61,62 and 63；(iv) 39,40,41,42,43,44,68,70,71 and 72；(v)41,42,43,44,45,65, 66,67,69 and 73；Or there are multiple substitutions at (vi) 45,47,49,95,97,99,102,103 and 104.About four in one group It can be substituted to whole amino acid positions.For purposes of the present invention, substitution is determined referring to SEQ ID NO:1.Therefore, such as One amino acid of fruit is different from the corresponding amino acid in the position SEQ ID NO:1, it is considered that the amino acid is substitution, even if The amino acid is present at position described in naturally occurring CH3 or CH2 Domain Polypeptide.

Be also provided herein generate TfR combination polypeptide method, the method be by generation (i) group, (ii) at multiple positions of group, (iii) group, (iv) group, (v) group or (vi) group there is the variant polypeptide replaced to realize.It can survey The TfR of the fixed variant combines and as described herein, is mutated it further to enhance combination.

In another aspect, turn composition targeting provided herein is treatment method and using TfR combination polypeptide Human Placental Ferritin Receptor expression cell, such as the composition is delivered to the cell, or across endothelium, such as across blood-brain barrier delivering The method of composition.

II. it defines

Unless context separately clearly provides, and otherwise as used herein, singular " one (kind) " and " described " packet Include multiple (kind) indicants.Thus, for example, it may include two or more such molecules and similar for mentioning " polypeptide " Statement.

As used herein, term " about " and " about " instruction when for modifying with digital value or range specified amount, The digital value and the legitimate skew known to those skilled in the art with described value, for example, ± 20%, ± 10% or ± 5% is in the predetermined meanings of described value.

As used in the context of the invention, term " TfR " or " TfR " refer to TfR albumen Matter 1.1 polypeptide sequence of mankind's transferrins receptor is set forth in SEQ ID NO:235.It it is known that and turn iron egg from other species Polymeric immunoglobulin receptor protein 1 is (for example, chimpanzee, deposits number XP_003310238.1；Macaque, NP_001244232.1；Dog, NP_ 001003111.1；Ox, NP_001193506.1；Mouse, NP_035768.1；Rat, NP_073203.1；And chicken, NP_ 990587.1).Term " TfR " also covers the base by being located at 1 chromosomal loci of transferrin receptor protein Because of the allelic variant of the example reference sequence of coding, such as human sequence.Overall length transferrin receptor protein includes Short N-terminal intracellular space, transmembrane region and maxicell extracellular portion.The extracellular domain is characterized with three structural domains: Protease-like domain, helix domain and apical domains.The apical domains sequence of mankind's transferrins receptor 1 is set forth in In SEQ ID NO:107.

As used herein, term " CH3 structural domain " and " CH2 structural domain " refer to that constant region for immunoglobulin structural domain is more Peptide.In the case where IgG antibody, CH3 Domain Polypeptide, which refers to, is such as numbered according to EU numbering plan by from about 341 to about 447 The section of the Amino acid profile of position, and CH2 Domain Polypeptide refers to such as according to EU numbering plan number by from about 231 to about The section of 340 Amino acid profiles.CH2 and CH3 Domain Polypeptide is also using the number side IMGT (ImMunoGeneTics) Case number, wherein CH2 structure Field Number is 1-110 and CH3 is tied according to IMGT Scientific numbering of figure (website IMGT) Structure Field Number is 1-107.CH2 and CH3 structural domain is a part of immunoglobulin fc region.In the case where IgG antibody, the area Fc Refer to and is such as numbered according to EU numbering plan, the section for the Amino acid profile that You Congyue is 231 to about 447.As used herein, Term " area Fc " may also include at least part of antibody hinge region.Exemplary hinge region sequence is set forth in SEQ ID NO:234 In.

Term " wild type ", " natural " and " naturally occurring " about CH3 or CH2 structural domain has for referring to herein The structural domain of sequence present in nature.

In the context of the present invention, the term " mutant " and " variant " about mutant polypeptide or mutation polynucleotide can It is used interchangeably.Variant about given wild type CH3 or CH2 structural domain reference sequences may include naturally occurring allele Variant.CH3 or CH2 structural domain existing for " non-natural ", which refers to, to be not present in cell in nature and by for example using gene Variation that engineering technology or the natural CH3 structural domain of induced-mutation technique gene modification or CH2 structural domain polynucleotide or polypeptide generate or Mutation structure domain." variant " includes any structural domain for containing at least one amino acid mutation relative to wild type.Mutation can be with Including substitution, insertion and missing.

Term " amino acid " refers to naturally occurring and synthesis amino acid and to be similar to the side of naturally occurring amino acid The amino acid analogue and amino acid simulant that formula works.

Naturally occurring amino acid is the amino acid by genetic code encoding, and the amino acid then modified, such as hydroxyl Proline, γ-carboxyglutamic acid and O- phosphoserine." amino acid analogue ", which refers to, to be had and naturally occurring amino acid phase The compound of same basic chemical structure (that is, α carbon is integrated to hydrogen, carboxyl, amino and R group), such as homoserine, just bright ammonia Acid, methionine sulfoxide, methionine methyl sulfonium.The analog has the R group (such as nor-leucine) or modification of modification Peptide backbone, but retain identical with naturally occurring amino acid basic chemical structure." amino acid simulant ", which refers to, to be had not The compound for being same as the structure of the general chemical structure of amino acid but being acted in a manner of being similar to naturally occurring amino acid.

Naturally occurring a-amino acid includes but is 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), glutamy Amine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr) and their group It closes.The stereoisomer of naturally occurring a-amino acid includes but is not limited to D-alanine (D-Ala), D-Cys (D- Cys), D-Asp (D-Asp), D-Glu (D-Glu), D-phenylalanine (D-Phe), D-His (D-His), D- are different Leucine (D-Ile), D-Arg (D-Arg), D-Lys (D-Lys), D-Leu (D-Leu), D-Met (D- Met), D-Asn (D-Asn), D-PROLINE (D-Pro), D-Gln (D-Gln), D-Ser (D-Ser), D- Soviet Union Propylhomoserin (D-Thr), D-Val (D-Val), D-trp (D-Trp), D-Tyrosine (D-Tyr) and their combination.

Amino acid herein can be with its commonly known three letter symbols or by IUPAC-IUB biological chemical name committee member The one-letter symbol that meeting (IUPAC-IUB Biochemical Nomenclature Commission) is recommended is mentioned.

Term " polypeptide ", " peptide " and " protein " are used interchangeably herein, and refer to the polymer of amino acid residue.It is described The amino acid that term is suitable for manufactured chemical's analogies that one or more amino acid residues are corresponding naturally occurring amino acid is poly- Object is closed, and applies also for naturally occurring amino acid polymer and non-naturally occurring amino acid polymer.Amino acid polymerization Object can only include l-amino acid, only comprising D- amino acid, or the mixture comprising L and D amino acid.

Term " conservative substitution ", " conservative variants " or the variant of modification " conservative ", which refer to can be classified as, has similar spy The change of one another amino acid of amino acid substitution of sign.The example of the classification of the conserved amino acid group defined by this method can To include: " electrification/polarity group ", including 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)；" aromatic amino acid group ", packet Include Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W) and (histidine or H)；" aliphatic amino acid Group ", including Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile are (different bright Propylhomoserin or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T) and Cys (cysteine or C).In In each group, subgroup can be also identified.For example, electrification or polar amino acid group can be subdivided into several subgroups, comprising: " band Positive electricity subgroup " includes Lys, Arg and His；" negatively charged subgroup " includes Glu and Asp；" polarity subgroup ", comprising Asn and Gln.In another example, aromatics or cyclic amino acid group can be subdivided into including following subgroup: " azo-cycle subgroup ", comprising Pro, His and Trp；" phenyl subgroup " includes Phe and Tyr.In a further example, aliphatic amino acid group can be subdivided into several Asias Group, such as " aliphatic series nonpolarity subgroup ", include Val, Leu, Gly and Ala；" aliphatic series low polarity subgroup ", comprising Met, Ser, Thr and Cys.The example of the classification of conservative variants includes the amino acid substitution of the amino acid in the above subgroup, such as but unlimited In: Lys replaces Arg or vice versa, it is possible thereby to maintain positive charge；Glu replaces Asp or vice versa, it is possible thereby to maintain Negative electrical charge；Ser replaces Thr or vice versa, it is possible thereby to maintain free-OH；Replace Asn or vice versa with Gln, thus may be used To maintain free-NH₂.In some embodiments, replaced with hydrophobic amino acid naturally occurring in such as active site Hydrophobic amino acid, to keep hydrophobicity.

In the situation of two or more polypeptide sequences, term " same " or " identity " percentage refer to when than When compared with comparing and compare to reach maximum correspondence in window or specified region, such as compare algorithm using sequence or by comparing manually To identical or specified region in the amino acid residue identity with prescribed percentage measured with visual inspection, such as extremely Few 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% Or two or more sequences or the subsequence of greater percentage identity.

The sequence of polypeptide is compared, typically a sequence serves as reference sequences, to compared with candidate sequence.Than To the available various methods of one of ordinary skill in the art, such as range estimation comparison or the publicly available software of use can be used, make It is carried out with known algorithm, to realize high specific to property.This class package includes blast program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR).Reach high specific for comparing The parameter of property can be determined by one of ordinary skill in the art.For purposes of this application, for the sequence ratio of polypeptide sequence Compared with use compares the BLASTP algorithm standard protein BLAST of two protein sequences with parameter preset.

When for identifying the given amino acid residue in polypeptide sequence, term " corresponding to ", " reference ... determine " or " reference ... number " refers to when farthest comparing and being compared given amino acid sequence and specified reference sequences, The position of residue in the reference sequences.Thus, for example, the ammonia when most preferably being compared with SEQ ID NO:1, in polypeptide In the case where amino acid alignment in base acid residue and SEQ ID NO:1, the residue " corresponds to " amino in SEQ ID NO:1 Amino acid in the region of sour 114-220.And the length for the polypeptide that reference sequences compare is without identical as reference sequences.

As used herein, " binding affinity " refer to single binding site on two molecules, such as a polypeptide with Its target combined, such as the intensity of the noncovalent interaction between TfR.Therefore, unless otherwise instructed or from Context is clearly visible, otherwise for example, the term can refer to that the 1:1 between polypeptide and its target interacts.Binding affinity It can be by measuring equilibrium dissociation constant (K_D) quantitative, the equilibrium dissociation constant refers to dissociation rate constant (k_d, the time^-1) remove With association rate constants (k_a, the time^-1M^-1)。K_DCan by using surface plasma body resonant vibration (SPR) method, such as Biacore^TMSystem；Dynamics excludes measurement, such asWith biological membranous layer interferometry (such as usingPlatform) measurement compound is formed and the dynamics of dissociation determines.As used herein, " in conjunction with affine Power " not only includes formal binding affinity, and the combination of the 1:1 interaction such as between reflection polypeptide and its target is affine Power, but also including apparent affinity, for apparent affinity, the K of calculating_DIt can reflect desired combination.

When mentioning the polypeptide of the CH3 as described herein comprising modification and/or the CH2 structural domain of modification, phrase is " special The opposite sex combines " or " selective binding " to target, such as TfR is to instigate the polypeptide to be integrated to structure not than it With target, such as the higher affinity of target, the higher affinity and/or longer that are not present in TfR family Duration is integrated to the association reaction of the target.In typical embodiments, measured when under the conditions of identical affinity determination When, the polypeptide is at least 5 times, 6 times, 7 times, 8 times, 9 that the affinity of TfR is to the affinity of uncorrelated target Again, 10 times, 20 times, 25 times, 50 times or 100 times, or more high magnification numbe.In some embodiments, the CH3 of modification and/or modification CH2 Domain Polypeptide be specifically bound on TfR between each species and guard, such as in non-human primate The epitope guarded between human species.In some embodiments, polypeptide can be only combined to mankind's transferrins receptor.

Term " subject ", " individual " and " patient " is used interchangeably herein, and is meant to mammal, including but not It is limited to the mankind, non-human primate, rodent (such as rat, mouse and guinea pig), rabbit, ox, pig, horse and other food in one's mouths Newborn animal species.In one embodiment, patient is the mankind.

Term " treatment (treatment) ", " treatment (treating) " and similar statement, which are generally used for referring to herein, to be obtained Pharmacology needed for obtaining and/or physiological role." treatment " can refer to any in terms for the treatment of or improving damage, disease or illness Success indicator, including any either objectively or subjectively parameter, such as elimination, alleviation, the extension of patient's survival period, time-to-live or survival Rate increases, mitigates symptom or makes patient to be easier to endure the damage, disease or illness, slow down the rate of degeneration or decline, or changes The body or mental health of kind patient.The treatment or improvement of symptom can be based on either objectively or subjectively parameters.Therapeutic effect can be with The individual or one group of individual of the treatment, or the different time with same patient before the treatment or during treatment are not received Situation compare.

Term " pharmaceutically acceptable excipient " refers to biologically or pharmacologically suitable for mankind or animal Nonactive medical components, such as, but not limited to buffer, carrier or preservative.

As used herein, a kind of " therapeutic dose " or " therapeutically effective amount " of medicament is treatment, mitigates, eliminates subject's disease Disease, or reduce the pharmaceutical quantities of the severity of disease symptoms." therapeutic dose " or the medicament of " therapeutically effective amount " can improve patient Survival condition；Increase time-to-live or survival rate；Mitigate symptom；Damage, disease or illness is set to be easier to endure；Slow down degeneration or subtracts The rate moved back；Or improve the body or mental health of patient.

Term administering " refers to the method that medicament, compound or composition are delivered to desired biological effect position. These methods include but is not limited to, surface delivering, parenteral delivering, intravenous delivery, Intradermal delivery, intramuscular delivery, intrathecal Delivering, colonic delivery, rectal delivery or intraperitoneal delivery.In one embodiment, polypeptide described herein is through vein Interior application.

III. TfR combination polypeptide

The polypeptide according to the present invention that this section description is integrated to TfR and can transport across blood-brain barrier (BBB) Generation.

In an aspect, the polypeptide comprising CH3 or CH2 structural domain is provided, CH3 the or CH2 structural domain has permission The polypeptid specificity is integrated to the modification of TfR.The modification is at the surface introduced in CH3 or CH2 structural domain The amino acid of existing designated groups.In some embodiments, the polypeptid specificity knot of the CH3 or CH2 structural domain comprising modification Close the epitope in the apical domains of TfR.

One of ordinary skill in the art should be understood that can by identifying other immunoglobulin isotypes, such as IgM, IgA, The amino acid for corresponding to (i) described herein-(vi) group in CH2 the and CH3 structural domain of IgE, IgD etc., modifies institute in a similar manner State structural domain.It can also be to from other species, such as non-human primate, monkey, mouse, rat, rabbit, dog, pig, chicken and class Like species immunoglobulin in corresponding construction domain modified.

CH3 TfR combination polypeptide

In some embodiments, the structural domain of modification is mankind's Ig CH3 structural domain, such as IgG CH3 structural domain.Institute Stating CH3 structural domain may belong to any IgG hypotype, that is, come from IgG1, IgG2, IgG3 or IgG4.In the case where IgG antibody, CH3 structural domain, which refers to, such as numbers according to EU numbering plan, the section for the Amino acid profile that You Congyue is 341 to about 447.Unless It is otherwise indicated, otherwise for the purpose for identifying the corresponding amino acid position group combined for TfR, in CH3 structural domain Position be to be determined referring to the determining or amino acid 1 14-220 referring to SEQ ID NO:1 of SEQ ID NO:3.Replace also with reference to SEQ ID NO:1 is determined, that is, relative to the amino acid of the corresponding position in SEQ ID NO:1, considers amino to be replaced Acid.SEQ ID NO:1 includes part hinge region sequence, i.e. PCP, as amino acid 1-3.The CH3 carried out referring to SEQ ID NO:1 The number of each position includes first three amino acid in structural domain.

As indicated above, the residue group that can be modified according to the present invention in CH3 structural domain is herein defined as referring to SEQ ID NO:1 number.Any CH3 structural domain, such as IgG1, IgG2, IgG3 or IgG4 CH3 structural domain can correspond to SEQ There is modification, such as amino acid substitution in one or more groups of residues of the residue at position described in ID NO:1.SEQ ID NO:1 1 amino acid sequence of human IgG and the comparison of human IgG 2, IgG3 and IgG4 be shown in Figure 38.Described IgG2, IgG3 and The position of any given position in each sequence in IgG4 sequence corresponding to SEQ ID NO:1 can readily determine that.

In one embodiment, the CH3 Domain Polypeptide for specifically binding the modification of TfR is integrated to institute State 208 comprising overall length mankind's transferrins receptor sequence (SEQ ID NO:235) in the apical domains of TfR The epitope of position, the position correspond to the mankind's transferrins receptor apical domains sequence stated in SEQ ID NO:107 11.SEQ ID NO:107 correspond to mankind's transferrins receptor -1uniprotein sequence P02786 (SEQ ID NO: 235) amino acid 1 98-378.In some embodiments, the CH3 Domain Polypeptide of modification is integrated to TfR Position 158 in apical domains comprising overall length mankind's transferrins receptor sequence (SEQ ID NO:235), 188,199,207, 208,209,210,211,212,213,214,215 and/or 294 epitope.The CH3 Domain Polypeptide of the modification can combine To TfR, while not blocking or inhibiting in other ways the combination of transferrins and the receptor.In some implementations In scheme, the combination of transferrins and TfR are generally not inhibited.In some embodiments, for transferrins and TfR In conjunction with inhibiting effect below about 50% (for example, less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%).In some embodiments, 20% is below about (for example, less than about for the inhibiting effect of the combination of transferrins and TfR 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%).The exemplary CH3 Domain Polypeptide for showing this binding specificity includes as referring to SEQ ID NO:1 There is amino acid substitution at position 157,159,160,161,162,163,186,189 and 194 determined by amino acid 1 14-220 Polypeptide.

CH3 TfR combination group (i): 157,159,160,161,162,163,186,189 and 194

In some embodiments, the CH3 Domain Polypeptide of modification according to the present invention comprising 157,159,160, It 161, include at least three or at least four in 162,163,186,189 and 194 one group of amino acid position (i group), and allusion quotation Type five, six, seven, eight or nine substitution.The exemplary substitution that can be introduced at these locations is shown in table 6 In.In some embodiments, 161 and/or 194 amino acid is aromatic amino acid, such as Trp, Phe or Tyr.One In a little embodiments, 161 amino acid is Trp.In some embodiments, 161 amino acid is Gly.In some realities It applies in scheme, 194 aromatic amino acids are Trp or Phe.

In some embodiments, the CH3 Domain Polypeptide of the modification of TfR is specifically bound relative to SEQ ID NO:1 has the position replaced comprising at least one as follows: in 157 Leu, Tyr, Met or Val；159 Leu, Thr, His or Pro of position；In 160 Val, Pro or acidic amino acid；In 161 aromatic amino acids, such as Trp Or Gly (such as Trp)；In 162 Val, Ser or Ala；186 acidic amino acids, Ala, Ser, Leu, Thr or Pro；In 189 Thr or acidic amino acids；Or in 194 Trp, Tyr, His or Phe.In some embodiments, it repairs The CH3 Domain Polypeptide of decorations can described group one or more positions include specific amino acids conservative substitution, such as In same electrification grouping, hydrophobicity grouping, side chain ring structure grouping (such as aromatic amino acid) or size packets and/or polarity Or the amino acid in nonpolarity grouping.Thus, for example, Ile can reside in 157,159 and/or 186.In some realities It applies in scheme, one, two in position 160,186 and 189 or the acidic amino acid at all positions are Glu.Other In embodiment, one, two in position 160,186 and 189 or the acidic amino acid at all positions are Asp.One In a little embodiments, two, three, four, five, six in position 157,159,160,161,162,186,189 and 194 A, seven or all eight positions have the amino acid substitution as illustrated in this section.

In some embodiments, having the CH3 Domain Polypeptide modified in (i) group includes 163 natural A sn. In some embodiments, the CH3 Domain Polypeptide of modification 163 comprising Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala or Asp.In some embodiments, the CH3 Domain Polypeptide of the modification is also including 153,164,165 and 188 Position at comprising one, two, three or four substitution.In some embodiments, Trp, Tyr, Leu or Gln can be deposited It is 153.In some embodiments, Ser, Thr, Gln or Phe can reside in 164.In some embodiments, Gln, Phe or His can reside in 165.In some embodiments, Glu can reside in 188.

In some embodiments, the CH3 Domain Polypeptide of modification includes two selected from the following, three, four, five A, six, seven, eight, nine or ten positions: in 153 Trp, Leu or Glu；In 157 Tyr or Phe；In 159 Thr；In 160 Glu；In 161 Trp；In 162 Ser, Ala, Val or Asn；In 163 Ser or Asn；In 186 Thr or Ser；In 188 Glu or Ser；In 189 Glu；And/or in 194 Phe.Some In embodiment, the CH3 Domain Polypeptide of modification includes following all 11 positions: in 153 Trp, Leu or Glu；In 157 Tyr or Phe；In 159 Thr；In 160 Glu；In 161 Trp；In 162 Ser, Ala, Val or Asn；In 163 Ser or Asn；In 186 Thr or Ser；In 188 Glu or Ser；In 189 Glu；And/or In 194 Phe.

In some embodiments, the CH3 Domain Polypeptide of modification is included in 157 Leu or Met；At 159 Leu, His or Pro；In 160 Val；In 161 Trp；In 162 Val or Ala；In 186 Pro；At 189 Thr；And/or in 194 Trp.In some embodiments, the CH3 Domain Polypeptide of modification is also included in 164 Ser, Thr, Gln or Phe.In some embodiments, the CH3 Domain Polypeptide of modification be also included in 153 Trp, Tyr, Leu or Gln and/or in 165 Gln, Phe or His.In some embodiments, Trp is present in 153 and/or Gln is deposited It is 165.In some embodiments, the CH3 Domain Polypeptide of modification does not have Trp at 153.

In other embodiments, the CH3 Domain Polypeptide of modification is included in 157 Tyr；In 159 Thr；In 160 Glu or Val；In 161 Trp；In 162 Ser；In 186 Ser or Thr；In 189 Glu；With/ Or in 194 Phe.In some embodiments, the CH3 Domain Polypeptide of modification is included in 163 natural A sn.One In a little embodiments, the CH3 Domain Polypeptide of modification is also included in 153 Trp, Tyr, Leu or Gln；And/or at 188 Glu.In some embodiments, the CH3 Domain Polypeptide of modification is also included in 153 Trp and/or at 188 Glu。

In some embodiments, the CH3 structural domain of modification includes one or more of following substitution: at 153 Trp；In 159 Thr；In 161 Trp；In 162 Val；In 186 Ser or Thr；In 188 Glu；With/ Or in 194 Phe.

In a further embodiment, the CH3 structural domain of the modification also includes one, two or three selected from the following Position: 187 are Lys, Arg, Gly or Pro；197 are Ser, Thr, Glu or Lys；It is Ser, Trp or Gly with 199.

In some embodiments, the CH3 Domain Polypeptide and SEQ ID of the modification of TfR are specifically bound The amino acid 1 14-220 of any of NO:4-29,236-299 and 422-435 have at least 70% identity, at least 75% together One property, at least 80% identity, at least 85% identity, at least 90% identity or at least 95% identity.In some implementations In scheme, the CH3 Domain Polypeptide of such modification includes the ammonia of any of SEQ ID NO:4-29,236-299 and 422-435 Base acid 157-163 and/or 186-194.In some embodiments, the CH3 Domain Polypeptide of such modification includes SEQ ID The amino acid 1 53-163 and/or 186-194 of any of NO:4-29,236-299 and 422-435.In some embodiments, The CH3 Domain Polypeptide of modification includes the amino acid 1 53-163 of any of SEQ ID NO:4-29,236-299 and 422-435 And/or 186-199.

In some embodiments, the CH3 Domain Polypeptide and SEQ ID of the modification of TfR are specifically bound The amino acid 1 14-220 of NO:1 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, restrictive condition are that homogeneity percentage does not include described group of position Set 157,159,160,161,162,163,186,189 and 194.In some embodiments, the CH3 structural domain of the modification is more Peptide includes such as any of SEQ ID NO:4-29,236-299 and 422-435 amino acid 1 57-163 stated and/or amino Sour 186-194.

In some embodiments, the CH3 Domain Polypeptide Yu SEQ ID NO:4-29,236-299 and 422-435 of modification Any of there is at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, extremely Few 90% identity or at least 95% identity, restrictive condition are corresponding to SEQ ID NO:4-29,236-299 and 422- Any of 435 position 153,157,159,160,161,162,163,164,165,186,187,188,189,194, There are at least five, six, seven, eight, nine, ten, 11,12,13,14 in 197 and 199 position It is a, 15 or 16 do not lack or unsubstituted.

In some embodiments, the CH3 Domain Polypeptide Yu SEQ ID NO:4-29,236-299 and 422-435 of modification Any of there is at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity or extremely Few 95% identity, and also comprising at least five, six in following position, seven, eight, nine, ten, 11, 12,13,14,15 or 16: in 153 Trp, Tyr, Leu, Gln or Glu；At 157 Leu, Tyr, Met or Val；In 159 Leu, Thr, His or Pro；In 160 Val, Pro or acidic amino acid；161 The aromatic amino acid of position, such as Trp；In 162 Val, Ser or Ala；In 163 Ser or Asn；164 Ser, Thr, Gln or Phe；In 165 Gln, Phe or His；Acidic amino acid, Ala, Ser, Leu, Thr or Pro at 186； In 187 Lys, Arg, Gly or Pro；In 188 Glu or Ser；In 189 Thr or acidic amino acids；At 194 Trp, Tyr, His or Phe；In 197 Ser, Thr, Glu or Lys；With in 199 Ser, Trp or Gly.

In some embodiments, the CH3 Domain Polypeptide of modification according to the present invention comprising 153,157,159, 160, replace at 162,163,186,188,189,194,197 and 199 one group of amino acid position comprising one or more；And Wherein the substitution and the position are that the sequence of reference SEQ ID NO:13 determines.In some embodiments, the modification CH3 structural domain be included in 153 Glu, Leu, Ser, Val, Trp or Tyr；157 aromatic amino acids (such as Tyr, Phe or Trp), Met, Pro or Val；In 159 Thr, Asn or Val；In 160 Glu, Ile, Pro or Val；162 Aliphatic amino acid (such as Ala, Ile or Val), Ser or the Thr of position；In 163 Ser, Asn, Arg or Thr；At 186 Thr, His or Ser；In 188 Glu, Ser, Asp, Gly, Thr, Pro, Gln or Arg；In 189 Glu or Arg；In 194 Phe, His, Lys, Tyr or Trp；In 197 Ser, Thr or Trp；With in 199 Ser, Cys, Pro, Met Or Trp.The CH3 Domain Polypeptide of modification can have sequence: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VX₁WESX₂GX₃X₄WX₅X₆YKTTPPVLDSDGSFFLYSKLTVX₇KX₈X₉WQQGX₁₀VFX₁₁CX₁₂VMHEALHNHYTQKSLSLS PGK (SEQ ID NO:556), wherein X₁It is E, L, S, V, W or Y；X₂It is aromatic amino acid (such as Y, F or W), M, P or V；X₃It is T, N or V；X₄It is E, I, P or V；X₅It is aliphatic amino acid (such as A, I or V), S or T；X₆It is S, N, R or T；X₇It is T, H or S；X₈ It is E, S, D, G, T, P, Q or R；X₉It is E or R；X₁₀It is F, H, K, Y or W；X₁₁It is S, T or W；And X₁₂It is S, C, P, M or W.In In certain embodiments, the CH3 Domain Polypeptide of modification may include sequence: X₁WESX₂GX₃X₄WX₅X₆(SEQ ID NO: 554), wherein X₁It is E, L, S, V, W or Y；X₂It is aromatic amino acid (such as Y, F or W), M, P or V；X₃It is T, N or V；X₄Be E, I, P or V；X₅It is aliphatic amino acid (such as A, I or V), S or T；And X₆It is S, N, R or T.In certain embodiments, it modifies CH3 Domain Polypeptide may include sequence: X₁KX₂X₃WQQGX₄VFX₅CX₆(SEQ ID NO:555), wherein X₁It is T, H or S； X₂It is E, S, D, G, T, P, Q or R；X₃It is E or R；X₄It is F, H, K, Y or W；X₅It is S, T or W；And X₆It is S, C, P, M or W.

In some embodiments, the CH3 Domain Polypeptide of the modification is included in 153 Glu, Leu or Trp；In 157 aromatic amino acids；In 159 Thr；In 160 Glu；In 162 aliphatic amino acids or Ser；At 163 Ser or Asn；In 186 Thr or Ser；In 188 Glu or Ser；In 189 Glu；194 Phe, His, Tyr or Trp；In 197 Ser；With in 199 Ser, wherein the substitution and the position are referring to SEQ ID NO:13 Sequence determine.In specific embodiments, the aromatic amino acid at 157 is Tyr or Phe and described at 162 Aliphatic amino acid is Ala or Val.The CH3 Domain Polypeptide of modification can have sequence: GQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVX₁WESX₂GX₃X₄WX₅X₆YKTTPPVLDSDGSFFLYSKLTVX₇KX₈X₉WQQGX₁₀VFX₁₁CX₁₂VMH EALHNHYTQKSLSLSPGK (SEQ ID NO:559), wherein X₁It is E, L or W；X₂It is aromatic amino acid (such as Y or F)；X₃It is T；X₄It is E；X₅It is aliphatic amino acid (such as A or V) or S；X₆It is S or N；X₇It is T or S；X₈It is E or S；X₉It is E；X₁₀It is F, H, Y Or W；X₁₁It is S；And X₁₂It is S.In certain embodiments, the CH3 Domain Polypeptide of modification may include sequence: X₁WESX₂GX₃X₄WX₅X₆(SEQ ID NO:557), wherein X₁It is E, L or W；X₂It is aromatic amino acid (such as Y or F)；X₃It is T； X₄It is E；X₅It is aliphatic amino acid (such as A or V) or S；And X₆It is S or N.In certain embodiments, the CH3 structure of modification Domain polypeptide may include sequence: X₁KX₂X₃WQQGX₄VFX₅CX₆(SEQ ID NO:558), wherein X₁It is T or S；X₂It is E or S；X₃ It is E；X₄It is F, H, Y or W；X₅It is S；And X₆It is S.

In a further embodiment, the CH3 Domain Polypeptide of the modification may be embodied in 153 Glu, Leu or Trp；In 157 Tyr or Phe；In 159 Thr；In 160 Glu；In 162 Ala, Val or Ser；At 163 Ser or As n；In 186 Thr or Ser；In 188 Glu or Ser；In 189 Glu；In 194 Phe；In 197 Ser；With in 199 Ser, wherein the substitution and the position are determined referring to the sequence of SEQ ID NO:13. The CH3 Domain Polypeptide of modification can have sequence: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVX₁WESX₂GX₃X₄WX₅X₆YKTTPPVLDSDGSFFLYSKLTVX₇KX₈X₉WQQGX₁₀VFX₁₁CX₁₂VMHEALHNHYTQKSLSLSPGK(SEQ ID NO:562), wherein X₁It is E, L or W；X₂It is Y or F；X₃It is T；X₄It is E；X₅It is S, A or V；X₆It is S or N；X₇It is T or S；X₈It is E or S；X₉It is E；X₁₀It is F；X₁₁It is S；And X₁₂It is S.In certain embodiments, the CH3 Domain Polypeptide of modification can wrap Containing sequence: X₁WESX₂GX₃X₄WX₅X₆(SEQ ID NO:560), wherein X₁It is E, L or W；X₂It is Y or F；X₃It is T；X₄It is E；X₅It is S, A or V；And X₆It is S or N.In certain embodiments, the CH3 Domain Polypeptide of modification may include sequence: X₁KX₂X₃WQQGX₄VFX₅CX₆(SEQ ID NO:561), wherein X₁It is T or S；X₂It is E or S；X₃It is E；X₄It is F；X₅It is S；And X₆It is S.

In some embodiments, the CH3 Domain Polypeptide of modification according to the present invention comprising 153,157,159, 160, only comprising a substitution in 162,163,186,188,189,194,197 and 199 one group of amino acid position；And wherein The substitution and the position are that the sequence of reference SEQ ID NO:238 determines.In some embodiments, the CH3 knot of modification Structure domain polypeptide is included in 153 Glu, Leu, Ser, Val, Trp or Tyr.The CH3 Domain Polypeptide of modification may be embodied in 153 Glu.The CH3 Domain Polypeptide of modification may be embodied in 153 Leu.The CH3 Domain Polypeptide of modification can wrap It is contained in 153 Ser.The CH3 Domain Polypeptide of modification may be embodied in 153 Val.The CH3 Domain Polypeptide of modification can To be included in 153 Trp.The CH3 Domain Polypeptide of modification may be embodied in 153 Tyr.In some embodiments, The CH3 Domain Polypeptide of modification is included in 157 Tyr, Phe, Trp, Met, Pro or Val.The CH3 Domain Polypeptide of modification It may be embodied in 157 Tyr.The CH3 Domain Polypeptide of modification may be embodied in 157 Phe.The CH3 structural domain of modification Polypeptide may be embodied in 157 Trp.The CH3 Domain Polypeptide of modification may be embodied in 157 Met.The CH3 of modification is tied Structure domain polypeptide may be embodied in 157 Pro.The CH3 Domain Polypeptide of modification may be embodied in 157 Val.In some realities It applies in scheme, the CH3 Domain Polypeptide of modification is included in 159 Thr, Asn or Val.The CH3 Domain Polypeptide of modification can be with Included in 159 Thr.The CH3 Domain Polypeptide of modification may be embodied in 159 Asn.The CH3 Domain Polypeptide of modification It may be embodied in 159 Val.In some embodiments, the CH3 Domain Polypeptide of modification included in 160 Glu, Ile, Pro or Val.The CH3 Domain Polypeptide of modification may be embodied in 160 Glu.The CH3 Domain Polypeptide of modification can be with Included in 160 Ile.The CH3 Domain Polypeptide of modification may be embodied in 160 Pro.The CH3 Domain Polypeptide of modification It may be embodied in 160 Val.In some embodiments, the CH3 Domain Polypeptide of modification included in 162 Ala, Ile, Val, Ser or Thr.The CH3 Domain Polypeptide of modification may be embodied in 162 Ala.The CH3 Domain Polypeptide of modification It may be embodied in 162 Ile.The CH3 Domain Polypeptide of modification may be embodied in 162 Val.The CH3 structural domain of modification Polypeptide may be embodied in 162 Ser.The CH3 Domain Polypeptide of modification may be embodied in 162 Thr.In some embodiment party In case, the CH3 Domain Polypeptide of modification is included in 163 Ser, Asn, Arg or Thr.The CH3 Domain Polypeptide of modification can be with Included in 163 Ser.The CH3 Domain Polypeptide of modification may be embodied in 163 Asn.The CH3 Domain Polypeptide of modification It may be embodied in 163 Arg.The CH3 Domain Polypeptide of modification may be embodied in 163 Thr.In some embodiments In, the CH3 Domain Polypeptide of modification is included in 186 Thr, His or Ser.The CH3 Domain Polypeptide of modification may be embodied in 186 Thr.The CH3 Domain Polypeptide of modification may be embodied in 186 His.The CH3 Domain Polypeptide of modification can wrap It is contained in 186 Ser.In some embodiments, the CH3 Domain Polypeptide of modification included in 188 Glu, Ser, Asp, Gly, Thr, Pro, Gln or Arg.The CH3 Domain Polypeptide of modification may be embodied in 188 Glu.The CH3 structural domain of modification Polypeptide may be embodied in 188 Ser.The CH3 Domain Polypeptide of modification may be embodied in 188 Asp.The CH3 of modification is tied Structure domain polypeptide may be embodied in 188 Gly.The CH3 Domain Polypeptide of modification may be embodied in 188 Thr.Modification CH3 Domain Polypeptide may be embodied in 188 Pro.The CH3 Domain Polypeptide of modification may be embodied in 188 Gln.It repairs The CH3 Domain Polypeptide of decorations may be embodied in 188 Arg.In some embodiments, the CH3 Domain Polypeptide packet of modification It is contained in 189 Glu or Arg.The CH3 Domain Polypeptide of modification may be embodied in 189 Glu.The CH3 structural domain of modification is more Peptide may be embodied in 189 Arg.In some embodiments, the CH3 Domain Polypeptide of modification included in 194 Phe, His, Lys, Tyr or Trp.The CH3 Domain Polypeptide of modification may be embodied in 194 Phe.The CH3 Domain Polypeptide of modification It may be embodied in 194 His.The CH3 Domain Polypeptide of modification may be embodied in 194 Lys.The CH3 structural domain of modification Polypeptide may be embodied in 194 Tyr.The CH3 Domain Polypeptide of modification may be embodied in 194 Trp.In some embodiment party In case, the CH3 Domain Polypeptide of modification is included in 197 Ser, Thr or Trp.The CH3 Domain Polypeptide of modification may include In 197 Ser.The CH3 Domain Polypeptide of modification may be embodied in 197 Thr.The CH3 Domain Polypeptide of modification can be with Included in 197 Trp.In some embodiments, the CH3 Domain Polypeptide of modification included in 199 Ser, Cys, Pro, Met or Trp.The CH3 Domain Polypeptide of modification may be embodied in 199 Ser.The CH3 Domain Polypeptide of modification can be with Included in 199 Cys.The CH3 Domain Polypeptide of modification may be embodied in 199 Pro.The CH3 Domain Polypeptide of modification It may be embodied in 199 Met.The CH3 Domain Polypeptide of modification may be embodied in 199 Trp.The CH3 structural domain of modification Polypeptide can have the sequence of any of SEQ ID NO:563-574.

In some embodiments, the CH3 Domain Polypeptide of modification according to the present invention comprising 153,157,159, 160, replace at 162,163,164,186,189 and 194 one group of amino acid position comprising one or more；And it is wherein described Substitution and the position are that the sequence of reference SEQ ID NO:9 determines.In some embodiments, the CH3 structure of the modification Domain is included in 153 Glu or Trp；In 157 Val, Trp, Leu or Tyr；In 159 Leu, Pro, Phe, Thr or His；In 160 Pro, Val or Glu；In 162 Ala, Ser, Val or Gly；163 Leu, His, Gln, Gly, Val, Ala, Asn, Asp, Thr or Glu；In 164 Thr, Phe, Gln, Val or Tyr；186 Leu, Ser, Glu, Ala or Pro；In 189 Glu, Asp, Thr or Asn；With in 194 Trp, Tyr, Phe or His.The CH3 structure of modification Domain polypeptide can have sequence: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVX₁WESX₂GX₃X₄WX₅X₆X₇K TTPPVLDSDGSFFLYSKLTVX₈KSX₉WQQGX₁₀VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:577), Middle X₁It is E or W；X₂It is V, W, L or Y；X₃It is L, P, F, T or H；X₄It is P, V or E；X₅It is A, S, V or G；X₆It is L, H, Q, G, V, A N, D, T or E；X₇It is T, F, Q, V or Y；X₈It is L, S, E, A or P；X₉It is E, D, T or N；And X₁₀It is W, Y, H or F.In certain realities It applies in scheme, the CH3 Domain Polypeptide of modification may include sequence: X₁WESX₂GX₃X₄WX₅X₆X₇(SEQ ID NO:575), Middle X₁It is E or W；X₂It is V, W, L or Y；X₃It is L, P, F, T or H；X₄It is P, V or E；X₅It is A, S, V or G；X₆Be L, H, Q, G, V, A, N, D, T or E；And X₇It is T, F, Q, V or Y.In certain embodiments, the CH3 Domain Polypeptide of modification may include sequence: X₁KSX₂WQQGX₃(SEQ ID NO:576), wherein X₈It is L, S, E, A or P；X₉It is E, D, T or N；And X₁₀It is W, Y, H or F.

In some embodiments, the CH3 Domain Polypeptide of the modification is included in 153 Glu or Trp；At 157 Trp, Leu or Tyr；In 159 Thr or His；In 160 Val；In 162 Ala, Ser or Val；At 163 Val, Asn or Thr；In 164 Gln or Tyr；In 186 Pro；In 189 Thr or Asn；With 194 Trp, Tyr, Phe or His, wherein the sequence that the substitution and the position are reference SEQ ID NO:9 determines.The CH3 structure of modification Domain polypeptide can have sequence: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVX₁WESX₂GX₃X₄WX₅X₆X₇K TTPPVLDSDGSFFLYSKLTVX₈KSX₉WQQGX₁₀VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:580), Middle X₁It is E or W；X₂It is W, L or Y；X₃It is T or H；X₄It is V；X₅It is A, S or V；X₆It is V, T or N；X₇It is Y or Q；X₈It is P；X₉It is T Or N；And X₁₀It is W, Y, H or F.In certain embodiments, the CH3 Domain Polypeptide of modification may include sequence: X₁WESX₂GX₃X₄WX₅X₆X₇(SEQ ID NO:578), wherein X₁It is E or W；X₂It is W, L or Y；X₃It is T or H；X₄It is V；X₅It is A, S Or V；X₆It is V, T or N；And X₇It is Y or Q.In certain embodiments, the CH3 Domain Polypeptide of modification may include sequence: X₁KSX₂WQQGX₃(SEQ ID NO:579), wherein X₁It is P；X₂It is T or N；And X₃It is W, Y, H or F.

In some embodiments, TfR combination polypeptide includes any of SEQ ID NO:116-130's Amino acid sequence.In other embodiments, TfR combination polypeptide includes any of SEQ ID NO:116-130 Amino acid sequence, but wherein have one or two amino acid be substituted.In some embodiments, the polypeptide includes SEQ The amino acid sequence of any of ID NO:116-130, but wherein there are three amino acid to be substituted.

In some embodiments, TfR combination polypeptide includes any of SEQ ID NO:131-139's Amino acid sequence.In other embodiments, TfR combination polypeptide includes any of SEQ ID NO:131-139 Amino acid sequence, but wherein have one or two amino acid be substituted.In some embodiments, the polypeptide includes SEQ The amino acid sequence of any of ID NO:131-139, but wherein there are three or four amino acid be substituted.

In some embodiments, TfR combination polypeptide includes any of SEQ ID NO:303-339's Amino acid sequence.In other embodiments, TfR combination polypeptide includes any of SEQ ID NO:303-339 Amino acid sequence, but wherein have one or two amino acid be substituted.In some embodiments, the polypeptide includes SEQ The amino acid sequence of any of ID NO:303-339, but wherein there are three amino acid to be substituted.

In some embodiments, TfR combination polypeptide includes in SEQ ID NO:136,138 and 340-345 The amino acid sequence of any one.In other embodiments, TfR combination polypeptide includes SEQ ID NO:136,138 With the amino acid sequence of any of 340-345, but wherein have one or two amino acid be substituted.In some embodiments In, the polypeptide includes the amino acid sequence of any of SEQ ID NO:136,138 and 340-345, but wherein there are three or Four amino acid are substituted.

In a further embodiment, transferrins combination polypeptide includes SEQ ID NO:4-29,236-299 and 422- Any of 435 amino acid 1 57-194, amino acid 1 53-194 or amino acid 1 53-199.In a further embodiment, institute Stating polypeptide includes amino acid 1 57-194 with any of SEQ ID NO:4-29,236-299 and 422-435, or with SEQ ID The amino acid 1 53-194 or amino acid 1 53-199 of any of NO:4-29,236-299 and 422-435 are same at least 75% The amino acid sequence of one property, at least 80% identity, at least 85% identity, at least 90% identity or at least 95% identity Column.

In some embodiments, the polypeptide includes any in SEQ ID NO:4-29,236-299 and 422-435 It is a.In a further embodiment, the polypeptide includes any of SEQ ID NO:4-29,236-299 and 422-435, But without first three amino acid " PCP " at amino terminal.In a further embodiment, the polypeptide can with such as in no amino Appointing in identified SEQ ID NO:4-29,236-299 and 422-435 in the case of first three amino acid " PCP " of end One has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity or at least 95% Identity.

CH3 TfR combination group (ii): 118,119,120,122,210,211,212 and 213

In some embodiments, the CH3 Domain Polypeptide of modification according to the present invention comprising 118,119,120, It 122, include at least three or at least four in 210,211,212 and 213 one group of amino acid position (ii group), and typically Five, six, seven or eight substitutions.The exemplary substitution that can be introduced at these locations is shown in table 5.In some realities It applies in scheme, the CH3 Domain Polypeptide of modification is included in 210 Gly；In 211 Phe；And/or in 213 Asp. In some embodiments, Glu is to be present in 213.In certain embodiments, the CH3 Domain Polypeptide of modification is included in At least one substitution at following position: in 118 Phe or Ile；In 119 Asp, Glu, Gly, Ala or Lys；In 120 Tyr, Met, Leu, Ile or Asp；In 122 Thr or Ala；In 210 Gly；In 211 Phe；212 His, Tyr, Ser or Phe of position；Or in 213 Asp.In some embodiments, position 118,119,120,122, 210, two, three, four, five, six, seven in 211,212 and 213 or all eight positions have such as institute in this section The substitution of explanation.In some embodiments, the CH3 Domain Polypeptide of modification can be in described group of one or more positions Conservative substitution comprising specific amino acids, for example, same electrification grouping, hydrophobicity grouping, side chain ring structure grouping (such as virtue Race's amino acid) or size packets and/or polarity or nonpolarity grouping in amino acid.

In some embodiments, the CH3 Domain Polypeptide and SEQ ID of the modification of TfR are specifically bound The amino acid 1 14-220 of any of NO:30-46 has at least 70% identity, at least 75% identity, at least 80% same Property, at least 85% identity, at least 90% identity or at least 95% identity.In some embodiments, such modification CH3 Domain Polypeptide includes the amino acid 1 18-122 and/or amino acid 210-213 of any of SEQ ID NO:30-46.

In some embodiments, the CH3 Domain Polypeptide of modification and the amino acid 1 14-220 of SEQ ID NO:1 have 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, restrictive condition be homogeneity percentage do not include described group of position 118,119,120,122,210, 211,212 and 213.In some embodiments, the CH3 Domain Polypeptide of the modification includes as in SEQ ID NO:30-46 The amino acid 1 18-122 and/or amino acid 210-213 stated in any one.

In some embodiments, TfR combination polypeptide includes any of SEQ ID NO:140-153's Amino acid sequence.In other embodiments, TfR combination polypeptide includes any of SEQ ID NO:140-153 Amino acid sequence, but wherein have one or two amino acid be substituted.

In some embodiments, TfR combination polypeptide includes any of SEQ ID NO:154-157's Amino acid sequence.In other embodiments, TfR combination polypeptide includes any of SEQ ID NO:154-157 Amino acid sequence, but wherein have an amino acid be substituted or in which there are two amino acid be substituted.

In a further embodiment, TfR combination polypeptide includes any of SEQ ID NO:30-46's Amino acid 1 18-213.In some embodiments, the polypeptide may include the ammonia with any of SEQ ID NO:30-46 Base acid 118-213 have at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity or At least amino acid sequence of 95% identity.

In some embodiments, the polypeptide includes any of SEQ ID NO:30-46.In other embodiment party In case, the polypeptide includes any of SEQ ID NO:30-46, but without first three amino acid " PCP " at amino terminal. In a further embodiment, the polypeptide can with any of SEQ ID NO:30-46 or with such as in no amino terminal Any of identified SEQ ID NO:30-46 is same at least 75% in the case of first three amino acid " PCP " at place Property, at least 80% identity, at least 85% identity, at least 90% identity or at least 95% identity.

CH2 TfR combination polypeptide

In some embodiments, the structural domain of modification is mankind's Ig CH2 structural domain, such as IgG CH2 structural domain.Institute Stating CH2 structural domain may belong to any IgG hypotype, that is, come from IgG1, IgG2, IgG3 or IgG4.In the case where IgG antibody, CH2 structural domain, which refers to, such as numbers according to EU numbering plan, the section for the Amino acid profile that You Congyue is 231 to about 340.For Identify the purpose of the corresponding amino acid position group combined for TfR, the position in CH2 structural domain is referring to SEQ ID NO:2 determining or referring to SEQ ID NO:1 amino acid 4-113 is determined.Replace and determined also with reference to SEQ ID NO:1, that is, Relative to the amino acid of the corresponding position in SEQ ID NO:1, amino acid to be replaced is considered.SEQ ID NO:1 includes portion Divide hinge legion sequence, i.e. PCP, as amino acid 1-3.Three residues are not a part in the area Fc；However, referring to SEQ ID The number of each position includes first three described amino acid in the CH2 structural domain that NO:1 is carried out.

As indicated above, the residue group that can be modified according to the present invention in CH2 structural domain is herein defined as referring to SEQ ID NO:1 number.Any CH2 structural domain, such as IgG1, IgG2, IgG3 or IgG4 CH2 structural domain can correspond to SEQ There is modification, such as amino acid substitution in one or more groups of residues of the residue at position described in ID NO:1.SEQ ID NO:1 1 amino acid sequence of human IgG and the comparison of human IgG 2, IgG3 and IgG4 be shown in Figure 38.Described IgG2, IgG3 and The position of any given position in each sequence in IgG4 sequence corresponding to SEQ ID NO:1 can readily determine that.

In one embodiment, the CH2 structural domain for specifically binding the modification of TfR, which is integrated to, turns iron egg Epitope in polymeric immunoglobulin receptor apical domains.Mankind's transferrins receptor apical domains are set forth in SEQ ID NO:107, right It should be in the amino acid 1 98-378 of mankind's transferrins receptor -1uniprotein sequence P02786.The CH2 structural domain of the modification Polypeptide can be incorporated into TfR, while not block or inhibiting in other ways the knot of transferrins Yu the receptor It closes.In some embodiments, the combination of transferrins and TfR are generally not inhibited.In some embodiments, for turning The inhibiting effect of the combination of ferritin and TfR below about 50% (for example, less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%).In some embodiments, the inhibiting effect of the combination of transferrins and TfR is below about 20% (for example, less than about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%).

CH2 TfR combination group (iii): 47,49,56,58,59,60,61,62 and 63

In some embodiments, the CH2 Domain Polypeptide of modification according to the present invention comprising 47,49,56,58,59, It 60, include at least three or at least four in 61,62 and 63 one group of amino acid position (iii group), and typically five, six A, seven, eight or nine substitutions.The exemplary substitution that can be introduced at these locations is shown in table 1.In some implementations In scheme, CH2 the Domain Polypeptide Glu included in 60 and/or the Trp at 61 of the modification.In some embodiments In, the CH2 Domain Polypeptide of modification includes at least one substitution at the following location: 47 Glu, Gly, Gln, Ser, Ala, Asn, Tyr or Trp；In 49 Ile, Val, Asp, Glu, Thr, Ala or Tyr；56 Asp, Pro, Met, Leu, Ala, Asn or Phe；In 58 Arg, Ser, Ala or Gly；In 59 Tyr, Trp, Arg or Val；At 60 Glu；In 61 Trp or Tyr；In 62 Gln, Tyr, His, Ile, Phe, Val or Asp；Or 63 Leu, Trp, Arg, Asn, Tyr or Val.In some embodiments, two in position 47,49,56,58,59,60,61,62 and 63, Three, four, five, six, seven, eight or all nine positions have the substitution as illustrated in this section.In some realities Apply in scheme, the CH2 Domain Polypeptide of modification can described group of one or more positions include specific amino acids guarantor Substitution is kept, such as in same electrification grouping, hydrophobicity grouping, side chain ring structure grouping (such as aromatic amino acid) or size point Amino acid in group and/or polarity or nonpolarity grouping.

In some embodiments, the CH2 Domain Polypeptide of the modification included in 47 Glu, Gly, Gln, Ser, Ala, Asn or Tyr；In 49 Ile, Val, Asp, Glu, Thr, Ala or Tyr；In 56 Asp, Pro, Met, Leu, Ala Or Asn；In 58 Arg, Ser or Ala；In 59 Tyr, Trp, Arg or Val；In 60 Glu；In 61 Trp； In 62 Gln, Tyr, His, Ile, Phe or Val；And/or in 63 Leu, Trp, Arg, Asn or Tyr.In some implementations In scheme, the CH2 Domain Polypeptide of the modification is included in 58 Arg；In 59 Tyr or Trp；In 60 Glu；In 61 Trp；And/or in 63 Arg or Trp.

In some embodiments, the CH2 Domain Polypeptide and SEQ ID of the modification of TfR are specifically bound The amino acid 4-113 of any of NO:47-62 has at least 70% identity, at least 75% identity, at least 80% same Property, at least 85% identity, at least 90% identity or at least 95% identity.In some embodiments, such modification CH2 Domain Polypeptide includes the amino acid 47-49 and/or amino acid 56-63 of any of SEQ ID NO:47-62.

In some embodiments, the amino acid 4- of the CH2 Domain Polypeptide of modification of the invention and SEQ ID NO:1 113 have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% together One property or at least 95% identity, restrictive condition be homogeneity percentage do not include described group of position 47,49,56,58,59, 60,61,62 and 63.In some embodiments, the CH2 Domain Polypeptide of the modification includes as in SEQ ID NO:47-62 The amino acid 47-49 and/or amino acid 56-63 stated in any one.

In some embodiments, TfR combination polypeptide includes any of SEQ ID NO:158-171's Amino acid sequence.In other embodiments, TfR combination polypeptide includes any of SEQ ID NO:158-171 Amino acid sequence, but wherein have an amino acid be substituted.

In some embodiments, TfR combination polypeptide includes any of SEQ ID NO:172-186's Amino acid sequence.In other embodiments, TfR combination polypeptide includes any of SEQ ID NO:172-186 Amino acid sequence, but wherein have an amino acid be substituted or in which there are two amino acid be substituted.In other embodiments In, TfR combination polypeptide includes the amino acid sequence of any of SEQ ID NO:172-186, but wherein there are three Or four amino acid are substituted.

In a further embodiment, TfR combination polypeptide includes any of SEQ ID NO:47-62's Amino acid 47-63.In some embodiments, the polypeptide may include the amino with any of SEQ ID NO:47-62 Sour 47-63 has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity or at least The amino acid sequence of 95% identity.

In some embodiments, the polypeptide includes any of SEQ ID NO:47-62.In other embodiment party In case, the polypeptide includes any of SEQ ID NO:47-62, but without first three amino acid " PCP " at amino terminal. In a further embodiment, the polypeptide can with any of SEQ ID NO:47-62 or with such as in no amino terminal Any of identified SEQ ID NO:47-62 is same at least 75% in the case of first three amino acid " PCP " at place Property, at least 80% identity, at least 85% identity, at least 90% identity or at least 95% identity.

CH2 TfR combination group (iv): 39,40,41,42,43,44,68,70,71 and 72

In some embodiments, the CH2 Domain Polypeptide of modification according to the present invention comprising 39,40,41,42,43, It 44, include at least three or at least four in 68,70,71 and 72 one group of amino acid position (iv group), and typically five, Six, seven, eight, nine or ten substitutions.The exemplary substitution that can be introduced at these locations is shown in table 2.In In some embodiments, the CH2 Domain Polypeptide of the modification is included in 43 Pro, in 68 Glu and/or at 70 Tyr.In some embodiments, the CH2 Domain Polypeptide of modification includes at least one substitution at the following location: 39 Pro, Phe, Ala, Met or Asp of position；In 40 Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp or Tyr；In 41 Thr, Ser, Gly, Met, Val, Phe, Trp or Leu；In 42 Pro, Val, Ala, Thr or Asp；At 43 Pro, Val or Phe；In 44 Trp, Gln, Thr or Glu；In 68 Glu, Val, Thr, Leu or Trp；At 70 Tyr, His, Val or Asp；In 71 Thr, His, Gln, Arg, Asn or Val；Or 72 Tyr, Asn, Asp, Ser or Pro.In some embodiments, two in position 39,40,41,42,43,44,68,70,71 and 72, three, four, Five, six, seven, eight, nine or all ten positions have the substitution as illustrated in this section.In some embodiments In, the CH2 Domain Polypeptide of modification can described group of one or more positions include specific amino acids conservative substitution, Such as in same electrification grouping, hydrophobicity grouping, side chain ring structure grouping (such as aromatic amino acid) or size packets, and/or Amino acid in polarity or nonpolarity grouping.

In some embodiments, the CH2 Domain Polypeptide of modification is included in 39 Pro, Phe or Ala；At 40 Gln, Pro, Arg, Lys, Ala or Ile；In 41 Thr, Ser, Gly, Met, Val, Phe or Trp；In 42 Pro, Val Or Ala；In 43 Pro；In 44 Trp or Gln；In 68 Glu；In 70 Tyr；In 71 Thr, His or Gln；And/or in 72 Tyr, Asn, Asp or Ser.

In some embodiments, the CH2 Domain Polypeptide of modification is included in 39 Met；In 40 Leu or Glu； In 41 Trp；In 42 Pro；In 43 Val；In 44 Thr；In 68 Val or Thr；In 70 His； In 71 His, Arg or Asn；And/or in 72 Pro.

In some embodiments, the CH2 Domain Polypeptide of modification is included in 39 Asp；In 40 Asp；41 The Leu of position；In 42 Thr；In 43 Phe；In 44 Gln；In 68 Val or Leu；In 70 Val；71 The Thr of position；And/or in 72 Pro.

In some embodiments, the CH2 Domain Polypeptide and SEQ ID of the modification of TfR are specifically bound The amino acid 4-113 of any of NO:63-85 has at least 70% identity, at least 75% identity, at least 80% same Property, at least 85% identity, at least 90% identity or at least 95% identity.In some embodiments, such modification CH2 Domain Polypeptide includes the amino acid 39-44 and/or amino acid 68-72 of any of SEQ ID NO:63-85.

In some embodiments, the amino acid 4- of the CH2 Domain Polypeptide of modification of the invention and SEQ ID NO:1 113 have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% together One property or at least 95% identity, restrictive condition be homogeneity percentage do not include described group of position 39,40,41,42,43, 44,68,70,71 and 72.In some embodiments, the CH2 Domain Polypeptide of the modification includes such as SEQ ID NO:63-85 Any of in the amino acid 39-44 and/or amino acid 68-72 that are stated.

In some embodiments, TfR combination polypeptide includes any of SEQ ID NO:187-204's Amino acid sequence.In other embodiments, TfR combination polypeptide includes any of SEQ ID NO:187-204 Amino acid sequence, but wherein have one or two amino acid be substituted.In other embodiments, TfR combines Polypeptide includes the amino acid sequence of any of SEQ ID NO:187-204, but wherein there are three amino acid to be substituted.

In some embodiments, TfR combination polypeptide includes any of SEQ ID NO:205-215's Amino acid sequence.In other embodiments, TfR combination polypeptide includes any of SEQ ID NO:205-215 Amino acid sequence, but wherein have an amino acid be substituted or in which there are two amino acid be substituted.

In a further embodiment, TfR combination polypeptide includes any of SEQ ID NO:63-85's Amino acid 39-72.In some embodiments, the polypeptide includes the amino acid 39- with any of SEQ ID NO:63-85 72 have at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity or at least 95% same The amino acid sequence of one property.

In some embodiments, the polypeptide includes any of SEQ ID NO:63-85.In other embodiment party In case, the polypeptide includes any of SEQ ID NO:63-85, but without first three amino acid " PCP " at amino terminal. In a further embodiment, the polypeptide can with any of SEQ ID NO:63-85 or with such as in no amino terminal Any of identified SEQ ID NO:63-85 is same at least 75% in the case of first three amino acid " PCP " at place Property, at least 80% identity, at least 85% identity, at least 90% identity or at least 95% identity.

CH2 TfR combination group (v): 41,42,43,44,45,65,66,67,69 and 73

In some embodiments, the CH2 Domain Polypeptide of modification according to the present invention comprising 41,42,43,44,45, It 65, include at least three or at least four in 66,67,69 and 73 one group of amino acid position (v group), and typically five, Six, seven, eight, nine or ten substitutions.The exemplary substitution that can be introduced at these locations is shown in table 3.In In some embodiments, the CH2 Domain Polypeptide of modification includes at least one substitution at the following location: in 41 Val Or Asp；In 42 Pro, Met or Asp；In 43 Pro or Trp；In 44 Arg, Trp, Glu or Thr；At 45 Met, Tyr or Trp；In 65 Leu or Trp；In 66 Thr, Val, Ile or Lys；In 67 Ser, Lys, Ala or Leu；In 69 His, Leu or Pro；Or in 73 Val or Trp.In some embodiments, position 41,42,43, 44, two, three, four, five, six, seven, eight, nine in 45,65,66,67,69 and 73 or all ten positions It sets with the substitution as illustrated in this section.In some embodiments, the CH2 Domain Polypeptide of modification can be at described group One or more positions include the conservative substitution of specific amino acids, such as in same electrification grouping, hydrophobicity grouping, side links Structure is grouped the amino acid in (such as aromatic amino acid) or size packets and/or polarity or nonpolarity grouping.

In some embodiments, the CH2 Domain Polypeptide of modification is included in 41 Val；In 42 Pro；43 The Pro of position；In 44 Arg or Trp；In 45 Met；In 65 Leu；In 66 Thr；In 67 Ser；69 The His of position；And/or in 73 Val.

In some embodiments, the CH2 Domain Polypeptide of modification is included in 41 Asp；In 42 Met or Asp； In 43 Trp；In 44 Glu or Thr；In 45 Tyr or Trp；In 65 Trp；In 66 Val, Ile or Lys；In 67 Lys, Ala or Leu；In 69 Leu or Pro；And/or in 73 Trp.

In some embodiments, the CH2 Domain Polypeptide and SEQ ID of the modification of TfR are specifically bound The amino acid 4-113 of any of NO:86-90 has at least 70% identity, at least 75% identity, at least 80% same Property, at least 85% identity, at least 90% identity or at least 95% identity.In some embodiments, such modification CH3 Domain Polypeptide includes the amino acid 41-45 and/or amino acid 65-73 of any of SEQ ID NO:86-90.

In some embodiments, the amino acid 4- of the CH2 Domain Polypeptide of modification of the invention and SEQ ID NO:1 113 have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% together One property or at least 95% identity, restrictive condition be homogeneity percentage do not include described group of position 41,42,43,44,45, 65,66,67,69 and 73.In some embodiments, the CH2 Domain Polypeptide of the modification includes such as SEQ ID NO:86-90 Any of in the amino acid 41-45 and/or amino acid 65-73 that are stated.

In some embodiments, TfR combination polypeptide includes any of SEQ ID NO:216-220's Amino acid sequence.In other embodiments, TfR combination polypeptide includes any of SEQ ID NO:216-220 Amino acid sequence, but wherein have one or two amino acid be substituted.

In some embodiments, TfR combination polypeptide includes any of SEQ ID NO:221-224's Amino acid sequence.In other embodiments, TfR combination polypeptide includes any of SEQ ID NO:221-224 Amino acid sequence, but wherein have an amino acid be substituted or in which there are two amino acid be substituted.In other embodiments In, TfR combination polypeptide includes the amino acid sequence of any of SEQ ID NO:221-224, but wherein there are three Or four amino acid are substituted.

In a further embodiment, TfR combination polypeptide includes any of SEQ ID NO:86-90's Amino acid 41-73.In a further embodiment, the polypeptide may include the ammonia with any of SEQ ID NO:86-90 Base acid 41-73 has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity or extremely The amino acid sequence of few 95% identity.

In some embodiments, the polypeptide includes any of SEQ ID NO:86-90.In other embodiment party In case, the polypeptide includes any of SEQ ID NO:86-90, but without first three amino acid " PCP " at amino terminal. In a further embodiment, the polypeptide can with any of SEQ ID NO:86-90 or with such as in no amino terminal Any of identified SEQ ID NO:86-90 is same at least 75% in the case of first three amino acid " PCP " at place Property, at least 80% identity, at least 85% identity, at least 90% identity or at least 95% identity.

CH2 TfR combination group (vi): 45,47,49,95,97,99,102,103 and 104

In some embodiments, the CH2 Domain Polypeptide of modification according to the present invention comprising 45,47,49,95,97, It 99, include at least three or at least four in 102,103 and 104 one group of amino acid position (vi group), and typically five, Six, seven, eight or nine substitutions.The exemplary substitution that can be introduced at these locations is shown in table 4.In some realities It applies in scheme, the CH2 Domain Polypeptide of modification includes Trp at 103.In some embodiments, the CH2 structural domain of modification is more Peptide includes at least one substitution at the following location: in 45 Trp, Val, Ile or Ala；In 47 Trp or Gly；In 49 Tyr, Arg or Glu；In 95 Ser, Arg or Gln；In 97 Val, Ser or Phe；In 99 Ile, Ser Or Trp；In 102 Trp, Thr, Ser, Arg or Asp；In 103 Trp；Or in 104 Ser, Lys, Arg or Val. In some embodiments, two in position 45,47,49,95,97,99,102,103 and 104, three, four, five, Six, seven, eight or all nine positions have the substitution as illustrated in this section.In some embodiments, modification CH2 Domain Polypeptide can described group of one or more positions include specific amino acids conservative substitution, such as same One electrification grouping, hydrophobicity grouping, side chain ring structure grouping (such as aromatic amino acid) or size packets and/or polarity or non- Amino acid in polarity grouping.

In some embodiments, the CH2 Domain Polypeptide of modification includes two selected from the following, three, four, five A, six, seven, eight or nine positions: 45 are Trp, Val, Ile or Ala；47 are Trp or Gly；49 be Tyr, Arg or Glu；95 are Ser, Arg or Gln；97 are Val, Ser or Phe；99 are Ile, Ser or Trp；102 be Trp, Thr, Ser, Arg or Asp；103 are Trp；It is Ser, Lys, Arg or Val with 104.

In some embodiments, the CH2 Domain Polypeptide of modification is included in 45 Val or Ile；In 47 Gly； In 49 Arg；In 95 Arg；In 97 Ser；In 99 Ser；In 102 Thr, Ser or Arg；At 103 Trp；And/or in 104 Lys or Arg.

In some embodiments, the CH2 Domain Polypeptide and SEQ ID of the modification of TfR are specifically bound The amino acid 4-113 of any of NO:91-95 has at least 70% identity, at least 75% identity, at least 80% same Property, at least 85% identity, at least 90% identity or at least 95% identity.In some embodiments, such modification CH3 Domain Polypeptide includes the amino acid 45-49 and/or amino acid 95-104 of any of SEQ ID NO:91-95.

In some embodiments, the amino acid 4- of the CH2 Domain Polypeptide of modification of the invention and SEQ ID NO:1 113 have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% together One property or at least 95% identity, restrictive condition be homogeneity percentage do not include described group of position 45,47,49,95,97, 99,102,103 and 104.In some embodiments, the CH2 Domain Polypeptide of the modification includes such as SEQ ID NO:91-95 Any of in the amino acid 45-49 and/or amino acid 95-104 that are stated.

In some embodiments, TfR combination polypeptide includes any of SEQ ID NO:225-228's Amino acid sequence.In other embodiments, TfR combination polypeptide includes any of SEQ ID NO:225-228 Amino acid sequence, but wherein have one or two amino acid be substituted.

In some embodiments, TfR combination polypeptide includes any of SEQ ID NO:229-233's Amino acid sequence.In other embodiments, TfR combination polypeptide includes any of SEQ ID NO:229-223 Amino acid sequence, but wherein have an amino acid be substituted or in which there are two amino acid be substituted.In other embodiments In, TfR combination polypeptide includes the amino acid sequence of any of SEQ ID NO:229-233, but wherein has three A, four or five amino acid are substituted.

In a further embodiment, TfR combination polypeptide includes any of SEQ ID NO:91-95's Amino acid 45-104.In a further embodiment, the polypeptide can be with the amino acid of any of SEQ ID NO:91-95 45-104 has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity or at least 95% identity.

In some embodiments, the polypeptide includes any of SEQ ID NO:91-95.In other embodiment party In case, the polypeptide includes any of SEQ ID NO:91-95, but without first three amino acid " PCP " at amino terminal. In a further embodiment, the polypeptide can with any of SEQ ID NO:91-95 or with such as in no amino terminal Any of identified SEQ ID NO:91-95 is same at least 75% in the case of first three amino acid " PCP " at place Property, at least 80% identity, at least 85% identity, at least 90% identity or at least 95% identity.

The Exemplary polypeptide of CH3 or CH2 structural domain comprising modification

CH3 the or CH2 Domain Polypeptide of modification of the invention can be joined to another structural domain in the area Fc.In some implementations In scheme, the CH3 Domain Polypeptide of modification of the invention is joined to CH2 structural domain, and the CH2 structural domain can be naturally occurring CH2 structural domain or variant CH2 structural domain, typically at the C-terminal of the CH2 structural domain.In some embodiments, originally The CH2 Domain Polypeptide of the modification of invention is joined to CH3 structural domain, and the CH3 structural domain can be naturally occurring CH3 structure Domain or CH3 variant structure domain, typically at the N-terminal of the CH3 structural domain.In some embodiments, comprising modification CH2 structural domain is joined to the polypeptide of CH3 structural domain, or the CH3 structural domain comprising modification is joined to the polypeptide of CH2 structural domain and also wraps Thus partial or complete hinge area containing antibody generates the CH3 Domain Polypeptide of modification or the CH2 Domain Polypeptide conduct of modification The form of a part in the area Fc with partial or complete hinge area.The hinge area can come from any immunoglobulin subclass Or homotype.Exemplary immunoglobulin hinge is IgG hinge area, such as IgG1 hinge area, such as 1 hinge area amino of human IgG Acid sequence EPKSCDKTHTCPPCP (SEQ ID NO:234).It in a further embodiment, can be in contain hinge area or portion Divide the polypeptide of the Fc form of hinge area to be further joined to another part, such as Fab segment, thus generates TfR knot Close Fc-Fab fusions.In some embodiments, the TfR combination Fc-Fab fusions include the CH3 of modification Domain Polypeptide or the CH2 Domain Polypeptide of modification, hinge area and Fab segment.Fab segment can be directed to any target of interest, Such as treatment nerve target passes through the CH3 Domain Polypeptide of modification or the CH2 Domain Polypeptide knot of modification in this case Fab is delivered to target by close that TfR mediated wear born of the same parents across blood-brain barrier and transport.

In some embodiments, the Fab segment for being joined to TfR combination polypeptide can be incorporated into tau protein (such as mankind's tau protein) or its segment.In some embodiments, Fab segment can be incorporated into phosphorylation tau protein, non-phosphoric acid The tau protein of change, the truncated tau protein of montage isoform, N-terminal of tau protein, the truncated tau protein of C-terminal and/or its segment.

In some embodiments, the Fab segment for being joined to TfR combination polypeptide can be incorporated into beta-secretase Enzyme 1 (BACE1) albumen (such as mankind BACE1 albumen) or its segment.In some embodiments, Fab segment can be incorporated into The one or more isoforms or its segment of BACE1 albumen.

In some embodiments, being joined to the Fab segment of TfR combination polypeptide, to can be incorporated into marrow thin Triggering receptor 2 (TREM2) albumen (such as mankind TREM2 albumen) expressed on born of the same parents or its segment.

In some embodiments, the Fab segment for being joined to TfR combination polypeptide can be incorporated into alpha-synapse Nucleoprotein (such as mankind's alpha-synapse nucleoprotein) or its segment.In some embodiments, Fab segment can be incorporated into monomer α- Synapse nucleoprotein, oligomeric alpha-synapse nucleoprotein, alpha-synapse nucleoprotein fibrinogen, soluble alpha-synapse nucleoprotein and/or its segment.

In some embodiments, the Fc-Fab fusions of the CH2 or CH3 Domain Polypeptide comprising modification of the invention are The sub-cell of dimer.In some embodiments, dimer is heterodimer.In some embodiments, dimer is same Dimer.In some embodiments, dimer includes the single polypeptide for being integrated to TfR, that is, for turning iron egg Polymeric immunoglobulin receptor is combined into unit price.In some embodiments, dimer includes the second polypeptide for being integrated to TfR.Institute State the second polypeptide may include identical modification present in the Fc-Fab fusions CH3 Domain Polypeptide (or modification CH2 Domain Polypeptide) with provide divalent combination homodimer or of the invention second modification CH3 Domain Polypeptide (or modification CH2 Domain Polypeptide) the second TfR binding site can be provided.In some embodiments, dimer includes containing First sub-cell of the CH2 Domain Polypeptide of the CH3 Domain Polypeptide or modification of modification, and the structural domain containing CH2 and CH3 second Sub-cell, CH2 the and CH3 structural domain do not combine TfR.

TfR combination polypeptide can also peptide fusions of interest different from addition to Fab.For example, some In embodiment, TfR combination polypeptide can be targeting transferrins from different peptide fusions, the not homopolypeptide Receptor-expressing cells are transported by wearing born of the same parents across needed for endothelium, such as blood-brain barrier delivering.In some embodiments, turn iron Protein receptor binding polypeptide and soluble protein, such as the extracellular domain of receptor or growth factor, cell factor or enzyme Fusion.

In other embodiments, TfR combination polypeptide can be with the peptide or egg that can be used for protein purification again White matter (such as polyhistidyl), epitope tag (such as FLAG, c-Myc, Hemagluttinin tags and analog), glutathione S transfer Enzyme (GST), thioredoxin, a-protein, protein G or maltose-binding protein (MBP) fusion.In some cases, with turn The peptide or protein matter of Human Placental Ferritin Receptor combination peptide fusion may include protease cracking site, and such as factor Xa or fibrin ferment are split Solve site.

TfR combination polypeptide of the invention can have the combination of wider range for example based on the form of polypeptide Affinity.For example, in some embodiments, the polypeptide pair of the CH3 structural domain comprising modification or the CH2 structural domain of modification The binding affinity of TfR is in the range of 1pM to 10 μM.In some embodiments, affinity can be by unit price Form measurement.In other embodiments, affinity can be by bivalent form, such as with two comprising polypeptide-Fab fusion protein Dimer form measurement.

Method for measuring binding affinity, binding kinetics and cross reactivity is known in technique.This A little methods include but is not limited to solid phase binding assay (such as ELISA measurement), immuno-precipitation, surface plasmon resonance (such as Biacore^TM(GE Healthcare, Piscataway, NJ)), dynamics exclude measurement (such as), stream Formula cell measurement art, fluorescence activated cell sorts (FACS), biological membranous layer interferometry (such as(FortéBio,Inc., Menlo Park, CA)) and Western measurement.In some embodiments, binding affinity and/or friendship are determined using ELISA Fork reactivity.For carry out ELISA method for measuring be in technique it is known, and in following embodiment partially also Description.In some embodiments, using surface plasmon resonance (SPR) determine binding affinity, binding kinetics and/ Or cross reactivity.In some embodiments, using dynamics exclude measure determine binding affinity, binding kinetics and/ Or cross reactivity.In some embodiments, interfere to measure using biological membranous layer and determine binding affinity, binding kinetics And/or cross reactivity.

Other mutation in the area Fc of CH3 or CH2 Domain Polypeptide comprising modification

It is provided in this article to be modified to combine TfR and starting also to may include in addition across the polypeptide that BBB is transported Mutation, such as to increase serum stability, mediating effect+6 function, influence glycosylation, reduce immunogenicity in human body, And/or provide the section and hole heterodimerization of polypeptide.

In some embodiments, polypeptide according to the present invention and the area corresponding wild type Fc (such as human IgG 1, The area IgG2, IgG3 or IgG4 Fc) have 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.

Polypeptide according to the present invention can also introduce other mutation outside designated amino acid group range, such as to influence glycosyl Change, increase serum half-life, or for CH3 structural domain, provides the different dimerization of section and hole of the polypeptide of the CH3 structural domain comprising modification Change.In general, the method is related to being introduced into protrusion (" section ") in the interface of the first polypeptide and draw in the second polypeptide interface Enter respective cavities (" hole "), thus the protrusion can be placed in cavity to promote heterodimer to be formed and hinder homodimer It is formed.Protrusion is the p1 amino acid by substituting the interface from the first polypeptide with larger side chain (such as tyrosine or tryptophan) Side chain constructs.By being substituted with compared with small amino acid side chains (such as alanine or threonine) compared with big amino acid side chains, second The compensatory cavity of size same or like with the protrusion is generated in the interface of polypeptide.Such other mutation is in polypeptide Do not have the position of negative effect to the combination of the CH3 or CH2 structural domain and TfR of modification.

In an exemplary implementation scheme of section and hole dimerization method, in the first Fc polypeptide sub-cell of dimerization There is 139 positions corresponding to SEQ ID NO:1 tryptophan to substitute natural threonine, and the 2nd Fc polypeptide of dimer At 180 corresponding to SEQ ID NO:1 there is valine to substitute native tyrosine.Second sub-cell of Fc polypeptide can be also Comprising replacing, wherein the natural threonine serine at 139 positions corresponding to SEQ ID NO:1 replaces and right It should replace in the natural leucine at the position of 141 of SEQ ID NO:1 through alanine.

Polypeptide described herein can also be by engineered at the other modifications contained for heterodimerization, such as CH3- The electrostatic of contact residues is engineered in the interface CH3, and the contact residues are natively electrification or hydrophobic patch modification.

In some embodiments, the modification for increasing serum half-life can be introduced.For example, in some embodiments In, the area Fc includes at 25 positions corresponding to SEQ ID NO:1 comprising Tyr, in 27 corresponding to SEQ ID NO:1 Position at comprising Thr and corresponding to SEQ ID NO:1 29 positions at include Glu CH2 structural domain.

In some embodiments, as determined referring to SEQ ID NO:1, in position 17-30,52-57,80-90,156- Mutation is introduced at one or more positions in 163 and 201-208, such as is replaced.In some embodiments, as referring to SEQ ID NO:1 determined, position 24,25,27,28,29,80,81,82,84,85,87,158,159,160,162,201,206, One or more mutation are introduced at 207 or 209.In some embodiments, as determined referring to SEQ ID NO:1,25, Mutation is introduced in one, two or three in 27 and 29.In some embodiments, as referring to SEQ ID NO:1 institute Number, the mutation is M25Y, S27T and T29E.In some embodiments, polypeptide as described herein also include mutation M25Y, S27T and T29E.In some embodiments, as determined referring to SEQ ID NO:1, one in 201 and 207 or Mutation is introduced in two.In some embodiments, as numbered referring to SEQ ID NO:1, it is described mutation be M201L and N207S.In some embodiments, polypeptide as described herein also includes N207S and presence or absence of M201L.In some realities It applies in scheme, as numbered referring to SEQ ID NO:1, polypeptide as described herein one in position T80, E153 and N207, Comprising replacing at two or all three.In some embodiments, mutation is T80Q and N207A.In some embodiments, Polypeptide as described herein includes mutation T 80A, E153A and N207A.In some embodiments, as referring to SEQ ID NO:1 institute Number, polypeptide as described herein is at position T23 and M201 comprising replacing.In some embodiments, polypeptide as described herein Include mutation T 23Q and M201L.In some embodiments, as numbered referring to SEQ ID NO:1, polypeptide as described herein exists Comprising replacing at position M201 and N207.In some embodiments, polypeptide as described herein includes to replace M201L and N207S. In some embodiments, polypeptide as described herein includes that N207S or N207A replaces.

Fc effector function

In some embodiments, the area Fc of the CH2 or CH3 structural domain comprising modification has effector function, that is, it can Certain biological functions are induced in the Fc receptor expressed on being integrated to the effector cell for mediating effector function.Effector cell include but It is not limited to, monocyte, macrophage, neutrophil(e) cell, Dendritic Cells, thermophilic Yihong haemocyte, mast cell, blood platelet, B Cell, large granular lymphocyte, Langerhans' cells (Langerhans'cell), natural killer (NK) cell and cytotoxic T Cell.

The example of antibody mediated effect function includes but is not limited to, C1q is combined and complement-dependent cytotoxicity (CDC), Fc by Body combination, the phagocytosis (ADCP) of the cytotoxicity (ADCC) of antibody dependent cellular mediation, antibody dependent cellular mediation, The B cell that reconciles under cell surface receptor (such as B-cell receptor) activates.Effector function can change with antibody isotype.Citing For, natural mankind IgG1 and IgG3 antibody can cause when being integrated to appropriate Fc receptor present on immune system cell ADCC and CDC activity；And the natural mankind IgG1, IgG2, IgG3 and IgG4 are being integrated to appropriate Fc present on immunocyte It can cause ADCP function when receptor.

In some embodiments, polypeptide as described herein may include the other modification of mediating effect+6 function.Alternatively, In some embodiments, the polypeptide of the CH2 or CH3 structural domain comprising modification of the invention may include enhancement effect function In addition modification.

The exemplary Fc polypeptide mutation of mediating effect+6 function includes but is not limited to, in CH2 structural domain, such as corresponding to Substitution at 7 and 8 of SEQ ID NO:1.In some embodiments, the substitution in the CH2 structural domain of the modification The Ala at 7 and 8 of SEQ ID NO:1.In some embodiments, taking in the CH2 structural domain of the modification The generation Ala and Gly at 102 at 7 and 8 of SEQ ID NO:1.

The other Fc polypeptide mutation of mediating effect+6 function includes but is not limited to, position 238,265,269,270,297, 327 and 329 (EU numbering plans, corresponding to 11,38,42,43,70,100 and of position such as numbered referring to SEQ ID NO:1 102) the one or more of place replace.It is exemplary to replace and (such as numbered according to EU numbering plan) include the following: 329 can have The mutation that proline is replaced by glycine or arginine or the sufficiently large amino acid residue to destroy Fc/Fc γ acceptor interface, institute State Fc/Fc γ acceptor interface be formed at Fc proline 3 29 and Fc γ RIII trp residue Trp 87 and Trp 110 it Between.In addition exemplary substitution includes S228P, E233P, L235E, N297A, N297D and P331S.It is taken there is likely to be multiple Generation, such as the L234A and L235A in 1 area Fc of human IgG；L234A, L235A and the P329G in the area human IgG 1Fc；Human IgG 4 The S228P and L235E in the area Fc；The L234A and G237A in 1 area Fc of human IgG；L234A, the L235A in 1 area Fc of human IgG and G237A；The V234A and G237A in 2 area Fc of human IgG；L235A, G237A and the E318A in 4 area Fc of human IgG；And the mankind The S228P and L236E in the area IgG4 Fc.In some embodiments, polypeptide of the invention can have one or more adjustings The amino acid substitution of ADCC, such as the substitution according to EU numbering plan, at position 298,333 and/or 334.

In some embodiments, polypeptide as described herein can have one or more amino increased or weaken ADCC Acid, which replaces or can have, changes C1q combination and/or the mutation of CDC.

Exemplary polypeptide comprising other mutation

Polypeptide as described herein (such as clone 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.23.2.1 Any of with CH3C.35.23.1.1) it may include other mutation, including section mutation (for example, as referring to SEQ ID NO:1 number T139W), hole mutation (for example, as referring to SEQ ID NO:1 number T139S, L141A and Y180V), adjust The mutation (for example, such as L7A, L8A and/or P102G (such as L7A and L8A) referring to SEQ ID NO:1 number) of effector function, And/or increase serum stability mutation (for example, (i) as referring to SEQ ID NO:1 number M25Y, S27T and T29E, or (ii) such as the N207S referring to SEQ ID NO:1 number and presence or absence of M201L).

In some embodiments, polypeptide as described herein (such as clone 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、 Any of CH3C.35.20.1.1, CH3C.23.2.1 and CH3C.35.23.1.1) it can have section mutation (for example, as joined According to the T139W of SEQ ID NO:1 number) and have with the sequence of any of SEQ ID NO:4-95,236-299 and 422-435 There are at least 85% identity, at least 90% identity or at least 95% identity.In some embodiments, there is SEQ ID The polypeptide of the sequence of any of NO:4-95,236-299 and 422-435 can be modified to have section mutation.

In some embodiments, polypeptide as described herein (such as clone 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、 Any of CH3C.35.20.1.1, CH3C.23.2.1 and CH3C.35.23.1.1) it can have section mutation (for example, as joined According to the T139W of SEQ ID NO:1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and sequence with any of SEQ ID NO:4-95,236-299 and 422-435 With at least 85% identity, at least 90% identity or at least 95% identity.In some embodiments, there is SEQ ID The polypeptide of the sequence of any of NO:4-95,236-299 and 422-435 can be modified to have section mutation and mediating effect+6 function The mutation of energy.

In some embodiments, polypeptide as described herein (such as clone 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、 Any of CH3C.35.20.1.1, CH3C.23.2.1 and CH3C.35.23.1.1) it can have section mutation (for example, as joined According to SEQ ID NO:1 number T139W), increase serum stability mutation (for example, (i) as referring to SEQ ID NO:1 number M25Y, S27T and T29E, or (ii) as referring to SEQ ID NO:1 number N207S and presence or absence of M201L) and with The sequence of any of SEQ ID NO:4-95,236-299 and 422-435 have at least 85% identity, at least 90% same Property or at least 95% identity.In some embodiments, have any in SEQ ID NO:4-95,236-299 and 422-435 The polypeptide of a sequence can be modified to section mutation and increase the mutation of serum stability.

In some embodiments, polypeptide as described herein (such as clone 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、 Any of CH3C.35.20.1.1, CH3C.23.2.1 and CH3C.35.23.1.1) it can have section mutation (for example, as joined According to the T139W of SEQ ID NO:1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, (i) as referring to SEQ ID NO:1 compile Number M25Y, S27T and T29E, or (ii) as referring to SEQ ID NO:1 number N207S and presence or absence of M201L) and There is at least 85% identity, at least 90% together with the sequence of any of SEQ ID NO:4-95,236-299 and 422-435 One property or at least 95% identity.In some embodiments, have in SEQ ID NO:4-95,236-299 and 422-435 and appoint The polypeptide of one sequence can be modified to section mutation, the mutation of mediating effect+6 function and increase the prominent of serum stability Become.

In some embodiments, polypeptide as described herein (such as clone 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、 Any of CH3C.35.20.1.1, CH3C.23.2.1 and CH3C.35.23.1.1) it can have hole mutation (for example, as joined According to T139S, L141A and Y180V of SEQ ID NO:1 number) and appoint with SEQ ID NO:4-95,236-299 and 422-435 One sequence has at least 85% identity, at least 90% identity or at least 95% identity.In some embodiments, The polypeptide of sequence with any of SEQ ID NO:4-95,236-299 and 422-435 can be modified to hole mutation.

In some embodiments, polypeptide as described herein (such as clone 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、 Any of CH3C.35.20.1.1, CH3C.23.2.1 and CH3C.35.23.1.1) it can have hole mutation (for example, as joined According to T139S, L141A and Y180V of SEQ ID NO:1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and with SEQ ID NO:4-95,236-299 and 422-435 Any of sequence have at least 85% identity, at least 90% identity or at least 95% identity.In some embodiment party In case, the polypeptide of the sequence with any of SEQ ID NO:4-95,236-299 and 422-435 can be modified to hole The mutation of mutation and mediating effect+6 function.

In some embodiments, polypeptide as described herein (such as clone 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、 Any of CH3C.35.20.1.1, CH3C.23.2.1 and CH3C.35.23.1.1) it can have hole mutation (for example, as joined According to T139S, L141A and Y180V of SEQ ID NO:1 number), increase the mutation of serum stability (for example, (i) as referring to SEQ M25Y, S27T and T29E of ID NO:1 number, or (ii) such as the N207S referring to SEQ ID NO:1 number and existence or non-existence M201L) and with the sequence of any of SEQ ID NO:4-95,236-299 and 422-435 there is at least 85% identity, extremely Few 90% identity or at least 95% identity.In some embodiments, have SEQ ID NO:4-95,236-299 and The polypeptide of the sequence of any of 422-435 can be modified to that there is hole to be mutated and increase the mutation of serum stability.

In some embodiments, polypeptide as described herein (such as clone 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、 Any of CH3C.35.20.1.1, CH3C.23.2.1 and CH3C.35.23.1.1) it can have hole mutation (for example, as joined According to T139S, L141A and Y180V of SEQ ID NO:1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, (i) as join According to SEQ ID NO:1 number M25Y, S27T and T29E, or (ii) as referring to SEQ ID NO:1 number N207S and exist or There is no M201L) and it is same at least 85% with the sequence of any of SEQ ID NO:4-95,236-299 and 422-435 Property, at least 90% identity or at least 95% identity.In some embodiments, there is SEQ ID NO:4-95,236-299 It with the polypeptide of the sequence of any of 422-435 can be modified to that there is hole mutation, the mutation of mediating effect+6 function and increase blood The mutation of clear stability.

Clone CH3C.35.20.1

In some embodiments, clone CH3C.35.20.1 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number) and there is at least 85% identity, at least 90% identity or at least with the sequence of SEQ ID NO:349 95% identity.In some embodiments, there is the sequence of SEQ ID NO:349 with the clone CH3C.35.20.1 of section mutation Column.

In some embodiments, clone CH3C.35.20.1 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and there is at least 85% identity, at least 90% together with the sequence of SEQ ID NO:350 or 351 One property or at least 95% identity.In some embodiments, there is the clone of section mutation and the mutation of mediating effect+6 function CH3C.35.20.1 has the sequence of SEQ ID NO:350 or 351.

In some embodiments, clone CH3C.35.20.1 can have section mutation (for example, as referring to SEQ ID NO: 1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:352 have at least 85% identity, at least 90% identity or at least 95% same Property.In some embodiments, the clone CH3C.35.20.1 with section mutation and the mutation for increasing serum stability has SEQ The sequence of ID NO:352.

In some embodiments, clone CH3C.35.20.1 can have section mutation (for example, as referring to SEQ ID NO: 1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and exist or There is no M201L) and there is at least 85% identity, at least 90% identity or at least with the sequence of SEQ ID NO:485 95% identity.In some embodiments, there is section mutation and increase the clone of the mutation of serum stability CH3C.35.20.1 has the sequence of SEQ ID NO:485.

In some embodiments, clone CH3C.35.20.1 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:353 or 354 Few 95% identity.In some embodiments, there is section mutation, the mutation of mediating effect+6 function and increase serum stability The clone CH3C.35.20.1 of mutation has the sequence of SEQ ID NO:353 or 354.

In some embodiments, clone CH3C.35.20.1 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and Presence or absence of M201L) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:486 or 487 Property or at least 95% identity.In some embodiments, have section mutation, the mutation of mediating effect+6 function and increase serum steady The clone CH3C.35.20.1 being qualitatively mutated has the sequence of SEQ ID NO:486 or 487.

In some embodiments, clone CH3C.35.20.1 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number) and there is at least 85% identity, at least 90% with the sequence of SEQ ID NO:355 Identity or at least 95% identity.In some embodiments, the clone CH3C.35.20.1 with hole mutation has SEQ The sequence of ID NO:355.

In some embodiments, clone CH3C.35.20.1 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and it is same at least 85% with the sequence of SEQ ID NO:356 or 357 Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation and mediating effect+6 function The clone CH3C.35.20.1 of mutation has the sequence of SEQ ID NO:356 or 357.

In some embodiments, clone CH3C.35.20.1 can have hole mutation (for example, as referring to SEQ ID NO: 1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:358 Few 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.20.1 has the sequence of SEQ ID NO:358.

In some embodiments, clone CH3C.35.20.1 can have hole mutation (for example, as referring to SEQ ID NO: 1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence or absence of M201L) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:488 One property or at least 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.20.1 has the sequence of SEQ ID NO:488.

In some embodiments, clone CH3C.35.20.1 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 M25Y, S27T and T29E of number) and there is at least 85% identity, at least 90% with the sequence of SEQ ID NO:359 or 360 Identity or at least 95% identity.In some embodiments, with hole mutation, the mutation of mediating effect+6 function and increase blood The clone CH3C.35.20.1 of the mutation of clear stability has the sequence of SEQ ID NO:359 or 360.

In some embodiments, clone CH3C.35.20.1 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 The N207S of number and presence or absence of M201L) and have with the sequence of SEQ ID NO:489 or 490 at least 85% same Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation, mediating effect+6 function The clone CH3C.35.20.1 of mutation and the mutation of increase serum stability has the sequence of SEQ ID NO:489 or 490.

Clone CH3C.35.23.2

In some embodiments, clone CH3C.35.23.2 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number) and there is at least 85% identity, at least 90% identity or at least with the sequence of SEQ ID NO:361 95% identity.In some embodiments, there is the sequence of SEQ ID NO:361 with the clone CH3C.35.23.2 of section mutation Column.

In some embodiments, clone CH3C.35.23.2 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and there is at least 85% identity, at least 90% together with the sequence of SEQ ID NO:362 or 363 One property or at least 95% identity.In some embodiments, there is the clone of section mutation and the mutation of mediating effect+6 function CH3C.35.23.2 has the sequence of SEQ ID NO:362 or 363.

In some embodiments, clone CH3C.35.23.2 can have section mutation (for example, as referring to SEQ ID NO: 1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:364 have at least 85% identity, at least 90% identity or at least 95% same Property.In some embodiments, the clone CH3C.35.23.2 with section mutation and the mutation for increasing serum stability has SEQ The sequence of ID NO:364.

In some embodiments, clone CH3C.35.23.2 can have section mutation (for example, as referring to SEQ ID NO: 1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and exist or There is no M201L) and there is at least 85% identity, at least 90% identity or at least with the sequence of SEQ ID NO:492 95% identity.In some embodiments, there is section mutation and increase the clone of the mutation of serum stability CH3C.35.23.2 has the sequence of SEQ ID NO:492.

In some embodiments, clone CH3C.35.23.2 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:365 or 366 Few 95% identity.In some embodiments, there is section mutation, the mutation of mediating effect+6 function and increase serum stability The clone CH3C.35.23.2 of mutation has the sequence of SEQ ID NO:365 or 366.

In some embodiments, clone CH3C.35.23.2 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and Presence or absence of M201L) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:493 or 494 Property or at least 95% identity.In some embodiments, have section mutation, the mutation of mediating effect+6 function and increase serum steady The clone CH3C.35.23.2 being qualitatively mutated has the sequence of SEQ ID NO:493 or 494.

In some embodiments, clone CH3C.35.23.2 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number) and there is at least 85% identity, at least 90% with the sequence of SEQ ID NO:367 Identity or at least 95% identity.In some embodiments, the clone CH3C.35.23.2 with hole mutation has SEQ The sequence of ID NO:367.

In some embodiments, clone CH3C.35.23.2 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and it is same at least 85% with the sequence of SEQ ID NO:368 or 369 Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation and mediating effect+6 function The clone CH3C.35.23.2 of mutation has the sequence of SEQ ID NO:368 or 369.

In some embodiments, clone CH3C.35.23.2 can have hole mutation (for example, as referring to SEQ ID NO: 1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:370 Few 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.23.2 has the sequence of SEQ ID NO:370.

In some embodiments, clone CH3C.35.23.2 can have hole mutation (for example, as referring to SEQ ID NO: 1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence or absence of M201L) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:495 One property or at least 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.23.2 has the sequence of SEQ ID NO:495.

In some embodiments, clone CH3C.35.23.2 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 M25Y, S27T and T29E of number) and there is at least 85% identity, at least 90% with the sequence of SEQ ID NO:371 or 372 Identity or at least 95% identity.In some embodiments, with hole mutation, the mutation of mediating effect+6 function and increase blood The clone CH3C.35.23.2 of the mutation of clear stability has the sequence of SEQ ID NO:371 or 372.

In some embodiments, clone CH3C.35.23.2 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 The N207S of number and presence or absence of M201L) and have with the sequence of SEQ ID NO:496 or 497 at least 85% same Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation, mediating effect+6 function The clone CH3C.35.23.2 of mutation and the mutation of increase serum stability has the sequence of SEQ ID NO:496 or 497.

Clone CH3C.35.23.3

In some embodiments, clone CH3C.35.23.3 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number) and there is at least 85% identity, at least 90% identity or at least with the sequence of SEQ ID NO:373 95% identity.In some embodiments, there is the sequence of SEQ ID NO:373 with the clone CH3C.35.23.3 of section mutation Column.

In some embodiments, clone CH3C.35.23.3 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and there is at least 85% identity, at least 90% together with the sequence of SEQ ID NO:374 or 375 One property or at least 95% identity.In some embodiments, there is the clone of section mutation and the mutation of mediating effect+6 function CH3C.35.23.3 has the sequence of SEQ ID NO:374 or 375.

In some embodiments, clone CH3C.35.23.3 can have section mutation (for example, as referring to SEQ ID NO: 1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:376 have at least 85% identity, at least 90% identity or at least 95% same Property.In some embodiments, the clone CH3C.35.23.3 with section mutation and the mutation for increasing serum stability has SEQ The sequence of ID NO:376.

In some embodiments, clone CH3C.35.23.3 can have section mutation (for example, as referring to SEQ ID NO: 1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and exist or There is no M201L) and there is at least 85% identity, at least 90% identity or at least with the sequence of SEQ ID NO:499 95% identity.In some embodiments, there is section mutation and increase the clone of the mutation of serum stability CH3C.35.23.3 has the sequence of SEQ ID NO:499.

In some embodiments, clone CH3C.35.23.3 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:377 or 378 Few 95% identity.In some embodiments, there is section mutation, the mutation of mediating effect+6 function and increase serum stability The clone CH3C.35.23.3 of mutation has the sequence of SEQ ID NO:377 or 378.

In some embodiments, clone CH3C.35.23.3 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and Presence or absence of M201L) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:500 or 501 Property or at least 95% identity.In some embodiments, have section mutation, the mutation of mediating effect+6 function and increase serum steady The clone CH3C.35.23.3 being qualitatively mutated has the sequence of SEQ ID NO:500 or 501.

In some embodiments, clone CH3C.35.23.3 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number) and there is at least 85% identity, at least 90% with the sequence of SEQ ID NO:379 Identity or at least 95% identity.In some embodiments, the clone CH3C.35.23.3 with hole mutation has SEQ The sequence of ID NO:379.

In some embodiments, clone CH3C.35.23.3 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and it is same at least 85% with the sequence of SEQ ID NO:380 or 381 Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation and mediating effect+6 function The clone CH3C.35.23.3 of mutation has the sequence of SEQ ID NO:380 or 381.

In some embodiments, clone CH3C.35.23.3 can have hole mutation (for example, as referring to SEQ ID NO: 1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:382 Few 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.23.3 has the sequence of SEQ ID NO:382.

In some embodiments, clone CH3C.35.23.3 can have hole mutation (for example, as referring to SEQ ID NO: 1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence or absence of M201L) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:502 One property or at least 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.23.3 has the sequence of SEQ ID NO:502.

In some embodiments, clone CH3C.35.23.3 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 M25Y, S27T and T29E of number) and there is at least 85% identity, at least 90% with the sequence of SEQ ID NO:383 or 384 Identity or at least 95% identity.In some embodiments, with hole mutation, the mutation of mediating effect+6 function and increase blood The clone CH3C.35.23.3 of the mutation of clear stability has the sequence of SEQ ID NO:383 or 384.

In some embodiments, clone CH3C.35.23.3 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 The N207S of number and presence or absence of M201L) and have with the sequence of SEQ ID NO:503 or 504 at least 85% same Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation, mediating effect+6 function The clone CH3C.35.23.3 of mutation and the mutation of increase serum stability has the sequence of SEQ ID NO:503 or 504.

Clone CH3C.35.23.4

In some embodiments, clone CH3C.35.23.4 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number) and there is at least 85% identity, at least 90% identity or at least with the sequence of SEQ ID NO:385 95% identity.In some embodiments, there is the sequence of SEQ ID NO:385 with the clone CH3C.35.23.4 of section mutation Column.

In some embodiments, clone CH3C.35.23.4 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and there is at least 85% identity, at least 90% together with the sequence of SEQ ID NO:386 or 387 One property or at least 95% identity.In some embodiments, there is the clone of section mutation and the mutation of mediating effect+6 function CH3C.35.23.4 has the sequence of SEQ ID NO:386 or 387.

In some embodiments, clone CH3C.35.23.4 can have section mutation (for example, as referring to SEQ ID NO: 1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:388 have at least 85% identity, at least 90% identity or at least 95% same Property.In some embodiments, the clone CH3C.35.23.4 with section mutation and the mutation for increasing serum stability has SEQ The sequence of ID NO:388.

In some embodiments, clone CH3C.35.23.4 can have section mutation (for example, as referring to SEQ ID NO: 1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and exist or There is no M201L) and there is at least 85% identity, at least 90% identity or at least with the sequence of SEQ ID NO:506 95% identity.In some embodiments, there is section mutation and increase the clone of the mutation of serum stability CH3C.35.23.4 has the sequence of SEQ ID NO:506.

In some embodiments, clone CH3C.35.23.4 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:389 or 390 Few 95% identity.In some embodiments, there is section mutation, the mutation of mediating effect+6 function and increase serum stability The clone CH3C.35.23.4 of mutation has the sequence of SEQ ID NO:389 or 390.

In some embodiments, clone CH3C.35.23.4 can have section mutation (for example, as referring to SEQ ID NO: The T139W of 1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and Presence or absence of M201L) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:507 or 508 Property or at least 95% identity.In some embodiments, have section mutation, the mutation of mediating effect+6 function and increase serum steady The clone CH3C.35.23.4 being qualitatively mutated has the sequence of SEQ ID NO:507 or 508.

In some embodiments, clone CH3C.35.23.4 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number) and there is at least 85% identity, at least 90% with the sequence of SEQ ID NO:391 Identity or at least 95% identity.In some embodiments, the clone CH3C.35.23.4 with hole mutation has SEQ The sequence of ID NO:391.

In some embodiments, clone CH3C.35.23.4 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and it is same at least 85% with the sequence of SEQ ID NO:392 or 393 Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation and mediating effect+6 function The clone CH3C.35.23.4 of mutation has the sequence of SEQ ID NO:392 or 393.

In some embodiments, clone CH3C.35.23.4 can have hole mutation (for example, as referring to SEQ ID NO: 1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:394 Few 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.23.4 has the sequence of SEQ ID NO:394.

In some embodiments, clone CH3C.35.23.4 can have hole mutation (for example, as referring to SEQ ID NO: 1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence or absence of M201L) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:509 One property or at least 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.23.4 has the sequence of SEQ ID NO:509.

In some embodiments, clone CH3C.35.23.4 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 M25Y, S27T and T29E of number) and there is at least 85% identity, at least 90% with the sequence of SEQ ID NO:395 or 396 Identity or at least 95% identity.In some embodiments, with hole mutation, the mutation of mediating effect+6 function and increase blood The clone CH3C.35.23.4 of the mutation of clear stability has the sequence of SEQ ID NO:395 or 396.

In some embodiments, clone CH3C.35.23.4 can have hole mutation (for example, as referring to SEQ ID NO: T139S, L141A and Y180V of 1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 The N207S of number and presence or absence of M201L) and have with the sequence of SEQ ID NO:510 or 511 at least 85% same Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation, mediating effect+6 function The clone CH3C.35.23.4 of mutation and the mutation of increase serum stability has the sequence of SEQ ID NO:510 or 511.

Clone CH3C.35.21.17.2

In some embodiments, clone CH3C.35.21.17.2 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:397 Few 95% identity.In some embodiments, the clone CH3C.35.21.17.2 with section mutation has SEQ ID NO: 397 sequence.

In some embodiments, clone CH3C.35.21.17.2 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and there is at least 85% identity, at least 90% together with the sequence of SEQ ID NO:398 or 399 One property or at least 95% identity.In some embodiments, there is the clone of section mutation and the mutation of mediating effect+6 function CH3C.35.21.17.2 has the sequence of SEQ ID NO:398 or 399.

In some embodiments, clone CH3C.35.21.17.2 can have section mutation (for example, as referring to SEQ ID NO:1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:400 have at least 85% identity, at least 90% identity or at least 95% same Property.In some embodiments, the clone CH3C.35.21.17.2 for having section mutation and increasing the mutation of serum stability has The sequence of SEQ ID NO:400.

In some embodiments, clone CH3C.35.21.17.2 can have section mutation (for example, as referring to SEQ ID NO:1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence Or M201L is not present) and there is at least 85% identity, at least 90% identity or at least with the sequence of SEQ ID NO:513 95% identity.In some embodiments, there is section mutation and increase the clone of the mutation of serum stability CH3C.35.21.17.2 has the sequence of SEQ ID NO:513.

In some embodiments, clone CH3C.35.21.17.2 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:401 or 402 Few 95% identity.In some embodiments, there is section mutation, the mutation of mediating effect+6 function and increase serum stability The clone CH3C.35.21.17.2 of mutation has the sequence of SEQ ID NO:401 or 402.

In some embodiments, clone CH3C.35.21.17.2 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and Presence or absence of M201L) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:514 or 515 Property or at least 95% identity.In some embodiments, have section mutation, the mutation of mediating effect+6 function and increase serum steady The clone CH3C.35.21.17.2 being qualitatively mutated has the sequence of SEQ ID NO:514 or 515.

In some embodiments, clone CH3C.35.21.17.2 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number) and there is at least 85% identity, at least with the sequence of SEQ ID NO:403 90% identity or at least 95% identity.In some embodiments, with the clone CH3C.35.21.17.2 tool of hole mutation There is the sequence of SEQ ID NO:403.

In some embodiments, clone CH3C.35.21.17.2 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and it is same at least 85% with the sequence of SEQ ID NO:404 or 405 Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation and mediating effect+6 function The clone CH3C.35.21.17.2 of mutation has the sequence of SEQ ID NO:404 or 405.

In some embodiments, clone CH3C.35.21.17.2 can have hole mutation (for example, as referring to SEQ ID NO:1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:406 have at least 85% identity, at least 90% identity or At least 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.21.17.2 has the sequence of SEQ ID NO:406.

In some embodiments, clone CH3C.35.21.17.2 can have hole mutation (for example, as referring to SEQ ID NO:1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence or absence of M201L) and with the sequence of SEQ ID NO:516 have at least 85% identity, at least 90% Identity or at least 95% identity.In some embodiments, gram with hole mutation and the mutation for increasing serum stability Grand CH3C.35.21.17.2 has the sequence of SEQ ID NO:516.

In some embodiments, clone CH3C.35.21.17.2 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 M25Y, S27T and T29E of number) and there is at least 85% identity, at least 90% with the sequence of SEQ ID NO:407 or 408 Identity or at least 95% identity.In some embodiments, with hole mutation, the mutation of mediating effect+6 function and increase blood The clone CH3C.35.21.17.2 of the mutation of clear stability has the sequence of SEQ ID NO:407 or 408.

In some embodiments, clone CH3C.35.21.17.2 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 The N207S of number and presence or absence of M201L) and have with the sequence of SEQ ID NO:517 or 518 at least 85% same Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation, mediating effect+6 function The clone CH3C.35.21.17.2 of mutation and the mutation of increase serum stability has the sequence of SEQ ID NO:517 or 518.

Clone CH3C.35.23

In some embodiments, clone CH3C.35.23 can have section mutation (for example, as referring to SEQ ID NO:1 The T139W of number) and there is at least 85% identity, at least 90% identity or at least with the sequence of SEQ ID NO:409 95% identity.In some embodiments, there is the sequence of SEQ ID NO:409 with the clone CH3C.35.23 of section mutation Column.

In some embodiments, clone CH3C.35.23 can have section mutation (for example, as referring to SEQ ID NO:1 The T139W of number), the mutation of mediating effect+6 function is (for example, such as L7A, L8A and/or P102G referring to SEQ ID NO:1 number (such as L7A and L8A)) and there is at least 85% identity, at least 90% identity with the sequence of SEQ ID NO:410 or 411 Or at least 95% identity.In some embodiments, there is the clone of section mutation and the mutation of mediating effect+6 function CH3C.35.23 has the sequence of SEQ ID NO:410 or 411.

In some embodiments, clone CH3C.35.23 can have section mutation (for example, as referring to SEQ ID NO:1 The T139W of number), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:412 have at least 85% identity, at least 90% identity or at least 95% same Property.In some embodiments, the clone CH3C.35.23 with section mutation and the mutation for increasing serum stability has SEQ The sequence of ID NO:412.

In some embodiments, clone CH3C.35.23 can have section mutation (for example, as referring to SEQ ID NO:1 The T139W of number), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and exist or not There are M201L) and there is at least 85% identity, at least 90% identity or at least 95% with the sequence of SEQ ID NO:520 Identity.In some embodiments, the clone CH3C.35.23 for having section mutation and increasing the mutation of serum stability has The sequence of SEQ ID NO:520.

In some embodiments, clone CH3C.35.23 can have section mutation (for example, as referring to SEQ ID NO:1 The T139W of number), the mutation of mediating effect+6 function is (for example, such as L7A, L8A and/or P102G referring to SEQ ID NO:1 number (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:413 or 414 there is at least 85% identity, at least 90% identity or at least 95% Identity.In some embodiments, there is section mutation, the mutation of mediating effect+6 function and the mutation of increase serum stability Clone the sequence that CH3C.35.23 has SEQ ID NO:413 or 414.

In some embodiments, clone CH3C.35.23 can have section mutation (for example, as referring to SEQ ID NO:1 The T139W of number), the mutation of mediating effect+6 function is (for example, such as L7A, L8A and/or P102G referring to SEQ ID NO:1 number (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence Or be not present M201L) and with the sequence of SEQ ID NO:521 or 522 have at least 85% identity, at least 90% identity or At least 95% identity.In some embodiments, there is section mutation, the mutation of mediating effect+6 function and increase serum stability Mutation clone CH3C.35.23 have SEQ ID NO:521 or 522 sequence.

In some embodiments, clone CH3C.35.23 can have hole mutation (for example, as referring to SEQ ID NO:1 T139S, L141A and Y180V of number) and there is at least 85% identity, at least 90% together with the sequence of SEQ ID NO:415 One property or at least 95% identity.In some embodiments, the clone CH3C.35.23 with hole mutation has SEQ ID The sequence of NO:415.

In some embodiments, clone CH3C.35.23 can have hole mutation (for example, as referring to SEQ ID NO:1 T139S, L141A and Y180V of number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and there is at least 85% identity, extremely with the sequence of SEQ ID NO:416 or 417 Few 90% identity or at least 95% identity.In some embodiments, the mutation with hole mutation and mediating effect+6 function Clone CH3C.35.23 have SEQ ID NO:416 or 417 sequence.

In some embodiments, clone CH3C.35.23 can have hole mutation (for example, as referring to SEQ ID NO:1 T139S, L141A and Y180V of number), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:418 Few 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.23 has the sequence of SEQ ID NO:418.

In some embodiments, clone CH3C.35.23 can have hole mutation (for example, as referring to SEQ ID NO:1 T139S, L141A and Y180V of number), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence or absence of M201L) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:523 One property or at least 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.23 has the sequence of SEQ ID NO:523.

In some embodiments, clone CH3C.35.23 can have hole mutation (for example, as referring to SEQ ID NO:1 T139S, L141A and Y180V of number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:419 or 420 Property or at least 95% identity.In some embodiments, steady with hole mutation, the mutation of mediating effect+6 function and increase serum The clone CH3C.35.23 being qualitatively mutated has the sequence of SEQ ID NO:419 or 420.

In some embodiments, clone CH3C.35.23 can have hole mutation (for example, as referring to SEQ ID NO:1 T139S, L141A and Y180V of number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence or absence of M201L) and there is at least 85% identity, extremely with the sequence of SEQ ID NO:524 or 525 Few 90% identity or at least 95% identity.In some embodiments, with hole mutation, mediating effect+6 function mutation and The clone CH3C.35.23 for increasing the mutation of serum stability has the sequence of SEQ ID NO:524 or 525.

Clone CH3C.35.21

In some embodiments, clone CH3C.35.21 can have section mutation (for example, as referring to SEQ ID NO:1 The T139W of number) and there is at least 85% identity, at least 90% identity or at least with the sequence of SEQ ID NO:436 95% identity.In some embodiments, there is the sequence of SEQ ID NO:436 with the clone CH3C.35.21 of section mutation Column.

In some embodiments, clone CH3C.35.21 can have section mutation (for example, as referring to SEQ ID NO:1 The T139W of number), the mutation of mediating effect+6 function is (for example, such as L7A, L8A and/or P102G referring to SEQ ID NO:1 number (such as L7A and L8A)) and there is at least 85% identity, at least 90% identity with the sequence of SEQ ID NO:437 or 438 Or at least 95% identity.In some embodiments, there is the clone of section mutation and the mutation of mediating effect+6 function CH3C.35.21 has the sequence of SEQ ID NO:437 or 438.

In some embodiments, clone CH3C.35.21 can have section mutation (for example, as referring to SEQ ID NO:1 The T139W of number), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:439 have at least 85% identity, at least 90% identity or at least 95% same Property.In some embodiments, the clone CH3C.35.21 with section mutation and the mutation for increasing serum stability has SEQ The sequence of ID NO:439.

In some embodiments, clone CH3C.35.21 can have section mutation (for example, as referring to SEQ ID NO:1 The T139W of number), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and exist or not There are M201L) and there is at least 85% identity, at least 90% identity or at least 95% with the sequence of SEQ ID NO:527 Identity.In some embodiments, the clone CH3C.35.21 for having section mutation and increasing the mutation of serum stability has The sequence of SEQ ID NO:527.

In some embodiments, clone CH3C.35.21 can have section mutation (for example, as referring to SEQ ID NO:1 The T139W of number), the mutation of mediating effect+6 function is (for example, such as L7A, L8A and/or P102G referring to SEQ ID NO:1 number (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:440 or 441 there is at least 85% identity, at least 90% identity or at least 95% Identity.In some embodiments, there is section mutation, the mutation of mediating effect+6 function and the mutation of increase serum stability Clone the sequence that CH3C.35.21 has SEQ ID NO:440 or 441.

In some embodiments, clone CH3C.35.21 can have section mutation (for example, as referring to SEQ ID NO:1 The T139W of number), the mutation of mediating effect+6 function is (for example, such as L7A, L8A and/or P102G referring to SEQ ID NO:1 number (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence Or be not present M201L) and with the sequence of SEQ ID NO:528 or 529 have at least 85% identity, at least 90% identity or At least 95% identity.In some embodiments, there is section mutation, the mutation of mediating effect+6 function and increase serum stability Mutation clone CH3C.35.21 have SEQ ID NO:528 or 529 sequence.

In some embodiments, clone CH3C.35.21 can have hole mutation (for example, as referring to SEQ ID NO:1 T139S, L141A and Y180V of number) and there is at least 85% identity, at least 90% together with the sequence of SEQ ID NO:442 One property or at least 95% identity.In some embodiments, the clone CH3C.35.21 with hole mutation has SEQ ID The sequence of NO:442.

In some embodiments, clone CH3C.35.21 can have hole mutation (for example, as referring to SEQ ID NO:1 T139S, L141A and Y180V of number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and there is at least 85% identity, extremely with the sequence of SEQ ID NO:443 or 444 Few 90% identity or at least 95% identity.In some embodiments, the mutation with hole mutation and mediating effect+6 function Clone CH3C.35.21 have SEQ ID NO:443 or 444 sequence.

In some embodiments, clone CH3C.35.21 can have hole mutation (for example, as referring to SEQ ID NO:1 T139S, L141A and Y180V of number), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:445 Few 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.21 has the sequence of SEQ ID NO:445.

In some embodiments, clone CH3C.35.21 can have hole mutation (for example, as referring to SEQ ID NO:1 T139S, L141A and Y180V of number), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence or absence of M201L) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:530 One property or at least 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.21 has the sequence of SEQ ID NO:530.

In some embodiments, clone CH3C.35.21 can have hole mutation (for example, as referring to SEQ ID NO:1 T139S, L141A and Y180V of number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:446 or 447 Property or at least 95% identity.In some embodiments, steady with hole mutation, the mutation of mediating effect+6 function and increase serum The clone CH3C.35.21 being qualitatively mutated has the sequence of SEQ ID NO:446 or 447.

In some embodiments, clone CH3C.35.21 can have hole mutation (for example, as referring to SEQ ID NO:1 T139S, L141A and Y180V of number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence or absence of M201L) and there is at least 85% identity, extremely with the sequence of SEQ ID NO:531 or 532 Few 90% identity or at least 95% identity.In some embodiments, with hole mutation, mediating effect+6 function mutation and The clone CH3C.35.21 for increasing the mutation of serum stability has the sequence of SEQ ID NO:531 or 532.

Clone CH3C.35.20.1.1

In some embodiments, clone CH3C.35.20.1.1 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:448 Few 95% identity.In some embodiments, the clone CH3C.35.20.1.1 with section mutation has SEQ ID NO:448 Sequence.

In some embodiments, clone CH3C.35.20.1.1 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and there is at least 85% identity, at least 90% together with the sequence of SEQ ID NO:449 or 450 One property or at least 95% identity.In some embodiments, there is the clone of section mutation and the mutation of mediating effect+6 function CH3C.35.20.1.1 has the sequence of SEQ ID NO:449 or 450.

In some embodiments, clone CH3C.35.20.1.1 can have section mutation (for example, as referring to SEQ ID NO:1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:451 have at least 85% identity, at least 90% identity or at least 95% same Property.In some embodiments, the clone CH3C.35.20.1.1 for having section mutation and increasing the mutation of serum stability has The sequence of SEQ ID NO:451.

In some embodiments, clone CH3C.35.20.1.1 can have section mutation (for example, as referring to SEQ ID NO:1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence Or M201L is not present) and there is at least 85% identity, at least 90% identity or at least with the sequence of SEQ ID NO:534 95% identity.In some embodiments, there is section mutation and increase the clone of the mutation of serum stability CH3C.35.20.1.1 has the sequence of SEQ ID NO:534.

In some embodiments, clone CH3C.35.20.1.1 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:452 or 453 Few 95% identity.In some embodiments, there is section mutation, the mutation of mediating effect+6 function and increase serum stability The clone CH3C.35.20.1.1 of mutation has the sequence of SEQ ID NO:452 or 453.

In some embodiments, clone CH3C.35.20.1.1 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and Presence or absence of M201L) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:535 or 536 Property or at least 95% identity.In some embodiments, have section mutation, the mutation of mediating effect+6 function and increase serum steady The clone CH3C.35.20.1.1 being qualitatively mutated has the sequence of SEQ ID NO:535 or 536.

In some embodiments, clone CH3C.35.20.1.1 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number) and there is at least 85% identity, at least with the sequence of SEQ ID NO:454 90% identity or at least 95% identity.In some embodiments, with the clone CH3C.35.20.1.1 tool of hole mutation There is the sequence of SEQ ID NO:454.

In some embodiments, clone CH3C.35.20.1.1 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and it is same at least 85% with the sequence of SEQ ID NO:455 or 456 Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation and mediating effect+6 function The clone CH3C.35.20.1.1 of mutation has the sequence of SEQ ID NO:455 or 456.

In some embodiments, clone CH3C.35.20.1.1 can have hole mutation (for example, as referring to SEQ ID NO:1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:457 have at least 85% identity, at least 90% identity or At least 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.20.1.1 has the sequence of SEQ ID NO:457.

In some embodiments, clone CH3C.35.20.1.1 can have hole mutation (for example, as referring to SEQ ID NO:1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence or absence of M201L) and with the sequence of SEQ ID NO:537 have at least 85% identity, at least 90% Identity or at least 95% identity.In some embodiments, gram with hole mutation and the mutation for increasing serum stability Grand CH3C.35.20.1.1 has the sequence of SEQ ID NO:537.

In some embodiments, clone CH3C.35.20.1.1 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 M25Y, S27T and T29E of number) and there is at least 85% identity, at least 90% with the sequence of SEQ ID NO:458 or 459 Identity or at least 95% identity.In some embodiments, with hole mutation, the mutation of mediating effect+6 function and increase blood The clone CH3C.35.20.1.1 of the mutation of clear stability has the sequence of SEQ ID NO:458 or 459.

In some embodiments, clone CH3C.35.20.1.1 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 The N207S of number and presence or absence of M201L) and have with the sequence of SEQ ID NO:538 or 539 at least 85% same Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation, mediating effect+6 function The clone CH3C.35.20.1.1 of mutation and the mutation of increase serum stability has the sequence of SEQ ID NO:538 or 539.

Clone CH3C.35.23.2.1

In some embodiments, clone CH3C.35.23.2.1 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:460 Few 95% identity.In some embodiments, the clone CH3C.35.23.2.1 with section mutation has SEQ ID NO:460 Sequence.

In some embodiments, clone CH3C.35.23.2.1 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and there is at least 85% identity, at least 90% together with the sequence of SEQ ID NO:461 or 462 One property or at least 95% identity.In some embodiments, there is the clone of section mutation and the mutation of mediating effect+6 function CH3C.35.23.2.1 has the sequence of SEQ ID NO:461 or 462.

In some embodiments, clone CH3C.35.23.2.1 can have section mutation (for example, as referring to SEQ ID NO:1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:463 have at least 85% identity, at least 90% identity or at least 95% same Property.In some embodiments, the clone CH3C.35.23.2.1 for having section mutation and increasing the mutation of serum stability has The sequence of SEQ ID NO:463.

In some embodiments, clone CH3C.35.23.2.1 can have section mutation (for example, as referring to SEQ ID NO:1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence Or M201L is not present) and there is at least 85% identity, at least 90% identity or at least with the sequence of SEQ ID NO:541 95% identity.In some embodiments, there is section mutation and increase the clone of the mutation of serum stability CH3C.35.23.2.1 has the sequence of SEQ ID NO:541.

In some embodiments, clone CH3C.35.23.2.1 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:464 or 465 Few 95% identity.In some embodiments, there is section mutation, the mutation of mediating effect+6 function and increase serum stability The clone CH3C.35.23.2.1 of mutation has the sequence of SEQ ID NO:464 or 465.

In some embodiments, clone CH3C.35.23.2.1 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and Presence or absence of M201L) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:542 or 543 Property or at least 95% identity.In some embodiments, have section mutation, the mutation of mediating effect+6 function and increase serum steady The clone CH3C.35.23.2.1 being qualitatively mutated has the sequence of SEQ ID NO:542 or 543.

In some embodiments, clone CH3C.35.23.2.1 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number) and there is at least 85% identity, at least with the sequence of SEQ ID NO:466 90% identity or at least 95% identity.In some embodiments, with the clone CH3C.35.23.2.1 tool of hole mutation There is the sequence of SEQ ID NO:466.

In some embodiments, clone CH3C.35.23.2.1 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and it is same at least 85% with the sequence of SEQ ID NO:467 or 468 Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation and mediating effect+6 function The clone CH3C.35.23.2.1 of mutation has the sequence of SEQ ID NO:467 or 468.

In some embodiments, clone CH3C.35.23.2.1 can have hole mutation (for example, as referring to SEQ ID NO:1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:469 have at least 85% identity, at least 90% identity or At least 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.23.2.1 has the sequence of SEQ ID NO:469.

In some embodiments, clone CH3C.35.23.2.1 can have hole mutation (for example, as referring to SEQ ID NO:1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence or absence of M201L) and with the sequence of SEQ ID NO:544 have at least 85% identity, at least 90% Identity or at least 95% identity.In some embodiments, gram with hole mutation and the mutation for increasing serum stability Grand CH3C.35.23.2.1 has the sequence of SEQ ID NO:544.

In some embodiments, clone CH3C.35.23.2.1 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 M25Y, S27T and T29E of number) and there is at least 85% identity, at least 90% with the sequence of SEQ ID NO:470 or 471 Identity or at least 95% identity.In some embodiments, with hole mutation, the mutation of mediating effect+6 function and increase blood The clone CH3C.35.23.2.1 of the mutation of clear stability has the sequence of SEQ ID NO:470 or 471.

In some embodiments, clone CH3C.35.23.2.1 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 The N207S of number and presence or absence of M201L) and have with the sequence of SEQ ID NO:545 or 546 at least 85% same Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation, mediating effect+6 function The clone CH3C.35.23.2.1 of mutation and the mutation of increase serum stability has the sequence of SEQ ID NO:545 or 546.

Clone CH3C.35.23.1.1

In some embodiments, clone CH3C.35.23.1.1 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:472 Few 95% identity.In some embodiments, the clone CH3C.35.23.1.1 with section mutation has SEQ ID NO:472 Sequence.

In some embodiments, clone CH3C.35.23.1.1 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and there is at least 85% identity, at least 90% together with the sequence of SEQ ID NO:473 or 474 One property or at least 95% identity.In some embodiments, there is the clone of section mutation and the mutation of mediating effect+6 function CH3C.35.23.1.1 has the sequence of SEQ ID NO:473 or 474.

In some embodiments, clone CH3C.35.23.1.1 can have section mutation (for example, as referring to SEQ ID NO:1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:475 have at least 85% identity, at least 90% identity or at least 95% same Property.In some embodiments, the clone CH3C.35.23.1.1 for having section mutation and increasing the mutation of serum stability has The sequence of SEQ ID NO:475.

In some embodiments, clone CH3C.35.23.1.1 can have section mutation (for example, as referring to SEQ ID NO:1 number T139W), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence Or M201L is not present) and there is at least 85% identity, at least 90% identity or at least with the sequence of SEQ ID NO:548 95% identity.In some embodiments, there is section mutation and increase the clone of the mutation of serum stability CH3C.35.23.1.1 has the sequence of SEQ ID NO:548.

In some embodiments, clone CH3C.35.23.1.1 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and there is at least 85% identity, at least 90% identity or extremely with the sequence of SEQ ID NO:476 or 477 Few 95% identity.In some embodiments, there is section mutation, the mutation of mediating effect+6 function and increase serum stability The clone CH3C.35.23.1.1 of mutation has the sequence of SEQ ID NO:476 or 477.

In some embodiments, clone CH3C.35.23.1.1 can have section mutation (for example, as referring to SEQ ID The T139W of NO:1 number), the mutation of mediating effect+6 function (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and Presence or absence of M201L) and there is at least 85% identity, at least 90% same with the sequence of SEQ ID NO:549 or 550 Property or at least 95% identity.In some embodiments, have section mutation, the mutation of mediating effect+6 function and increase serum steady The clone CH3C.35.23.1.1 being qualitatively mutated has the sequence of SEQ ID NO:549 or 550.

In some embodiments, clone CH3C.35.23.1.1 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number) and there is at least 85% identity, at least with the sequence of SEQ ID NO:478 90% identity or at least 95% identity.In some embodiments, with the clone CH3C.35.23.1.1 tool of hole mutation There is the sequence of SEQ ID NO:478.

In some embodiments, clone CH3C.35.23.1.1 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)) and it is same at least 85% with the sequence of SEQ ID NO:479 or 480 Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation and mediating effect+6 function The clone CH3C.35.23.1.1 of mutation has the sequence of SEQ ID NO:479 or 480.

In some embodiments, clone CH3C.35.23.1.1 can have hole mutation (for example, as referring to SEQ ID NO:1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number M25Y, S27T and T29E) and with the sequence of SEQ ID NO:481 have at least 85% identity, at least 90% identity or At least 95% identity.In some embodiments, the clone with hole mutation and the mutation for increasing serum stability CH3C.35.23.1.1 has the sequence of SEQ ID NO:481.

In some embodiments, clone CH3C.35.23.1.1 can have hole mutation (for example, as referring to SEQ ID NO:1 number T139S, L141A and Y180V), increase serum stability mutation (for example, as referring to SEQ ID NO:1 number N207S and presence or absence of M201L) and with the sequence of SEQ ID NO:551 have at least 85% identity, at least 90% Identity or at least 95% identity.In some embodiments, gram with hole mutation and the mutation for increasing serum stability Grand CH3C.35.23.1.1 has the sequence of SEQ ID NO:551.

In some embodiments, clone CH3C.35.23.1.1 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 M25Y, S27T and T29E of number) and there is at least 85% identity, at least 90% with the sequence of SEQ ID NO:482 or 483 Identity or at least 95% identity.In some embodiments, with hole mutation, the mutation of mediating effect+6 function and increase blood The clone CH3C.35.23.1.1 of the mutation of clear stability has the sequence of SEQ ID NO:482 or 483.

In some embodiments, clone CH3C.35.23.1.1 can have hole mutation (for example, as referring to SEQ ID T139S, L141A and Y180V of NO:1 number), the mutation of mediating effect+6 function is (for example, as referring to SEQ ID NO:1 number L7A, L8A and/or P102G (such as L7A and L8A)), increase the mutation of serum stability (for example, as referring to SEQ ID NO:1 The N207S of number and presence or absence of M201L) and have with the sequence of SEQ ID NO:552 or 553 at least 85% same Property, at least 90% identity or at least 95% identity.In some embodiments, with hole mutation, mediating effect+6 function The clone CH3C.35.23.1.1 of mutation and the mutation of increase serum stability has the sequence of SEQ ID NO:552 or 553.

VI. conjugate

In some embodiments, the TfR knot of the CH2 or CH3 structural domain according to the present invention comprising modification Closing polypeptide is medicament to be connected to via connexon, such as internalization, into cell and/or across endothelium, such as blood-brain barrier wears born of the same parents' transport Medicament.The connexon can be sub to any connection of polypeptide to be suitble to engage medicament.In some embodiments, described Connection is enzyme cleavable.In certain embodiments, the connection can pass through enzymatic lysis present in central nervous system.

In some embodiments, connexon is peptide connexon.Peptide connexon can be configured to that it is made to allow the medicament It is rotated relative to each other with TfR combination polypeptide, and/or resistant to protease digestion.In some embodiments In, the connexon can be flexible linker, such as the amino containing such as Gly, Asn, Ser, Thr, Ala and analog Acid.Such connexon is designed using known parameters.For example, the connexon can have repetitive sequence, such as Gly- Ser repetitive sequence.

In various embodiments, well known chemical linking agent can be used in conjugate and scheme generates.For example, Numerous chemical cross-linking agents known to those skilled in the art and can be used for are crosslinked polypeptide and medicament of interest.For example, it hands over Joining agent is heterobifunctional agents, can be used for connection molecule in a step-wise fashion.Heterobifunctional agents make it possible to design The conjugation methods for conjugated protein are more biased towards, undesired side reaction is thus reduced, such as homologous protein polymer Occur.A variety of heterobifunctional agents known in the art, including n-hydroxysuccinimide (NHS) or its water solubility it is similar Object n-Hydroxysulfosuccinimide (sulfo group-NHS), 4- (N- maleimidomethyl) hexamethylene -1- formic acid amber Imide ester (SMCC), maleimide benzoyl-N-hydroxysuccinimide eater (MBS)；(4- iodoacetamido Base) aminobenzoic acid N- succinimide ester (SIAB), 4- (to maleimide phenyl) butyric acid succinimide ester (SMPB), 1- ethyl -3- (3- dimethylaminopropyl) carbodiimide hydrochloride (EDC)；4- succinimide base oxygroup carbonyl Base-a- methyl-a- (2- pyridyidithio)-toluene (SMPT), 3- (2- pyridyidithio) propionic acid N- succinimide ester (SPDP) and 6- [3- (2- pyridyidithio) propionamido] caproic acid succinimide ester (LC-SPDP).With N- hydroxyl amber The crosslinking agent of amber imide moieties can in n-Hydroxysulfosuccinimide analog form obtain, generally have compared with Highly-water-soluble.In addition, in connection chain with disulphide bridges the crosslinking agent can transfer in the form of alkyl derivative obtain so as to Reduce the amount of internal connexon cracking.In addition to heterobifunctional agents, there is also a variety of other crosslinking agents, including same difunctionality to hand over Join agent and photoreactivity crosslinking agent.Suberic acid double amber imide ester (DSS), double maleimide hexanes (BMH) and Dimethyl-g imidodicarbonic diamide ester .2HCl (DMP) is the example of useful same bifunctional crosslinking agent, and double-[B- (4- azido water Yankee acylamino-) ethyl] disulphide (BASED) and 6 (4'- azido -2'- nitro-phenylamino) caproic acid N- succinimides Ester (SANPAH) is the example of useful photoreactivity crosslinking agent.

Reagent of interest can be therapeutic agent, including cytotoxic agent, DNA or RNA molecule, chemical part and analog. In some embodiments, the reagent can be peptide or small molecule therapy agent or developer.In some embodiments, described Small molecule is less than 1000Da, is less than 750Da or is less than 500Da.

Reagent of interest may be coupled to N-terminal or the C-terminal area of TfR combination polypeptide, or be connected to polypeptide Any region, as long as the reagent does not interfere the combination of TfR combination polypeptide and TfR.

V. FC polypeptide is engineered at the method for combining TfR

In another aspect, it provides CH2 or CH3 Domain Polypeptide is engineered special at combining with TfR Anisotropic method.In some embodiments, the modification of CH3 Domain Polypeptide include replace as described herein (i) group and/or (ii) the various amino acid in group.In some embodiments, the method includes the poly-nuclears of modification coding CH3 Domain Polypeptide Thuja acid is incorporated to ammonia at nine positions with three, four, five, six, seven, eight in CH3 structural domain (i) group or all The variation of base acid.In some embodiments, the method includes the polynucleotides of modification coding CH3 Domain Polypeptide in CH3 Three, four, five, six, seven in structural domain (ii) group or amino acid variation is all incorporated at eight positions.Introduce institute Need the amino acid of position that can generate by random mutation or part random mutation has amino at described group of various positions The CH3 Domain Polypeptide library that acid replaces generates.In some embodiments, CH3 Domain Polypeptide is in the case where the area Fc Part or all of complete hinge region can be contained or can be free of in experience mutation, the area Fc.

In an aspect, CH3 structural domain is engineered at being specifically bound to TfR in the following manner: (a) polynucleotide for encoding CH3 structural domain is modified into the position such as numbered referring to the amino acid 1 14-220 of SEQ ID NO:1 Setting at 153,157,159,160,161,162,163,164,165,186,187,188,189,194,197 or 199 has at least Five amino acid replaces；(b) express and recycle the polypeptide of the CH3 structural domain comprising modification.In some embodiments, CH3 Structural domain is modified into position 153,157,159,160,161,162,163,164,165,186,187,188,189,194,197 Or at 199 there is at least five to replace, wherein the substitution is selected from following: in 153 Trp, Tyr, Leu or Gln；157 Leu, Tyr, Met or Val of position；In 159 Leu, Thr, His or Pro；In 160 Val or acidic amino acids；161 The aromatic amino acid of position, such as Trp；In 162 Val, Ser or Ala；In 163 Ser；164 Ser, Thr, Gln or Phe；In 165 Gln, Phe or His；In 186 Glu, Ala, Ser, Leu, Thr or Pro；At 187 Arg, Gly or Pro；In 188 Glu；In 189 Thr or acidic amino acids；In 194 Trp, Tyr, His or Phe； In 197 Thr, Glu or Lys；With in 199 Trp or Gly.

In another aspect, CH3 structural domain is engineered at being specifically bound to TfR in the following manner: (a) polynucleotide for encoding CH3 structural domain is modified into the position such as numbered referring to the amino acid 1 14-220 of SEQ ID NO:1 Setting has at least five amino acid substitutions at 118,119,120,122,210,211,212 and 213；(b) it expresses and recycles packet The polypeptide of CH3 structural domain containing modification.In some embodiments, CH3 structural domain be modified into position 118,119,120, 122, there is at least five to replace at 210,211,212 and 213, wherein the substitution is selected from following: in 118 Phe or Ile；In 119 Asp, Glu, Gly, Ala, or Lys；In 120 Tyr, Met, Leu, Ile or Asp；In 122 Thr Or Ala；In 210 Gly；In 211 Phe；At 212；With in 213 Asp.

CH2 Domain Polypeptide can in a similar manner, by CH2 structural domain (iii) group, (iv) group, (v) group or (vi) Mutation is introduced in any of three of group extremely all positions, it is engineered in conjunction with TfR.Therefore, in some realities It applies in scheme, the modification of CH2 Domain Polypeptide includes to replace (iii) group, (iv) group, (v) group and/or (vi) as described herein Various amino acid in group.In some embodiments, the method includes the polynucleotides of modification coding CH2 Domain Polypeptide Amino is incorporated to three, four, five, six, seven, eight in CH2 structural domain (iii) group or all at nine positions Acid variation.In some embodiments, the method includes the polynucleotides of modification coding CH2 Domain Polypeptide to tie in CH2 Three, four, five, six, seven, eight, nine in structure domain (iv) group or amino acid change is all incorporated at ten positions Change.In some embodiments, the method includes the polynucleotides of modification coding CH2 Domain Polypeptide in CH2 structural domain (v) amino acid variation is incorporated at three in group, four, five, six, seven, eight, nine or whole ten positions.In In some embodiments, the method includes the polynucleotides of modification coding CH2 Domain Polypeptide in CH2 structural domain (vi) group In three, four, five, six, seven, eight or amino acid variation is all incorporated at nine positions.Position needed for introducing Amino acid can be generated by random mutation or part random mutation at described group of various positions have amino acid substitution CH2 Domain Polypeptide library generate.In some embodiments, CH2 Domain Polypeptide undergoes prominent in the case where the area Fc Become, part or all of complete hinge region can be contained or can be free of in the area Fc.

In an aspect, CH2 structural domain is engineered at being specifically bound to TfR in the following manner: (a) polynucleotide for encoding CH2 structural domain is modified into the position such as numbered referring to the amino acid 4-113 of SEQ ID NO:1 47, there are at 49,56,58,59,60,61,62 and 63 at least five amino acid substitutions；(b) it expresses and recycles comprising modification The polypeptide of CH2 structural domain.In some embodiments, CH2 structural domain is modified into position 47,49,56,58,59,60,61,62 With 63 at there is at least five to replace, wherein the substitution is selected from following: 47 Glu, Gly, Gln, Ser, Ala, Asn, Tyr or Trp；In 49 Ile, Val, Asp, Glu, Thr, Ala or Tyr；In 56 Asp, Pro, Met, Leu, Ala, Asn Or Phe；In 58 Arg, Ser, Ala or Gly；In 59 Tyr, Trp, Arg or Val；In 60 Glu；At 61 Trp or Tyr；In 62 Gln, Tyr, His, Ile, Phe, Val or Asp；With 63 Leu, Trp, Arg, Asn, Tyr or Val。

In another aspect, CH2 structural domain is engineered at being specifically bound to TfR in the following manner: (a) polynucleotide for encoding CH2 structural domain is modified into the position such as numbered referring to the amino acid 4-113 of SEQ ID NO:1 39, there are at 40,41,42,43,44,68,70,71 and 72 at least five amino acid substitutions；(b) it expresses and recycles comprising repairing The polypeptide of the CH2 structural domain of decorations.In some embodiments, CH2 structural domain be modified into position 39,40,41,42,43,44, 68, there is at least five to replace at 70,71 and 72, wherein the substitution is selected from following: in 39 Pro, Phe, Ala, Met Or Asp；In 40 Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp or Tyr；41 Thr, Ser, Gly, Met, Val, Phe, Trp or Leu；In 42 Pro, Val, Ala, Thr or Asp；In 43 Pro, Val or Phe；At 44 Trp, Gln, Thr or Glu；In 68 Glu, Val, Thr, Leu or Trp；In 70 Tyr, His, Val or Asp；71 Thr, His, Gln, Arg, Asn or Val of position；With in 72 Tyr, Asn, Asp, Ser or Pro.

In another aspect, CH2 structural domain is engineered at being specifically bound to TfR in the following manner: (a) polynucleotide for encoding CH2 structural domain is modified into the position such as numbered referring to the amino acid 4-113 of SEQ ID NO:1 41, there are at 42,43,44,45,65,66,67,69 and 73 at least five amino acid substitutions；(b) it expresses and recycles comprising repairing The polypeptide of the CH2 structural domain of decorations.In some embodiments, CH2 structural domain be modified into position 41,42,43,44,45,65, 66, there is at least five to replace at 67,69 and 73, wherein the substitution is selected from following: in 41 Val or Asp；At 42 Pro, Met or Asp；In 43 Pro or Trp；In 44 Arg, Trp, Glu or Thr；In 45 Met, Tyr or Trp；In 65 Leu or Trp；In 66 Thr, Val, Ile or Lys；In 67 Ser, Lys, Ala or Leu；At 69 His, Leu or Pro；With in 73 Val or Trp.

In another aspect, CH2 structural domain is engineered at being specifically bound to TfR in the following manner: (a) polynucleotide for encoding CH2 structural domain is modified into the position such as numbered referring to the amino acid 4-113 of SEQ ID NO:1 45, there are at 47,49,95,97,99,102,103 and 104 at least five amino acid substitutions；(b) it expresses and recycles comprising repairing The polypeptide of the CH2 structural domain of decorations.In some embodiments, CH2 structural domain be modified into position 45,47,49,95,97,99, 102, there is at least five to replace at 103 and 104, wherein the substitution is selected from following: in 45 Trp, Val, Ile or Ala；In 47 Trp or Gly；In 49 Tyr, Arg or Glu；In 95 Ser, Arg or Gln；97 Val, Ser or Phe；In 99 Ile, Ser or Trp；In 102 Trp, Thr, Ser, Arg or Asp；103 Trp；With 104 Ser, Lys, Arg or Val.

Multiple systems expression can be used in the polypeptide of CH3 and/or CH2 structural domain comprising mutation.For example, some In embodiment, mutant polypeptide is expressed in display systems.In other exemplary embodiments, mutant polypeptide is with host The soluble polypeptide form expression of cell secretion.In some embodiments, expression system is display systems, such as viral display System, cell surface display system such as yeast display systems, mRNA display systems or polysome display systems.Using known Method screens library to identify TfR conjugate, and the conjugate can be further characterized to determine and combine power It learns.In addition mutation can then be introduced into selected clone each position in initial group ((i) group or (ii) group)；Or described The other positions also existed in the selected combined area cloned outside group range.

VI. nucleic acid, carrier and host cell

The TfR combination polypeptide of modification described herein is typically prepared using recombination method.Therefore, In In some aspects, the present invention provides isolated nucleic acid, and it includes the nucleic acid of any polypeptide of the coding containing polypeptide described herein Sequence；And host cell, the nucleic acid are to be introduced into the host cell for replicating polypeptide encoding nucleic acid and/or expression institute State polypeptide.In some embodiments, host cell is eukaryocyte, such as human cell.

In another aspect, the polynucleotide of the nucleotide comprising encoding polypeptide described herein is provided.The polynucleotide It can be sub-thread or bifilar.In some embodiments, the polynucleotide is DNA.In specific embodiments, described poly- Nucleotide is cDNA.In some embodiments, the polynucleotide is RNA.

In some embodiments, the polynucleotide is included in nucleic acid construct body.In some embodiments, institute Stating construct is reproducible carrier.In some embodiments, the carrier is selected from plasmid, viral vectors, phasmid, ferment Female chromosome vector and non-free type mammalian vector.

In some embodiments, the polynucleotide is operably connected to one or more tune in expression construct Control nucleotide sequence.In a series of embodiments, expression of nucleic acid construct is suitable as surface expression library.In some implementations In scheme, the library is suitable for the surface expression in yeast.In some embodiments, the library is suitable for the table in bacteriophage Face expression.In another series of embodiments, expression of nucleic acid construct be suitable for be allow to separate milligram quantities or gram quantity polypeptide Polypeptide is expressed in system.In some embodiments, the system is mammalian cell expression system.In some embodiments In, the system is yeast expression system.

Expression medium for generating recombinant polypeptide includes plasmid and other carriers.For example, carrier is suitble to include with lower class The plasmid of type: in prokaryotic cell, pBR322 endogenous plasmid, the pEMBL endogenous plasmid, pEX of such as expression in escherichia coli Endogenous plasmid, pBTac endogenous plasmid and pUC endogenous plasmid.pcDNAI/amp,pcDNAI/neo,pRc/CMV,pSV2gpt, The source pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg property carrier is adapted for transfecting eukaryon The example of the mammalian expression vector of cell.Alternatively, virus derivatives can be used, such as bovine papilloma virus (BPV-1) Or Ai-bars of Er Shi virus (Epstein-Barr virus) (source pHEBo, pREP property and p205) transient expression in eukaryocyte Polypeptide.It in some embodiments, may be desirable using baculovirus expression system expression recombinant polypeptide.Such bar The example of shape virus expression systems includes the source pVL property carrier (such as pVL1392, pVL1393 and pVL941), the source pAcUW property load Body (such as pAcUW1) and the source pBlueBac property carrier.Other expression system includes adenovirus, adeno-associated virus and other diseases Malicious expression system.

Carrier can be converted in any suitable host cell.In some embodiments, host cell, for example, bacterium or Yeast cells may be suitable as surface expression library.In some cells, carrier is expressed opposite to express in host cell Larger amount of polypeptide.Such host cell includes mammalian cell, yeast cells, insect cell and prokaryotic cell.Some In embodiment, cell is mammalian cell, such as Chinese hamster ovary (CHO) cell, baby hamster kidney (BHK) cell, NS0 Cell, Y0 cell, HEK293 cell, COS cell, Vero cell or HeLa cell.

It can allow polypeptide expression with the host cell of the expression vector transfection of coding TfR combination polypeptide Under appropriate conditions of cultivate.The polypeptide can be secreted from cell and divide from the cell containing the polypeptide and culture medium mixture From.Alternatively, the polypeptide can be retained in cytoplasm or collect cell in membrane part and using required method, dissolution and separate Polypeptide out.

VII. treatment method

TfR combination polypeptide according to the present invention can be used for treating many indications.In some embodiments In, TfR combination polypeptide is the target cell for therapeutic agent to be delivered to expression TfR.In some realities It applies in scheme, TfR combination polypeptide can be used for across endothelium, such as blood brain barrier transport treatment part through brain to absorb.

In some embodiments, TfR combination polypeptide of the invention can be used for, such as be conjugated to treatment Agent delivers therapeutic agent to treat nervous disorders, such as disease of brain or central nervous system (CNS).Exemplary diseases include Ah Hereby extra large Mo's disease, Parkinson's disease, amyotrophic lateral sclerosis, Frontotemporal dementia, vascular dementia, dementia with Lewy body (Lewy Body dementia), Pick's disease (Pick's disease), the primary age correlation tau protein disease or progressive core on fiber crops Numbness.In some embodiments, the disease can for tau protein disease, prion disease (such as bovine spongiform encephalopathy, itch, gram- Refined two Cotard (Creutzfeldt-Jakob syndrome), kuru (kuru), Jie Ciman-Si Tuosile-Shi Yinke Sick (Gerstmann-Straussler-Scheinker disease), chronic wasting disease and Fatal familial insomnia Disease), bulbar paralysis, motor neuron disease or the different neuodegenerative disorder of nervous system (such as Ka Nafanshi disease (Canavan Disease), Huntington's disease (Huntington's disease), neuronal waxy lipofuscinosis, Ya Lishan great Shi disease (Alexander's disease), appropriate Reye syndrome (Tourette's syndrome), Meng Shi Menkes Ⅱ syndrome (Menkes Kinky hair syndrome), the triumphant engler's syndrome of section (Cockayne syndrome), hallerman-Streiff syndrome (Halervorden-Spatz syndrome), good fortune Laplace disease (lafora disease), Reiter's syndrome (Rett are drawn Syndrome), hepatolenticular degeneration, Lei-receive Xi Shi mutual aid in two Cotards (Lesch-Nyhan syndrome), Fred Lack of proper care (Friedreich's ataxia), two Cotard (Unverricht- of myeloid muscular dystrophy and You-orchid Lundborg syndrome)).In some embodiments, the disease is apoplexy or multiple sclerosis.In some embodiment party In case, patient can be asymptomatic, but have marker relevant to brain or CNS disease.In some embodiments, it provides TfR combination polypeptide of the invention is in manufacturing the purposes in drug for treating nervous disorders.

In some embodiments, TfR combination polypeptide of the invention is used for treating cancer.In some implementations In scheme, the cancer is primary CNS cancer, such as glioma, glioblastoma multiforme, meningioma, star Cytoma, acoustic neurinoma, chondroma, few dendron spongiocytoma, medulloblastoma, ganglioglioma, schwann's tumor (Schwannoma), neurofibroma, neuroblastoma or Epidural cavity, in myelin or intradural tumor.In some embodiment party In case, the cancer is entity tumor, or in other embodiments, and the cancer is non-solid tumors.Solid tumor-type cancers Including central nerve neuroma, breast cancer, prostate cancer, cutaneum carcinoma (including basal-cell carcinoma, cell cancer, squamous cell carcinoma and Melanoma), cervix cancer, uterine cancer, lung cancer, oophoroma, carcinoma of testis, thyroid cancer, astrocytoma, glioma, Cancer of pancreas, celiothelioma, gastric cancer, liver cancer, colon and rectum carcinoma, kidney (including nephroblastoma), bladder cancer, cancer of the esophagus, larynx It is cancer, carcinoma of parotid gland, cancer of bile ducts, carcinoma of endometrium, gland cancer, small cell carcinoma, neuroblastoma, adrenocortical carcinoma, epithelioma, tough Band sample tumor, Desmoplastic small round cell tumor, endocrine tumors, ewing's sarcoma family tumor (Ewing sarcoma Family tumor), germinoma, hepatoblastoma, hepatocellular carcinoma, non-rhabdomyosarcoma soft tissue sarcoma, osteosarcoma, Appearance of peripheral primitive neuroectodermal tumors, retinoblastoma and rhabdomyosarcoma.In some embodiments, the present invention is provided TfR combination polypeptide in manufacture for treating cancer drug in purposes.

In some embodiments, TfR combination polypeptide of the invention can be used for treating autoimmune or inflammation Property disease.The example of such disease includes but is not limited to ankylosing spondylitis, arthritis, osteoarthritis, rheumatoid joint Inflammation, arthritic psoriasis, asthma, chorionitis, apoplexy, atherosclerosis, Crohn's disease (Crohn's disease), Colitis, ulcerative colitis, dermatitis, diverticulitis, fibrosis, idiopathic pulmonary fibrosis, fibromyalgia, hepatitis, intestines easily swash Syndrome (IBS), lupus, systemic lupus erythematosus (SLE), ephritis, multiple sclerosis and ulcerative colitis.In some implementations In scheme, TfR combination polypeptide of the invention is provided in manufacturing the medicine for treating autoimmune or inflammatory disease Purposes in object.

In some embodiments, TfR combination polypeptide of the invention can be used for treating cardiovascular disease, all Such as coronary artery disease, heart attack, arrhythmia cordis or cardiac arrhythmia, heart failure, valvulopathy, congestive cardiac Disease, cardiomyopathies, cardiomyopathy, pericardial disease, arotic disease, Marfan's syndrome (marfan syndrome), vascular diseases (vascular disease) and vascular diseases (blood vessel disease).In some embodiments, this hair is provided Bright TfR combination polypeptide is in manufacturing the purposes in drug for treating cardiovascular disease.

In some embodiments, the method also includes applying one or more other therapeutic agents to subject.For example, In some embodiments for treating brain or central nervous system disease, the method may include apply mind to subject Through protective agent, such as cholilytic drug, dopaminergic agents, Glutamatergic agent, histon deacetylase (HDAC) (HDAC) inhibitor, hemp Alkali, Caspase inhibitors, melatonin, anti-inflammatory agent, hormone (such as estrogen or progesterone) or vitamin.In some implementations In scheme, the method includes to subject's application for treat nervous disorders cognition or behavior symptom medicament it is (such as anti- Down, dopamine agonist or major tranquilizer).

TfR combination polypeptide of the invention is with therapeutically effective amount or dosage application subject.Available example Property dosage include about 0.01mg/kg to about 500mg/kg, or about 0.1mg/kg to about 200mg/kg, or about 1mg/kg is to about 100mg/kg, or the daily dose range of about 10mg/kg to about 50mg/kg.However, dosage may change according to a number of factors, Including selected dosing way, the formulation of the composition, patient's reaction, the severity of the patient's condition, the weight of subject and place The judgement of square doctor.According to the requirement of few patients, the dosage can increase with time or reduce.In some embodiments In, patient initially gives relatively low-dose, then dosage is increased to the effective dose of patient tolerance.The determination of effective dose be In the limit of power of one of ordinary skill in the art.

In various embodiments, TfR combination polypeptide of the invention is non-enteral.In some implementations In scheme, the polypeptide is administered intraveniously.Intravenous application can be for example, by through about 10 to about 30 minutes, or warp At least 1 hour, 2 hours or 3 hours time, which was transfused, to carry out.In some embodiments, the polypeptide is through intravenous push Application.Infusion also can be used and inject the combination of dispensing.

In some parenteral embodiments, TfR combination polypeptide be intraperitoneally, subcutaneously, intradermal or muscle Interior application.In some embodiments, the polypeptide is applied through intradermal or intramuscular.In some embodiments, described more Peptide is through intrathecal, such as by applying in Epidural cavity dispensing or the ventricles of the brain.

In other embodiments, TfR combination polypeptide can with oral administration, transpulmonary application, intranasal administration, It is intraocular to apply or through surface applied.Lung application also can be used, such as applied by using inhalator or atomizer, and and aerosol It prepares together.

VIII. pharmaceutical composition and kit

In another aspect, pharmaceutical composition and examination comprising TfR combination polypeptide according to the present invention are provided Agent box.

Pharmaceutical composition

In relation to prepare can be seen for the guidance of the formulation in the present invention it is known to those skilled in the art related In medicine preparation and a variety of handbooks prepared.

In some embodiments, pharmaceutical composition includes TfR combination polypeptide as described herein and goes back Include one or more pharmaceutically acceptable carriers and/or excipient.Pharmaceutically acceptable carrier is included in physiologically phase Hold and does not preferably interfere or active any solvent, decentralized medium or the coating agent of inhibitory activity agent in other ways.Various medicines Acceptable excipient is well known on.

In some embodiments, the carrier is suitable in intravenous, the intrathecal, ventricles of the brain, intramuscular, oral, peritonaeum is interior, warp Skin, surface or subcutaneous administration.Pharmaceutically acceptable carrier containing one or more can be used to that composition for example to be made to stablize, or Person increases or decreases the physiologically acceptable compound of the absorption of polypeptide.Physiologically acceptable compound may include for example Carbohydrate, such as glucose, sucrose or glucan；Antioxidant, such as ascorbic acid or glutathione；Chelating agent；It is low Molecular weight protein matter；Reduce the composition of removing or the hydrolysis of activating agent；Or excipient or other stabilizers and/or buffering Agent.Other pharmaceutically acceptable carriers and its formulation can also be used in technique.

Pharmaceutical composition described herein can be in mode known to those skilled in the art, such as by means of known mixed It closes, dissolve, pelletizing, dragee processed, emulsify, be encapsulated, encapsulating or freeze drying process manufactures.Following methods and excipient are merely illustrative And certainly without limitation.

For oral administration, TfR combination polypeptide can be by can pharmaceutically connect with well known in technique The carrier received combines to prepare.The carrier compound can be formulated as tablet, pill, dragee, capsule, lotion, Lipophilicity and hydrophilic suspensions, liquid, gel, syrup, slurries, suspension and analog, so as to for patient mouthful to be treated Clothes.Gained mixture can be optionally ground, and when necessary by mixing polypeptide with solid excipient, be suitble in addition Adjuvant post-process granulate mixture, to obtain tablet or Dragee cores, to obtain oral medicinal preparation.Suitable excipient It is such as sugared including such as filler, including lactose, sucrose, mannitol or D-sorbite；Cellulose preparation, such as corn form sediment Powder, wheaten starch, rice starch, potato starch, gelatin, bassora gum, methylcellulose, hydroxypropyl methyl cellulose, carboxylic first Base sodium cellulosate and/or polyvinylpyrrolidone.When necessary, disintegrating agent, such as crosslinked polyvinylpyrrolidone, fine jade can be added Rouge or alginic acid or its salt, such as sodium alginate.

As disclosed above, TfR combination polypeptide described herein can be formulated for through injection, example As by injecting or the parenteral application of continuous infusion.It, can be by aqueous or non-aqueous solvent, such as vegetable oil for injection Or the polypeptide is dissolved, suspends or emulsified in other similar oil, synthctic fat acid glyceride, higher fatty acids or propylene glycol ester； And known additive when necessary, is added, such as solubilizer, isotonic agent, suspending agent, emulsifier, stabilizer and preservative, by it It is configured to preparation.It in some embodiments, can in aqueous solution, preferably in the buffer of physical compatibility, such as Han Shi Polypeptide is prepared in solution (Hanks's solution), Ringer's solution or normal saline solution buffer.Injection formulation can It is provided in for example with unit dosage forms added in the ampoule of preservative or multi-dose container.The composition can be in such as in oiliness Or the form of suspension in aqueous vehicles, solution or lotion, and it can contain preparaton, such as suspending agent, stabilizer and/ Or dispersing agent.

In some embodiments, TfR combination polypeptide is to be prepared into for example to dredge in the solid containing activating agent Sustained release, control release, extended release, time controlled released or sustained release formulation in the semi-permeable matrix of waterborne polymeric Form delivering.It has determined and one of ordinary skill in the art knows various types of sustained release materials.Extended release formulation Tablet, more particles or pellet system including film cladding, using hydrophily or lipophilic materials matrix technology and contain pore-forming The tablet based on wax of excipient.According to design, Sustained release delivery system can within a few hours or time a couple of days, such as through 4,6,8,10,12,16,20,24 hours or longer time release compound.In general, sustained release formulation can be used naturally In the presence of or synthetic polymer, such as polyvinyl pyrrolidone, such as polyvinylpyrrolidone；Carboxyvinyl hydrophilic polymerization Object；Hydrophobicity and/or hydrophilic hydrocolloid, such as methylcellulose, ethyl cellulose, hydroxypropyl cellulose and hydroxypropyl methyl Cellulose；It is prepared with carboxypolymethylene.

Typically, the pharmaceutical composition in vivo applying is sterile.Sterilizing can be according to known in the art Method, such as heat sterilization, steam sterilizing, aseptic filtration or irradiation realize.

The dosage of pharmaceutical composition of the invention and required drug concentration can depend on expected special-purpose and change. The determination of appropriate administration dosage or approach is within the scope of the technical ability of one of ordinary skill in the art.It is suitble to dosage in the above Section VII It is also described in part.

Kit

In some embodiments, the kit comprising TfR combination polypeptide as described herein is provided. In some embodiments, the kit is for preventing or treating nervous disorders, such as brain or central nervous system (CNS) Disease.

In some embodiments, the kit additionally comprises one or more other therapeutic agents.For example, In In some embodiments, the kit include TfR combination polypeptide as described herein and also comprising one kind or A variety of other therapeutic agents for being used to treat nervous disorders.In some embodiments, the kit, which additionally comprises, illustrates material Material, containing about practice approach described herein guidance (that is, scheme) (such as it is related use across the blood brain of the kit The specification of barrier application composition).Although described illustrate that material typically comprises written or printing material, it is not limited to This.Such specification can be stored and be conveyed to any media of terminal user and be encompassed by the present invention.Such media Including but not limited to, electronic storage medium (such as disk, tape, cartridge, chip), optical media (such as CD-ROM) and Analog.Such media may include providing the address of the Internet website of this class declaration material.

IX. embodiment

The present invention will be more fully described by means of specific embodiment.Following embodiment is provided to be for illustration purposes only, and And it is not intended to limit the present invention in any manner.One of ordinary skill in the art will readily recognize that, thus it is possible to vary or modification is a variety of Non-key parameter is to obtain substantially the same result.A large amount of make great efforts to ensure numerical value used (such as amount, temperature etc.) is made Accuracy, but there will still likely be some experimental errors and deviation.Unless otherwise instructed, otherwise practice of the invention will use this Protein chemistry, biochemistry, recombinant DNA technology and pharmacological prior art method within the scope of the technical ability of item technology.The skill Art will completely illustrate in document.In addition, one of ordinary skill in the art are it should be apparent that the engineering for being suitable for certain libraries changes The method of making can also be used in other libraries as described herein.